Pathobiology 1992:60(suppl 1): 1—38

Abstracts

LUMINOL-DEPENDENT CHEMILUMINESCENCE OF MONOCYTES, BUT NOT OF NEUTROPHILS, DEPENDS ENTIRELY ON THE TRIGGER-MEDIATED

A R E C E P T O R F O R C - R E A C T IV E P R O T E IN (C R P ). IS O L A T E D A N D P U R IF IE D F R O M R A T M A C R O P H A G E S

EXTRACELLULAR DEPLOYMENT OF CELL-DERIVED MYELOPEROXIDASE AND K athrin A lsd o rff and V icto ria Kolb-Bachofen

REACTIVE OXYGEN SPECIES. Institute fo r Immunobiology, Far. o f Medicine. Heinrich ■Heine •U n i versify, O CSSELDO RF,

Daniel Albrecht and Thomas W. Jungl, Institute of Veterinary Virology, University of

Gernutny.

Berne, Langgass-Str. 122, CH-3012 Berne, Switzerland. Rat K u p ffc r cells arc able to endocytosc particulate ligaiuls via a galaclosc-xpccifie binding activity. Wc could recently show, that this "particle receptor" is identical to a membraneLum inol-enhanced chem iluminescence is an often-used method to d em onstrate and quantitate an oxidative b u rs t It is more sensitive than measurement o f superoxide inhibitable cytochrom e C reduction. Its drawback, however, is its dependency on several metabolic events. It is dependent on both superoxide generation, and myeloperoxidase

associated form o f the w ell known acute phase protein C-rcactive protein (CRP). This CRP is a peripheral membrane protein attached to a hypothetical membrane receptor in a C'a**dependent way. We now have used a new method . the ‘gold a ffin ity chromatography", to isolate and purify this receptor fro m isolated rat K upffer cells.

activity. Furtherm ore, it has been shown that both intracellular and extracellular events are measured. This com plexity precludes the use of the method fo r studying biological

Freshly isolated rat K u p ffc r cells were incubated w ith galactosylated bovine scrum albumin

response modification, since modulatory effects may affect any of the diverse parameters

adsorbed onto colloidal gold particles as a specific ligand fo r membrane associated CRP. By

contributing to lum inol-enhanced chemiluminescence (LCL). We have used LC L to m onitor

lysing the cells w ith detergent, several centrifugation steps and separation fro m the gold-

for signal transduction in m onocytes by antibody crosslinking of either surface constituents

labelled ligand by adding E D T A . wc obtained a receptor protein preparation.

or of cell receptor-associated ligands We now have analysed the m etabolic events leading to LCL and provide evidence that in monocytes. LCL is an entirely extracellular event requiring b oth the release o f myeloperoxidase, and me generation of superoxide. This ts in contrast to LCL of '■¡eutrophils which has both an intracellular and an extracellular

For purification wc used rat CRP also adsorbed onto colloidal gold as a direct ligand fo r the receptor protein. As described fo r the isolation, the receptor was separated fro m the ligand by E D T A after centrifugation. In an SDS gel the enriched CRP receptor protein consisted o f two bands w ith relative molecular weights o f 59 and 6 1 kPa.

com ponent. The advent of adding H20 2 to the assay allows to measure the degranulationdependent m oiety separately. As described earlier, LCL in the presence o f horse radish peroxidase and azide represents extracellular H20 2 generation m ediated by an NADPH-

From peritoneal macrophages the receptor protein could also be isolated and purified by treating the cells sim ilar as described fo r the K u p ffcr cells.

Moreover, the form er procedure allows to study

In conclusion, rat K u p ffc r cells and peritoneal macrophages express a membrane receptor fo r

degranulation kineticalty and to investigate signal transduction cascades leading to

now be m easured independently

CRP. Future investigations w ill have to show, whether a ll macrophage subpopulations possess

degranulation a n d /o r frustrated phagocytosis.

this receptor protein.

C 1992 S. Karger A G . Basel 1015-2008/92/0607-0001 S2.75/0

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dependent oxidase. U sing these m odified LCL systems, m odulation o f either event can

M ea sur em ent

of

TNFa

m R N A p r o d u c t io n o f m o n o n u c l e a r c e l l s

IS O LA TE D FR O M M U CO SA I. BIOPSIES BY Q U A N T IT A T IV E R N A -P O L Y M E R A S E C H A IN R E A C T IO N . T . A n d u s . S .R . T a rg a n * . R . D e e m *, J. S c h ö lm c r ic h , H . T o y o d a * . M e d iz in is c h e K lin ik d e r U n iv e rs itä t R e g e n s b u rg I , ‘ U C L A and ‘ C e d a rs -S in a i M e d ic a l C e n te r. L o s A n g e le s . C A 9 0 0 4 8

In c re a s e d n u m b e rs o f a c tiv a te d m a cro p h a g e s in the m u co sa a re a c h a r a c te ris tic fe a tu re

in

in fla m e d

le s io n s o f p a tie n ts

w it h

in fla m m a to ry

b o w e l d isea se .

A c tiv a te d m o n o n u c le a r la m in a p ro p ria c e lls p ro d u c e tu m o r n e c ro s is fa c to r a ( T N F a ) , w h ic h has been s h o w n to k i l l h u m a n e p ith e lia l ta rg e ts s u ch as H T - 2 9 . T h e r e fo r e , w e e x a m in e d the r o le o f T N F a in the in s itu in fla m m a to r y p ro ce ss u s in g m u c o s a l b io p s ie s . Because o n ly a s m a ll n u m b e r o f c e lls c a n be o b ta in e d fro m

b io p s ie s ,

we

d e v e lo p e d

a

m e th o d

to

m e a sure

T N F a -m R N A

by

q u a n tita tiv e p o ly m e ra s e c h a in re a c tio n (P C R ) . T N F a m essage w a s q u a n tita te d by

c o -rc v c r e s tra n s c r ip tio n and c o -a m p lific a tio n

o f c e llu la r

RNA

w it h

an

a r t if ic ia l T N F a - R N A c o n ta in in g an in te rn a l d e le tio n a fte r re v e rs e tra n s c r ip tio n .

IN D U C T IO N O F T N F -a S E C R E T IO N BY M U R A M Y L D IP E P T ID E C O N JU G A T E D T O AN A N T I-M A C R O P H A G E M O N O C L O N A L A N T I­ B O D Y , bv Régine A U D R A N » . Patrick MIDOUX*>, Annie-Claude R O C H E b, Michel MONSIGNY*» and Louis T O L'JA S*. ( a : D e pa rte m e nt d c B io lo g ic , C entre R é gion a l de L u tte co ntre le Cancer, BP 6 27 9, 35062 Renncs-cedex. France and D épartem ent de B io c h im ie des g lycoco n ju gu és et le c tin e s endog è ne s. C e n tre de B io p h y s iq u e m o lé c u la ire . C N R S , IN S E R M et U n ive rsité , I , rue Haute. 45071 O rlé a ns-ce d cx 0 2 . France). M u ra m y l d ip e p tid e (M D P ) targe tte d w ith an a n ti-m acrop h ag e m o n o c lo n a l a n tib o d y ( M A b ) m ig h t be used as a cance r tre atm e n t. T h is h ypo the sis w as firs t in ve stig a te d using a mouse e xp e rim e n ta l m o d el. A ra t anti-m ou se M a c-1 M A b ( M A b 3 A 3 3 ) was ra d io la b e l le d . in je cte d in tra ve n o u sly in to m ice and fo u n d to b in d to m acrophages and to segregate p re d o m in a n tly in in fla m m a to ry f o c i and tu m o r g ra fts. ( C o lle t e t a l. C ancer Im m u n o l. Im m n n o th e r., 1988. 2 & 2 3 7 -2 42 ). M A b 3 A 3 3 was n ext substituted w ith 5 m o le cu le s o f a n eu tra l p o ly p e p tid e (p a r tia lly g lu c o n o v la te d p o ly - L - ly s in e ) b ea rin g 15 M D P m o ie tie s fo r m in g M A b 3 A 3 3 -(-S S -.G lr.A i8 0 * * M D P j$ -P L K )5 com plexes. T h is conjugate induced c y to to x ic ity o f mouse p eriton ea l macrophages, and w as m o re e ffic ie n t than the va rio u s re le v a n t c o n tro ls ( M id p u x et al. B io c o n ju g a tc C h em .. 1992, & 194-199). R e cen tly, a s im ila r co m p ou nd was prepared u sing a m ouse a n ti-h u m a n C D l l b M A b ( k in d ly g ive n b y Im m u n o tc c h ) as a v e c to r. A n t i- C D U b M A b -(-S S -. G lc A 6 5 * .M D P 3 0 -P L K ) 2 co n ju g a te was tested f o r its a b ility to in d u ce T N F - a s e c re tio n in c u ltu re d h u m a n m o n o c y te s . T N F - a w as assayed w it h a c y to to x ic ity test u sing a c tin o m y c in D -trcate d L 9 2 9 c e lls . M o n o c y te s o bta ine d fro m b lo o d m o n on uclea r c e lls were le ft to adhere fo r 8 hours, to a llo w spontaneous T N F - a release to be co m p le te d , b e fo re a d d in g M D P -c o n ju g a tc s . A n t i - C D l l b M A b -M D P c o n ju g a te in du ce d the se cre tio n o f a c y to to x ic fa c to r, the a c tiv ity o f w h ic h was abolished b y a n ti- T N T -a a n tib o d y. In co n tro l e xpe rim en ts using u n sp e cific a n tib o d yM D P conjugates, no such fa c to r c o u ld be detected. Taken together, the present resu lts suggest that the a n titu m o r a c tiv ity o f m o n ocyte -m a cro ph a ge m ig h t be stim u la te d in v iv o b y M D P ve cto rized b y sp e c ific antib od ie s.

U s in g o u r q u a n tita tiv e P C R m e th o d w e d e te cte d m e a s u ra b le T N F a R N A in as fe w as 104 la m in a p ro p ria m o n o n u c le a r c e lls . T h e le v e l o f T N F a m essage w a s a p p ro x im a te ly 8 fo ld

in cre a se d in c e lls

fr o m

in fla m m a to r y

le s io n s o f C D

p a tie n ts c o m p a re d to n o rm a l m u co sa . In c o n c lu s io n q u a n tita tiv e P C R a llo w s the m e a s u re m e n t o f m R N A c o n c e n tra tio n s o f c y to k in e s in m u c o s a l le s io n s a nd the s tu d y o f the r e g u la tio n o f c y to k in e m R N A s in m o n o n u c le a r c e lls is o la te d fro m m u c o s a l b io p s ie s .

IN FL U E N Z A A VIRUS IN FE C TIO N OF MACROPHAGES: IN D U C T IO N OF TWO DIFFERENT TYPES OF T N F -a mRNA

NF KB: A TRANSCRIPTIONAL ACTIVATO R A N D COORDINATOR OF IMMEDIATE-EARLY

GENE EXPRESSION IN

MACROPHAGES

Patrick A- Bacuciic. Ralf Schreck, Judith M üller and Loms Ziegler Hcttbrockl M.Bacher. H.Sprenger. A.Bender. A.Rischkowsky, M .N ain and D.Gemsa

Gene Center, Ludwig-Maxim llians-University Munich. Am Klopferspiiz 18a. D-8033 Martinsried,

Institute o f Immunology. Philipps University, Marburg. Germany

and 1Institute fo r Immunology. Gocthestr. 31, D-8000 Munich, Germany. Activation o f macrophages, for instance, in response to lipopolysaccharidc (LPS) and TNF, leads to

Infection o f murine PUS-1.8 macrophages and human monocytes by influenza A virus, strain Puerto Rico 8 ( H IN I) , is associated w ith release o f tumor necrosis factor-a (TN F-a) and other

a rapid and coordinate induction o f many genes encoding cytokines and other effector molecules of the inflammatory response. The coordinate induction o f a group of these genes appears to be achieved by the use o f the same inducible transcriptional activator. There is evidence that nuclear

cytokines. On a molecular level, Northern blot analysis detected a massive T N F -a mRNA

factor kappa B (NF-k B )

accumulation which consisted o f a regular and an additional, high molecular weight (hmw)

G-CSF, GM-CSF. M-CSF. IL-3, tissue factor and IL-2R a-chain. A ll these genes contain in

mRNA species. In this study we were interested in characterizing hmw T N F -a mRNA. We found that hmw T N F -a mRNA could be detected by plasmid-derived as w ell as by PCR-amplified, exon-specific cDNA probes. The presence o f genomic in iron sequences, which characterize a prim ary T N F-a mRNA transcript, was ruled out by failure to hybridize with intron-spccific

is

involved in the induction o f the genes encoding TN F-a. EL-1,11-6. IL -8.

enhancer elements functional binding sites for NF-kB cooperating with other factor binding sites in gene induction. NF- k B is an inducible transcription factor that is activated when many different cell types art exposed to TNF. IL-1. LPS. viruses. U V light, v rays and m ild oxidative stress. A hallmark o f NF-k B is that, in non-stimulated cells, it resides in the cytoplasm in complex with the inhibitory subunit I k B. Stimulation o f cells results in release o f I k B, nuclear transport o f N F- k B and finally

T N F -a cD N A probes. Several other possibilities were taken into consideration: the two T N F-a

binding o f NF- k B to promoter and enhancer elements in genes - an event causing the initiation o f

mRNA species may be generated by alternative splicing o r may deviate in different 3' and

mRNA synthesis from genes.

5'ends. W ith the use o f rapid amplification o f cD N A ends (R ACE) we amplified regions between

TNF is known to induce the production o f Superoxide and H 2O2 in cells. This prompted us to test whether H 2O2 itself is capapble o f inducing NF-k B. Micromolar concentrations o f H2O2 could

defined single point transcripts and 3’ o r 5'ends. Extension o f cDNAs from the end o f the

indeed potently activate N F- k B in a human T cell line and the activation was blocked by

message to specific primer sequences was accomplished by primers annealing to the natural 3'end

N-acctyl-L-cysteine (NAC). dithiocarbamatcs and non-sulfur irpn/copper chelators. This shows that

o r synthetically prolonged poly A 3 ' tails. Whereas the 3’ RACE showed no difference between

NF- k B is an oxidative stress-responsive factor. Various antioxidants also blocked induction o f NF-k B by LPS and TN F in tnacrophages/monocytes. 'Diese results suggest that reactive oxygen

PCR-amplified 3 'cD N A ends o f regular and hmw T N F -a mR NA, additional hmw products were

intermediates play a role as messengers or modulators in the activation o f NF-k B by LPS and other

detected during the 5'R ACE. These findings demonstrate that upon influenza A virus infection o f

agents. Antioxidants (N AC etc.) may thus turn out as phitrmceutics with the potential to suppress activation o f NF- k B and. thereby, activation o f macrophages and monocytes.

macrophages, a 5'end-elongated T N F -a mRNA was generated which w ill be further

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characterized with respect to base pair sequence and possible biological activity.

NITRIC OXIOE (NO) PRODUCTION BY SPLENIC MACROPHAGES IS RESPONSIBLE FOR THE DEPRESSION OF LYMPHOPROLIFERATIVE RESPONSES OBSERVEO AFTER BURN

Activated microglia in vivo rapidly synthesize Alzheimer/?A4amyloid precursor protein (APP)

INJURY IN RATS T- Bamberger*. J. M nlhliiu**. I

M asson*..M

De Sousa*. B. Chanaud. S. Strzalko*. R .B . B anati . J. G chrm a nn . C . Czech, U . M ö n nin g . L . L . Jonc.% O . K önig, K.

L ChauvekM-Moachon. J.P. Glroud and I. Florentin. •Dôpariomoni de Pharmacoloqio (URA CNRS 595). Hôpital Cochin. Paris. ’ ’ Département de Radlobiochlmte. Centre de Recherche du Sorvico do Santé des Armées. La Tronche. France. Impairemont ot cell-mediated Immunity dovolops early after burn injury and likely

B cyrcu th cr and G .W . K rcu tzb crg Department Of Neuromorphology, Max-Planck-In&titulc of Psychiatry, D-8033 Murtiiuric«] near Munich; Center for Molecular Biology Heidelberg (ZM BII). University o i lleulclhcrg. Im Ncucnhcimer Feld 282. IT6900 Heidelberg. Federal Republic of Germany.

is at tho origin of froquont septic complications. T suppressor cells and low molecular wolght suppressor factors, including prostaglandin E2, have been implicated. We show that tho defective lymphoproliloralivo responses to T coll mitogens (PHA and Con A) obsorved attor thermal injury In rats can bo restored by macrophage depletion (adherence) of spleen cells or by the action of chloroadenosine which is toxic for macrophages (Masson ot ai. abstract in the same issue). In tho present work, wo investigate whether nitric oxide (NO) could be tho mediator of macrophago supprossrve activity In tho first part of this work, we demonstrated that ihe NO donors, molsidomine and sodium nitroprussido. Inhibited, in a dose dependent manner, the proliferative responses to T coll mitogens ot normal spleen cells from Wistar rats. Spleen cells from somo rats responded poorly to mitogens. The addition to the cultures of inhibitors of the NO synthase. (N G -m onom ethyl-L-arglnine.

N n ilro L-arginlno

mothyt

ostor

and

L-

canavanine)

allowed the increase of the responses lo the level o l high responder rats Lymphoproliférative responses aro strongly doprossed 3-4 days after thermal in|ury. They are fully restored when spieon colls are cultured in presence of NO synthase inhibitors. In parallel. NO release was measured (by dosage ot nitrites with the Grless rcagont) in the supernatants of spleen cells stimulated with Con A. We observed a correlation between impaired responses to mitogens and increased production of NO. These results demonstrate that in rats. NO is an immunosupressivo agent that is responsible for impaired lymphocyle reactivity to mitogens In normal and pathological situations.

C E LLU LA R CO M I*O Sm O N IN TH E PERITONEAL C A V IT Y A N D CYTO TO XIC FUNCTION O F PERITONEAL MACROPHAGES FROM PA l l ENTS W ITH O VA R IA N CANCER; EFFECT

Upon acute activation microglia, the i mm uneffector ceils o f the brain parenchyma express the amyloid precursor protein (APP) that is otherwise prominent in pathological structures related to Alzheimer’s disease. In this disease complex amyloid-bearing neuritic plaques contain 0A4-amyloid protein, the amyloid precursor protein (APP) and inflammatory proteins. The accompanying activation o f microglia has mostly been viewed as a secondary reaction to amyloid deposits. Activation of microglia was performed in a graded fashion. Transection of peripheral nerves such as the facial or sciatic nerve causes a microglial reaction within hours in the nucleus of origin or in projection areas o f the C N S . A predominantly glial up-rcgulation of APP m RNA and protein could be detected as early as 6 h post lesion not only at the site of affected neuronal cell bodies but also in corresponding projection areas. Its time course suggests rapid transneuronal signalling to glial cells in the projection area. Light and electron microscopy demonstrate that microglia, which are ceils of mononuclear phagocyte lineage and comprise up to 20% of all glial cells, are the dominant source for non-neuronal APP-exprcssion. Ultrastructurally, brain perivascular cells within the basal lamina constitutively express APP and thus are a possible source o f vascular amyloid. Microglia express leukocyte-derived L-A PP m RNA and protein that have recently been described in mononuclear cells of the immune system. Increased L-APP-cxpression may serve as a potential marker for glial/microglial activation. Such immune-mediated amyloidogcncsis initiated by microglia might have implications for the treatment o f neurodcgcncrative diseases.

IS O L A T IO N A N D C H A R A C T E R IS A T IO N OF A N TIG EN PRESENTING CELLS FRO M THE H U M A N P ER ITO NE A L C A V IT Y M .G .H B elies. C .W . T u k . * D .G . S tru iik .

R .H J , B e e le r

O F T U M O R NECROSIS FA C TO R -o T R E A T M E N T IN V IV O

D e p t. C e ll b io lo g y . V rije U n iv e rs n o it. A m s te rd a m C e n te r. A m s te rd a m , T he N e th e rla n d s .

R H LJJcsJyn. C .D . Richters, A .A . van dc l.oosd re chr, C .W . Burger1. G J .M . Eppink,

T h e a im o f th is s tu d y w a s to a ssess the a n tig e n p re s e n tin g c a p a c ity o f h u m a n p e rito n e a l c e lls (PC) and e s p e c ia lly th e ro le o f d e n d ritic c e lls (DC) ve rsu s m a cro p h a g e s (M2'

releusc in differentiated but not in resting U-937 cell* (0.62 ± 0.13 nmoles O y /106 cells compared to

p e r i p h e r a l m a c ro p h a g e s . M o r e o v e r ,

o r. c y t o k i n e

in d ic a t e

fro m

to

ra ts ,

a n d m a c ro p h a g e s w i t h

fin d in g s

d e r iv e d

In

a n i»

15% FCS and 5% newborn calf scrum (NCS), and 5% PCS and 5% NCS. respectively. Cell

or

CD4 a n d MHO c l a s s

s u rfa c e m a rk e rs .

These

a lt h o u g h

( S IV )

c a l c i u m e l e v a t io n

or phorbol mynstatc acetate ol in press. 1992)

P2 1 * known t o p ro d u c e t h e I n d u c t i o n o f n e u r it is

(E A K ).

d o n y o l ln a t ln g p o ly n e u r o p a t h y t h a t , a nd in f la m m a to r y le s io n s ,

EAN i s

fo r it s

7 of which are also phosphorylated m response to the protein kinase C activator. PMA (M ol

s im ila r c l i n i c a l fe a tu r e s

Cell Biochem 86:163. 1989)

release was examined. w a n te d t o

t h o PNS d u r in g EAN, we

f i n d o u t w h e th e r m o n o n u c le a r p h a g o c y te s w e re a b le t o

g e n o r a te p r o in f la n s n a t o r y m e d ia t o r s upon I n t e r a c t i o n w it h

P2 .

For

t h i s p u r p o s e wn t r e a t e d human c u lt u r e d m o n o c y te s o r m o n o c y te - d e r iv e d m a cro ph a ge s

(MDM) w i t h p u r i f i e d b o v in e P2 p r o t e i n ,

c o m p a ris o n , w i t h

LPS.

p u r if ie d

r a b b i t P2 p r o t e i n , in

a b o lis h e d t h e a c t i o n o f LPS. u n a b le t o t r i g g e r t h e

I n a d d it io n ,

A maximal inhibition of 55% was observed at 100 pM H-7. the highest

concentration tested

Pretreatment of the AMO with staurosponne also resulted in the dose-

dependent inhibition of tannin-mediated C204 release

The EC50 for staurosponne inhibition

was 690 nM and a maximal inhibition of 65% was observed at 1 pM staurosporine. the Dose-response curves for the tannin-mediated release of C204

of C20 4 in the presence of staurosporine was uniform over the entire tannin dose range Finally, because exposure to tannin resulted m the loss of the ability of the AMO to reacylate C20 4. the effect of staurosporine on reacylation in tannin-treated cells was examined a

a n d was n o t I n f lu e n c e d b y p o ly m ix ln B, p a r a l l e l e x p e r im e n t s ,

The EC50 for this

mhiDition was 35 pM

highest concentration tested

(TNF) and

C y t o k in e p r o d u c t io n was a ls o o b t a in e d w i t h

w h ic h , on t h o o t h e r h a n d ,

Pretreatment of the AMO with H-7 resulted m the dose-dependent

inhibition of the subsequent ability of tannin to evoke the release of C20 4

performed in the presence or absence of 1 pM staurosponne indicated that percent inhibition

in d u c e d a tim e d e p e n d e n t raRNA

a c c u m u la t io n a nd s e c r e t i o n o f Tum or N e c r o s is F a c to r (1 1 .- 8 ) .

t o make a

N o r t h e r n b l o t a n a ly s is a nd s p e c i f l o ELISAs

d e m o n s tra te d t h a t t h e P2 p r o t e i n

I n t e r l o u k i n -8

and,

To test this hypothesis, the

effect of two protein kinase inhibitors. H-7 and staurosponne. on tannin-mediated C20 4

S in c e m a cro ph a ge s a r e th e

p re d o m in a n t I n f la m m a t o r y c e l l t y p e p r e s e n t I n

Together these data suggested that tannin-mediated release

of C204 from AMO may involve activation of protein kinases

r e p r e s e n ts a c lo s e a n im a l m o d e l o f a c u te

human G u i l i a i n - B a r r e • s y n d ro m e .

Other studies from our laboratory

have shown that exposure to tannin results m the phosphorylation of at least 9 AMO proteins.

an in f la n o n a t o r y

a lm o s t c o m p le t e ly

was paralleled by a restoration of reacyiatmg activity

In conclusion, these data suggest that

the mechanism by which tannin promotes the release of C2o 4 from AMO involves the action of a protein kinase and lhat the action of this kinase is involved Ihe inhibition of the ability of the tannin-exposed cells to reacylate C20 4 into ihe membrane lipids

P2 p r o t e i n was fo u n d

r e le a s e o f H2O2 b y m o n o c y te s o r b y MDM, even

These

studies revealed that staurosponne-dependent inhibition of tanmn-mediated C20 4 release

Although these studies

do not directly identify the protein kinase mvovled in mediation of C20 4 release, protein kinase C is a likely candidate based on the similarities in ihe previously observed pattern of

th o u g h p h a g o c y te s h ad b ee n p r e v io u s ly a c t i v a t e d b y an e x p o s u ro t o IF N - y f o r 2 4 -4 8 h , b e f o r e

O ur r e s u l t s

proteins phosphorylated by tannin and Ihe protein kinase C activator. PMA.

c o u ld s u g g e s t

r e le a s e o f p r o in f la n o n a t o r y c y t o k in e s b y human p h a g o c y te s ,

u pon s t i m u l a t i o n w i t h

P2 p r o t e i n ,

may have an im p o r t a n t r o l e i n

e f f o c t o r m e ch an ism s u n d e r ly in g d e m y e lin a t io n d u r in g

th e

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th a t th o

P2 s t i m u l a t i o n .

P R IM IN G

AND

REGULATION

OF

THE

SYNTHESIS

CYTOTOXIC MEDIATORS IN

AND

FORMATION

OF

ADOPTIVE IMMUNOTHERAPY OF WALKER-256 CARCINOSARCOMA WITH ACTIVATED MACROPHAGES BY C.PARVUM. INTERFERON AND PHOTO­

L IV E R MACROPHAGES

DYNAMIC THERAPY . P.

D ie te r .

P.

A m bs,

U.

A r lt,

J.

D u y s te r,

E.

F itz k e

and

H.

V.F.Oima* , V .V a siliu ^ , D .laky^ , P.Ionescu4

S ch w e n d e I n s t jt u t

fu r

F r e ib u r g .

M o lo k u l a r e

Z e llb io io g ie ,

M o o s w a Id a 1 l e e

1 -9 ,

CantacuzIno-InstUute (1 ), In stitu te o f Atomic Physics (2)

7800

V ictor Babes In stitu te (3 ), Central M ilita ry Hospital (4)

G e rm a n y

Bucharest - Romania . M a c ro p h a g e s

a ro

kn o w n

a c t i v e m e d ia t o r s n itr ic

o x id e .

fo r m a tio n in d u c e d

is

an

by is

LPS ,

and

s e ru m

is

A d d it io n

e ffe c t

to

of

on

in

t h e ir

to

in o s ito l

of

in o s ito l h o w e v e r,

LPS

to

p h o s p h a te s LPS l e d

The e f f e c t

th e

r e le a s e

is

under

of of

a nd

sh o w n

th e

th e

to

has and

been n itr ic

p h o rb o l

th e

th e

fo r m a tio n

of

to

s e ru m

c u lt u r e d and

a ro

le s s

not

and

p h o s p h a te s s e ru m . had

in f lu e n c e

p h o s p h a te s a n d PGE2 b u t p r im e d

no th e

in

th e

p re s e n c e

th e

fo r m a tio n o f

a 2 -5

LPS a n d

c u lt u r e d

s u p e r o x id e fo ld

w as

in c r e a s e

in

o th e r co m p ou nd s

e ic o s a n o i d s ,

in t r a c e llu la r

c o m p o n e n ts

p a th w a y s

PKC,

s u p e r o x id e ,

NADPH o x id a s e )

in w ill

s lig h tly th e

(e .g . n itr ic

A d d itio n a lly , in v o lv e d

The a ctiv a tio n degree o f macrophages was evaluated by means o f the four experimental parameters. We used 4 batches o f animals with Walker-256 carcino­ sarcoma. Control and treated animals were studied 45 days a fte r tran splant. The obtained resu lts revealed the follow ing : a) macrophages activated log ical a c tiv ity ( phagocytary, metabolic and cyto toxic ) ; b) they inhibited tumor evolution ; c) an increased survival r a te , e .g . 78.31 in the batch treated with C.parvum-AM0 ; 70.61 in IFN-AM0 as compared to 65.95 survival rate in animals within the batch treated with PDT-AM(5 and in contrast with the survival rate o f rats within the untreated ( control-batch ) Walker-256 tumours, which was 13.91 . Adoptive imuunotherapy with C.parvum, IFN and PDT activated macrophages induced tumor regression in 11* 3.5 days and th e ir disappearance a fte r 22* 5.3

th e p h a g o c y t ic a c t i v i t y ; and

therapy ( PDT-AM0 ) .

m a c ro p h a g e s

fo r m a tio n .

m a c ro p h a g e s

with Walker-256 tumours, 12 days a fte r transplant and activated by photodynamic

of

a c tiv e

p re s e n c e o f

macrophages ( C.parvum-AM{i ) as compared to macrophages prelevated from rats

with 3 inmjnopotentiators ( C.parvum, IFN-gamma and PDT ) had a strong b io­

e ic o s a n o i d s

m a c ro p h a g e s

d id

th e s e

e ffe c ts

in o s ito l

th e

s ig n a l

some o f

The paper aimed a t studying the antitumor e ffe c t s o f in v itro Interferon activated macrophages ( Mu rIFN-garma-AM0 ) or in vivo with C.parvum activated

PKC

m e d ia t o r s .

r e le a s e

in

be and

e s te r.

d ir e c t

th e s e

r e le a s e

a c tiv ity

of

and

absence o f

to

can

s u p e r o x id e

in t r a c e llu la r

p r im in g

and

th a t

o x id e

zym osan o r

s e ru m -fre e

in v e s tig a tio n .

(e .g .

s u p e r o x id e

it

s y n th e s is

c a p a c it y

p h a g o c y t ic

s e ru m h a d n o e f f e c t o n

-

th e

a n e n h a n c e d s u p e r o x id e

A d d it io n

to

b io lo g ic a lly

of

in

th e

of

e ic o s a n o id s .

m a c ro p h a g e s c u l t u r e d

LPS

th e

fo r m a tio n o f fo r -

le a d in g

a rra y

g e n e r a t io n

by

zym osan p a r t i c l e s

w h ile

c o m p a r a b le

e .g .

we d e s c r i b e

M a c ro p h a g e s c u l t u r e d

s u p e r o x id e

th e

com ponent

on

t o p h a g o c y to s e

w id u

e ic o s a n o i d s

w h ile

p a th w a y

H e re

-

LPS

e s s e n t ia l

tra n s d u c tio n

a

m a c ro p h a g e s

t r ig g e r e d

m e d ia t o r s .

-

liv e r

c y to k in e s ,

e .g .

e ic o s a n o i d s

r e le a s e

such as c y to k in e s ,

In

of

to

th e th e

enhancod;

r e le a s e T N F -a , o x id e

of

of

PGE5.

y -IF N ) and

T N F -a

e x p r e s s io n d iffe r e n t

on

of

s ig n a l

days since the beginning o f the treatment. Conclusions : in animals treated with adoptive immunotherapy the following were noticed : a) an increased b io lo g ica l a c tiv ity o f macrophages a c­ tivated with the 3 immunopotentiators (C.parvum, IFN and POT); b) an increased survival rate and c) a s ig n ific a n t antitumor a c tiv ity varying from a reduction of tumor volume to complete recovery . Being sim ple, safe and lacking side e ffe c ts a ctive immunotherapy with photobioactivated macrophages might be extensively used in oncologic c lin ic s fo r cancer treatment .

be s t u d ie d .

MEMBRANE ASSOCIATED C-REACTIVE PROTEIN EXPRESSION

MODULATION

BY MACROPHAGES

U937

Christine E gcuhofcr. Karin Fchscl and V ictoria Kolb-Bachofen I n s t it u t e f u r I m m u n o b io lo g y , F a c . o f M e d ic in e , H e in r ic h - H e in e - 1 J n iv e r s it y , D l/S S F . I . I X ) R F , G e rm a n y .

OF

INTERCELLULAR ADHESION MOLECULES

MONOCYTIC

R a c d e r,

E .1,

CELLS H o ff.

BY

GLUCOCORTICOIDS.

T . 2 . S p e n c k e r,

a n d G oppo I t —S t r t ib o , M. 2 * F ra u n h o fe r I n s t i t u t e H a n n o v e r, 2 M e d ic a l S c h o o l H a n n o v e r , D e p t , The

hum an

m o n o c y t ic

c e ll

lin e

T .2 .

ON

TP A-INDUCED

Emmc n d t t r f f o r . __ .

L o h n a n n - H a tth e s ,

M .-L .1,

D e p t , o f Im m u n o b io lo g y o f M o le c u l a r P h a rm a c o lo g y U937 s e r v e d

as

m odel

s y s te m

to

C-rcacttve protein (CRP) is an acute phase marker belonging to the pentraxin fam ily. During infections o r inflammations serum concentrations raise rapidly and early in man and to a lesser degree also in rats.

m o n it o r

th e

e ffe c ts

of

g lu c o c o r t ic o id s

on

d iffe r e n tia tio n .

D iffe r e n tia tio n

o f U937 c e l l s

and f u n c t i o n a l l y

m a c ro p h a g e - 1 ik e

c e lls

We could show, that CRP occurs us a membrane-associated protein (m CRP) constitutivcly

w ith

expressed on rat live r macrophages (K u p ffe r cells), representing the galactose-spccific receptor

a

p h o r b o 1c a t e r T P A .

th e

S in c e

th e to

m o n o c y t ic

m o r p h o lo g ic a lly

w as In d u c e d b y

in c u b a tio n

w ith

of

a d h e re n t,

activity on the surface o f rat live r macrophages. This receptor mediates effectively the

in v e s t ig a te d

by

f lo w

c y t o m e t r y w h e t h e r t h e e x p r e s s io n

of

endocytosis o f paniculate ligands exposing m ultiple galactosyl groups on their surface.

s u rfa c e

lik o

in t e r c e llu la r

(IC A M -1 )

Monoclonal antibodies, which recognize a neo-epitope on rat CRP present on protein subunits but not on native serum CRP (sCRP) and polyclonal antisera against native sCRP which do not

and

th e

common s u b u n i t

g p l5 0 / 9 5

(CD 1 1 c )

recognize subunits have been used to investigate the mCRP on K up ffe r cells: Indirect

th a t

immunopcroxida.se labelling reveals positive staining o f K upffer cells by the monoclonal

u p r e g u la to d .

d u r in g

antibodies against subunits but not by the polyclonal* against the native pentumcr. The same

d o x a ro e th a s o n e

mCRP expression was found on alveolar macrophages and activated peritoneal macrophages

1 0 ” 6M)

as w ell as on rat and human monocytes a fter culture on glass surface.

m o n it o r e d

d lffo r o n tia tio n

w ith

in f lu e n c e

p ro c e s s

of

of

L F A -1 ,

c e ll

M a c-1

and

The r e s u lt s

b o th

U937

we

show

r o o lo c u lo c

c o lls

w ith

( 1 0 ~ 7M

and an

ro u n d e d s t r u c t u r e I n c u b a t io n

c y to m e tr ic

and th o w it h

p r e d n is o lo n e

w e re o it h e r

t h e T P A - in d u c e d p h e n o t y p i c a l t e r a t i o n s

b y m o r p h o lo g y a n d f lo w

18 was r e d u c e d .

CD 18)

1 0~ 6M) o r w i t h

( 1 0 ~ yM a n d

c e lls ,

a d h e s io n m o l e c u l o - l

w as c h a n g e d c o n c o m i t t a n t l y .

P r e in c u b a t io n

In te rfe re d

ke p t t h o lr Rat K up ffe r cells synthesize their own mCRP: W ith R N A -R N A in situ hybridization using a human CRP-cIonc wc obtained positive signals in the macrophages. In addition a positive in

th o

(0 - c h a in ;

U937

In

m o n o la y e r

re c e p to rs

n o n - p r o lif o r a t in g

I n c u b a t io n

TPA r e o u l t o d

a n a ly s is .

e x p r e s s io n

p ro g e s te ro n e ,

t h o T P A - in d u c e d p h e n o t y p i c

The

ICAM- 1

of

h o w e v e r,

a lte r a tio n s .

c e lls and

CD

d id

not

F u rth e rm o re ,

situ hybridization signal was also found in cryostat sections o f rat liver in both: hepatocytcs as in c u b a tio n

of

fu lly

d iffe r e n tia te d

U937

c e lls

w ith

well as K up ffe r cells. 18 hours after i.v. injection o f C. parvum K up ffe r cells and hepatocytcs

alveolar macrophages and human monocytes.

CD

1 8,

c o rre s p o n d in g

c o n c lu s io n

th e s e

In conclusion wc here describe data which show that rat live r marrophages synthesize and

g lu c o c o r tic o id s

express membrane-associated mCRP w ith a conform ation different fro m scrum C.'RP (sCRP).

and a l t e r

The mode o f expression suggests, that mCRP can also serve as a marker fo r inflam matory

to

macrophages.

com pounds.

6

r e s u lte d

d a ta

in to

a d e c r e a s e d e x p r e s s io n re d u c e d

a d h e re n c e

of

sh ow t h a t b e s id e s s e v e r a l

m o d u la t e t h e d i f f e r e n t i a t i o n

e x p r e s s io n

of

a d h e s io n m o le c u l e s .

th e a n ti- in fla m m a to r y

a nd

of

ICAM- 1

th o

a nd

c e lls .

o th o r

In

o ffo c ts

o f m o n o c y t ic

c o lls

T h i s m ig h t c o n t r i b u t e

im m u n o s u p p re s s iv e e f f e c t s

of

th e s e

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showed an enhanced hybridization signal. Extrahcpatic synthesis o f CRP was found in rat

g lu c o c o r tic o id s

RM3/1 SURFACE EXPRESSION ON MONOCYTES IS DIRECTLY CORRELATED TO

FUNCTIONAL M ORPHOLOGY O F SUBARACHNOIDAL M ACROPHAGES IN AN

GLUCOCORTICOID RECEPTOR BINDING AFFINITY.

EXPERIMENTAL ACUTE MENINGITIS M ODEL

U. Erpenstein. P. R o h d o w a ld*. C. Sorg; In stitu te o f E xperim ental D erm atology, von-

P M Faust matin' . P Krause2, R D u x*. C W Zim inctm ann*. R Dermictzcl2

Esmarch S tr.5 6 , and • P harm aceutical C hem istry. 4 4 0 0 M ün ste r. FRG 'Departm ent o f Neurology. University o f Essen and “ Institute o f Anatomy. University o f The RM3/1 antig en is expressed on a m onocyte /m acrop ha g e su bp op u la tion during Regensburg the d ow n re g u la to ry phase o f in fla m m a tio n 1. This antigon sh ow s h ig h ly increased cellsurfaco expression In the presence o f g luco co rtico ids in v itro and in vivo 1>2. W o analyzed the relation b etw o en RM3/1

antigen expression and g lu co co rtico id s o f

d iffe re n t receptor bind in g a ffin itie s. M on ocyte s, isolatod fro m hum an b u ffy coats, wore cultured for 16 h w ith

five co n ce n tra tio n s each o f tria m cin o lon e (relative

re ce pto r a ffin ity ; 1). prednisolone (1 2 ). beclom ethasone (76). dexam ethasone (100),

Polymorphonuclear leukocytes (P M N ) and cells o f the mononuclear-macrophage lineage (M M L ) play an essential role in the manifestation o f acute meningeal inllammntory response Cells o f the M M L secrete endogenous mediators, c g cytokines, o f the inllammatory reaction

flunisolide (1 8 0 ), tria m cin o lon e acetonide (3 6 1). budesonide 0 3 5 ) . beclom ethasone

in the subarachnoidal space and stimulate P M N extravasation Subarachnoidal macrophages

m o n opropionate (1 2 0 0 ), and fluticasone- 1 7-propionate (1 8 00 ). resp ective ly.

(S A M ) are one subclass o f the M M L and have receptors fo r IgG and complement W c studied

The

antigen cellsurfaco expression w a s dete cte d by fluorescence flo w c y to m e try using

the role o f S AM in an experimental acute meningitis model Meningitis was induced by

the m onoclonal a n tib o d y R M 3/1 . For all tested storoids, a co n ce n tra tio n dependent

subarachnoidal application o f the complement-C5a-fragment in rabbits W ithin one hour o f

u pregulation o f the RM3/1 antig en ( m ax. 8 5 % ) w a s recorded as com pared to co n tro l cells ( 4 0 % l W e fo u n d a sig n ifica n t co rre la tion b otw een a d e fin ite increase of

RM 3/1

positive

cells

and

the

relative

re ceptor

a ffin ity

of

the

stimmulation increased numbers o f P M N and o f cells o f the M M L were detected in the cerebrospinal fluid (CSF) by flo w cytom etry This indicates an acute inflammatory reaction

d iffe re n t After intracardial perfusion w ith a fixative and preparation o f the tissue, subarachnoidal spaces

g lu co co rtico ids,

as s h o w n

by

a P earson's co rre la tion

c o iffic ie n t

o f 0 ,8 3 .

We and meninges were investigated using transmission electron microscopy S A M were identified

conclude th a t there is a co njo in in g m echanism o f g lu co co rtico id re ce pto r bind in g and

steroid responsivnoss o f m o n ocyte s, is a suitable in v itro param eter to investig a te d iffe re n t

Im m unm odulating

e ffe c ts o f g lu co co rtico id s,

especially

fro m

a clinical

between the collagen and the pial cells In serial sections S AM and PMN were found in interaction via pscudopodia In addition S AM could be found close to o i inserting psetidopodia into the basal lamina o f the marginal glia No interaction between the S AM and the pial cells

standpoint.

were observed w ithin this acute stage o f inflammation

1Z w a d lo G. Voegeli R, Schulze O sth o ff K, S org C (1 9 8 7 ) Expl Cell Biol 5 5 : 2 9 5 -3 0 4

We conclude that in the early stages o f acute meningitis intercellular actions o f S A M and

2 Zw a d lo K lärw ässer G, Bent S. Haubeck HD. Sorg C, S chm utzler W {1 9 9 0 ) Int A rch

PMN. as well as S A M and the basal lamina o f the marginal gJia play an im portant role in the

A lle rg y A ppl Im m unol 9 1 ; 1 75 -1 80

mediation o f the inflam matory response

C Y T O K IN E IN T E R A C T IO N S IN TH E G R O W T H O F M Y E L O ID PRECURSOR CELLS W illom E.Fibbe, J.C.KIum-Nolomans. J.H.F.Falkenburg and R.Willumzo Lab. of Experim ental Hem atology. Leiden U niversity Medical Center. The Netherlands A lthough the proliferative a ction of the CSF's on responding target cells is dire ct and mediated by specific receptors on tho cell surfaco. thero aro many exam ples of interactions b etw oen d iffe re n t CSF's Several examples of synergistic or in hibito ry interactions b otw e un GM-CSF and other cytokinos w ill bo discussod The indirect synergistic a ction betw een IL-1 and GM-CSF. The addition of IL -1 alpha to bone m arrow cu ltures stim ulated w ith GM-CSF results in highor num bers o f granu lo cy­ tic colonies, having no e ffe c t on m onocytic colony g ro w th This synorgistic a c tiv ity is not present anym ore w h on CD34-purified bono m arrow cell suspensions aro used Neutralization experim ents w ith an anti-lL-6 antiserum show ed no e ffe c t o f the antiserum on gra nu lo cytic co lon y gro w th. In contrast, tho addition of an anti-G-CSF m onoclonal a ntibody com p lete ly abrogates the synergy betw een i l -1 and GM-CSF These results indicate th a t the increase in CFU-G g ro w th is due to the synergistic action b etw een GM-CSF and IL-1-Induced G-CSF. Tho direct inhibito ry interactio n betw een IL-4 and GM-CSF. As a single factor, IL-4 has no colony-stim ulating a ctivity H ow ever, on hum an bone m arrow cells stim ulated w ith GM-CSF. oddition o f IL-4 results In a reduction to 1 0-20% of tho number of CFU-M. w hile the num ber of CFU-G colonies is not a ffo ctod . This e ffo c t is prosorvod in cultures of C 0 34 -p u nfie d cells, suggesting a dire ct e ffe c t on progenitor cells. Since IL-4 d ow n regulates the production of IL-6 in m on ocyto s, w o tostod w hether addition o f IL-6 could overcom e the IL-4 induced Inhibition. A partial reversal of inhibition was observed, indicating tha t d ow n regulated production o f IL-6 m ay contribute to the IL-4 induced inhibition of CFU-M g ro w th. The porm issivo role o f IL-6 on GM-CSF stim ulated CFU-M g ro w th . Since m onocytos are potent producers of IL-6 . the role of auto-and paracrine production o f IL6 on the developm ent of colonies w as studied. The addition of an anti-IL-6 a ntibody to cultures of bone m arrow cells stim ulated w ith GM-CSF resulted in o 50% reduction in tho num ber of CFU-M. whereas the num ber of granulocytic colonies w a s una ffe cted This e ffe c t w as preserved in cultures o f CD34-positivo bone m arrow cells and could bo reversed by the addition of excess concentrations of IL-6 . Interestingly, tho addition of an a nti-IL-6 -receptor (8 0 kd chain) blocking monoclonal antibody resultod in an alm ost com pleto inhibition of m on ocytic colony form ation. This inhibito ry e ffe c t could bo partiality overcom e by adding excess concentrations of IL-6 . A ddition of IL-6 w a s able to enhance m o n ocytic colony form ation in serum-deprived cultures. These results show tha t IL-6 is a porm issivo factor for m onocytic colony g ro w th. Conclusion: The d iffe re n t Interactions b etw een g ro w th facto rs in bone marrow cultures illustra te a com plex n e tw o rk, where enhancing or inhibiting activities depond on the lineage o f the p rogeni­ to r cell, the type of g ro w th facto r used to induce colony form ation, and tho prosortce of accessory colls. Many a c tivitie s of HGF are duo to in term ediate production of othor HGF. tha t m ay serve as synergistic, inhibitory or pormissivo factors.

EFFE C TS O F II.-1 0 O N H U M A N M O N O C Y T E F U N C T IO N . C a rl G. F ig d o r. R e né d c W a a l M a le f ijt * . A n je A . t c V e ld e . Ja n E. d e V r ie s ’ D iv is io n o f I m m u n o lo g y . T h e N e t h e r la n d s C a n c e r I n s t it u t e , P le s m a n la a n 121. 1066 C X a n d ’ D N A X R e s e a rc h

A m s te r d a m . T h e N e th e r la n d s .

I n s t it u t e o f M o le c u la r a n d C e llu la r B io lo g y .

P a lo A lt o . C A 9 43 04 -1 1 04 . U.S.A.

W c p r e v io u s ly o b s e r v e d t h a t IF N y a n d IL-4 h a v e d if f e r e n t r e g u la t o r y e ffe c ts

on

tw o

d if fe r e n t

m o n o c y te

m e d ia t e d

fu n c t io n s :

a n t ig e n

p r e s e n t a t io n , a n d a n t ib o d y m e d ia t e d c e llu la r c y t o t o x ic it y (A D C C ). T h is is r e fle c te d b y t h e c e ll s u rfa c e e x p r e s s io n o f M H C c la s s II a n d Fey r e c e p t o r s , r e s p e c t iv e ly .

IF N y s t im u la t e s e x p r e s s io n o f th e s e m o le c u le s a t t h e c e ll

s u rfa c e o f m o n o c y t e s w h e r e a s IL -4 s t im u la t e s M H C c la s s II e x p r e s s io n b u t re d u c e s

Fey

r e c e p t o r e x p r e s s io n . W c

now

o b s e rv e d

th a t

a ls o

IL - 1 0

d r a m a t ic a lly a lt e r s m o n o c y t e f u n c t io n . W e o b s e r v e d t h a t in c o n t r a s t t o IF N y a n d IL -4 t h i s c y t o k in e d o w n r e g u ia t e s M H C c la s s II e x p r e s s io n , w h ic h is a s s o c ia te d w it h d im in is h e d a n tig e n p r e s e n t in g c a p a c it y . In a d d it io n w e d e m o n s t r a t e d t h a t IL -1 0 is a ls o p r o d u c e d b y h u m a n m o n o c y t e s a n d t h a t it h a s a n a u t o r e g u la t o r y f u n c t io n w it h

re s p e c t t o t h e s y n th e s is o f o t h e r

m o n o c y t e p r o d u c e d c y t o k in e s . F in a lly w e o b s e r v e d t h a t IlrO s t im u la t e s Fey re c e p to r-1 e x p r e s s io n , w h ic h c o r r e la te s w it h in c re a s e d A D C C a c t iv it y . T o g e t h e r , th e s e r e s u lt s s h o w t h a t IF N y. IL-4 a n d IL -1 0 h a v e d if fe r e n t r e g u la t o r y e ffe c ts o n t h e a n tig e n p r e s e n tin g c a p a c it y a n d A D C C a c t i v it y o f h u m a n m o n o c y te s .

7

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RM 3/1 antigen surface e xpression.Thus, the RM 3/1 antig en , w h ic h e vid e n tly im plies

THE

HOVEL

SEPSIS

SUBSET OF

PATIENTS:

B lu m e n s te in 4 .

C D 14*/C D 16*

g,

M.

'I n s t it u t e

fo r

M e d ic in e .

K lin ik u m

BLOOD

F in a e r le » ,

A.

S t r ô b e l* ,

and

Im m u n o lo g y .

MONOCYTES

P fo rte * . H .W .L .

G o e th e s tr.

IS

B.

EXPANDED

IN

P a s s lic k * .

M.

MACROPHAGES AS TARGET AND EFFECTOR CELLS DURING IN V ITR O INFECTION BY TOXOPLASMA GONDII. THE INTERACTION W ITH HELPER T CELLS DETERMINES MACROPHAGE ANTIPARASITIC ACTIVITY

Z io g le r - H e it b r o c k ' .

31, •

*D e p .

In to rn a l

H.G, Fischer. G. Reichmann. B Nitzgen. K, Oick. W. Daubener and U. Haddlng, Inst. tOr Med, Mikrobiologie und Virologie. Heinrich-Heine-Universrtfit. D 4000 DOsseldorl, FRG.

4 Dep I n t e r n a l

3 D ep. S u r g e r y .

In n e n s ta d t.

M e d ic in e .

K lin ik u m

K lin ik u m

In n e n s ta d t

G ro B h a d e rn . U n i v e r s i t y

of

M u n ic h .

To analyze intercellular signals which form the im mune response In toxoplasm osis a murine in vitro model has been established by co-cultures of m acrophages (M©) with T

8000 M u n ic h .

Germany

S t a in in g w i t h 2 m o n o c y te

cells and viable parasites. Tachyzoites of the highly virulent Toxoplasma g ondii strain BK

CD14 a nd CD16 m o n o c lo n a l

s u b p o p u la t io n s

r e g u l a r m o n o c y te s ,

w h ic h

in

human

s tr o n g ly

a n t ib o d ie s

b lo o d :

a m a jo r

e x p re s s e s

th e

w i l l id e n t if y

were used at a lethal dose permitting at least one cycle of intracellular multiprication during

p o p u l a t io n

of

the assay. As appropriate targets peritoneal exudate Ms wore preferentially infected and proved to control parasite growth following interaction w ith T cells. A s regulatory elements

and a

within this 3-party system Toxoplasma-specific C D 44- T cell lines o r clones were introduced

CD14 a n t ig e n

which had been derived from mice Immunized with Toxoplasma lysate antigen: 3Tx T line m in o r

p o p u l a t io n

weak e x p r e s s io n In

s e p t ic e m ia

p o p u l a t io n a nd w i t h r e g u la r

(4 2 of

30 c e lls / m m * .

CD14 and s t r o n g

p a tie n ts ,

w it h

m ore

m ore th a n

m o n o c y te s

d e n s it y

t

in

th e s e

th a n

show ed

e x p r e s s io n

50 % o f

a

% of

CD 14*/C D 16*

250 c e l l s

9 /1 1

9 .7

in

a ll

7 /1 8

of

c e lls

cases.

m o n o c y te s )

th e

CD16 a n t ig e n .

in

3 /1 8

p a t ie n t s

The r e m a in in g

d e c re a s e

T h re e

w it h

c a n become a m a jo r

m o n o c y te s

s u b s t a n t ia l

p a t ie n t s .

a ll

c o lo r

in

CD14

CD14*4 a n t ig e n

im m u n o flu o re s c e n c e

cells recognize in the context of AabA«k MHC class II m olecules a < 31 kD common antigen of tachyzoites from both high- and low-vinjlent strains which is also contained in brain cysts of infected mice. According to their cytokine pattern, 3Tx cells and a series of clones tested represent typical T ^ 1 cells secreting high titers of IFN-r and LT/TNF-activity and no IL4 o r IL6 . whereas two T ^ 2 clones tested produced much IL4 and IL6 and only minute levels o f IFN* o r LT/TNF-activrty. W ithin the complete Infection culture, antigenic T cell activation at the one end of the reaction cascade was monitored by measuring IL3, while the growth of toxoplasm s at the other end was evaluated sim ultaneously via the uptake of tritiated uracil. As an im portant mechanism of M© toxoplasm ostatic effector function the synthesis of nitrogen oxides was

d e m o n s tr a te when

th a t

co m p a re d

th e

to

C D 14*/C D 16*

th e

C D 1 4 '•

a nd

a

m o n o c y te s

m o n o c y te s

in

s e p t ic e m ia

e x h ib it

a h ig h e r

p a t ie n t s

dem onstrated to cause total growth inhibition and to be induced by the presence of

le v e l

activated T cells. Graded paralysis of this antiparasitic pathway revealed a direct

of

correlation between the burden of infection and the level of M© activation required to c la s s

II

a n t ig e n

c o n s is t e n t L e v e ls

of

w it h

m ore

w it h

le v e ls

s e p t ic e m ia c o m p o s it io n

of may

w e re

th is

and

CD33

th e

in c r e a s e d

in

s e p t ic e m ia

C D 1 4 '/C D 1 6*

s u b s t a n t ia l may

C D llb of

( > 3 0 0 /U /m l> . to

of

n a tu re

n um be rs o f

I L -6 le a d

and

c y to k in e s l i k e

h ig h

le v e l

m a tu re

i n t e r l e u k i n -6

3 /4 p a t i e n t s h ig h

a

lo w e r

be

T h e se changes

r e la t e d

to

a n t ig e n s ,

C D 14*/C D 16*

c e lls .

p a tie n ts

and

c o lls

(>250/mm»>

had

d a ta

suggest

in

b lo o d

e le v a t e d

th a t

m o n o c y te le v e ls

of

IL -6.

A FIBROSIS INHIBITING THERAPY BY MACROPHAGE STIMULATION IN MYELOFIBROSIS AND LIVERFIBROSIS Fleischer J. W o lf H. Mohr B. Keck G: D epartm ent H aem atology/O ncology. Clinic Internal M edicine. Medical Faculty T. University, Dresden. Germany

We dem onstrated the antifib rotic and thioacetam ide liver cirrhosis-inhibiting

overcome it. The crucial role of T cell help was manifested by blocking T cell activation with an anti-la mab resulting In uncontrolled increase of toxoplasm s. In comparison, co ­ cultures of Infected M * with T ^ t cells showed a reduced parasite growth, whereas those with T ^ 2 cells failed to influence the progress of in vitro infection. Thereby the presence of T H i cells could be replaced by the cytokine cocktail produced upon mitogenic stimulation, however none of the Individual cytokines proved to provide the full antitoxoplasmic efficacy. Further studies to elucidate the cytokine-m ediated M© activation and to characterize the antiparasitic capacity of M© from different origins are in progress. Supported by the DFG. project B8 . SFB 194

ID E N T IF IC A T IO N OF MACROPHAGE ADHESION MOLECULES BY MONOCLONAL ANTIBODY STRATEGY. Ia in

F ra s e r,

S i r W illia m S o u th P a r k s

C e lls

o f th e

effe ct o f clofazim in (Cl) {Ciba-Geigy, Basel). This effe c t was referred to the

m o m b ra n e

stim ulation o f Kupffercellfunction. Therefore, Zym osan (Z) was applied

e n v ir o n m e n t ,

additionally as macrophage irritant and clearly intensified the clofazim in effect,

D e r r a ly n n

m o n o n u c le a r p h a g o c y t e

re c e p to rs as

im p lic a t e d

in

inhibitoring factor to leucotrienes or TGF Beta or these cytokines are inhibiting

m ig r a t io n

of

them selves functionally. W e determined superoxideanions, leucotriens, PG E2

s ig n a llin g ,

w orking hypothesis: on the top o f stim ulation the Kupffercells produce an

for characterisation o f Kupffercells. The levels of lipidperoxydate, hydroxyproline. N-acetyl-B-D glucosammidase. procollagen Ill-peptide in liver

H u g h e s a n d S ia m o n G o rd o n

D u nn S c h o o l o f P a t h o l o g y ; U n i v e r s i t y R oad ; O x f o r d ; 0X1 3 RE ; UK

to

w e ll

as

w it h

s y s te m

w it h

o th e r

p ro c e s s e s a s d iv e r s e m o n o c y te s

a n tig e n

in t e r a c t io n s

in te r a c t

w it h

fro m

o th e r

c e lls

u s e a r a n g e o f p la s m a

b o th

th e ir

e x tr a c e llu la r

c e lls .

These

re c e p to rs

as c o n s titu tiv e

th e

p r e s e n t a t io n

o f O x fo rd

c ir c u la tio n ,

and

(1 ).

le s s

w e ll

R a is in g

a re

and e l i c i t e d e n d o c y t o s is ,

d e f in e d

raAb t h a t

t r o p h ic

in te r fe r e

tissue confirm ed the cirrhosis-inhibiting effect of Cl and Z.

w it h th e s e f u n c t io n s p r o v id e s a s t r a t e g y f o r th e c h a r a c t e r is a tio n

W e used Cl alone in patients w ith m yelofibrosis. In single cases in cell-rich stage

of

the m egakaryocytosis and the cell content w ere diminished. The erythrocytopoiesis im proved in several patients w ith solitary loss o f necessity of blood transfusions. Now exam inations o f liver biopsy and peripheral blood especially, before and after Interferon: hydroxyproline. pro-collagen-lll-peptide. IL 1 and 6, PG E 2, TNF, leucotriens. Comparison w ith Interferon literatureresults.

th e

p a r tic ip a t in g

S c r e e n in g

h y b r id o m a

m a c ro p h a g e (2 ).

In

a d d it io n

in h ib it s have

expanded

in h ib it s

to

upon

m o le c u l e s

b a c te r io lo g ic th is

and

s tra te g y

in v o lv e d

in

m a c ro p h a g e a b ilit y

p la s t ic

in h ib ito r y

n e u t r o p h il

th is

th e th e ir

in h ib it

g e n e ra te d

mAb 5C6

a c tiv ity

r e c r u it m e n t to

id e n t if y

m a c ro p h a g e

s u rfa c e .

to

in in

v itr o . v iv o

a d d itio n a l,

a d h e s io n .

5C6

(3 ).

mAb

We

non2F8

t h e d i v a l e n t c a t i o n - i n d e p e n d e n t a d h e s io n o f m a c ro p h a g e s

tis s u e

b io c h e m ic a l a d h e s io n

on fo r

to

m a c ro p h a g e

in t e g r in

to

b in d in g

m o le c u l e s

s u p e rn a ta n ts

c u ltu r e s t u d ie s

p la s t ic of

re c e p to r w i l l

th e

s u rfa c e s . 2 F8

a n tig e n

O n g o in g as

a

fu n c t io n a l novel

and

m a c ro p h a g e

be p re s e n te d .

B g t e c f in s s s

8

S . G o r d o n e t a l ; C u r r . O p i n . I m m u n o l. 4 : 2 5 - 3 2 (1 9 9 2 ) H . R o s e n a n d S . G o r d o n ; J . E x p . M e d . 1 6 6 : 1 6 8 5 - 1 7 0 1 (1 9 8 7 ) H . R o s e n ; J . L e u k . B i o l . 4 8 : 4 6 5 - 4 6 9 (1 9 9 0 ) Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/18/2019 1:34:47 PM

1) 2) 3)

THE EFFICIENT BOVINE INSULIN PRESENTATION CAPACITY OF GM-CSF-ACTIVATED BONE

MARROW -DERIVED

MACROPHAGES

CORRELATES WITH

A

PURIFICATION AND CHARACTERIZATION OF MURINE LPS BINDING PROTEIN

DIMINISHED

P. Gallav. M P. Glauser. C. Barras. R.J. Ulevitch. P.S. Tobias. J.D Baumgartner and

PROTEASE ACTIVITY A ND A HIGH LEVEL OF INTRACELLULAR REDUCING AGENTS

D. Heumann Stofanle F ro sch . Ursula Bonifas. Hans-Peter E c k '. W D roego*. and A B. Reske-Kunz Institut für Im m unologio. J o h G utonborg Univorsität. O bere Zahlbacher Straße 67. 6500

Div.

Infectious

Diseases. CHUV,

Lausanne.

Switzerland and

Department of

M ainz 'A b te ilu n g Im m unchem ie. D eutsches K rebsforschungszentrum . Im N ouonholm or

Immunology, Scnpps Research Institute, La Jolla. CA

Feld 280. 6900 Heidelberg. B ono m arrow -derived m a cro ph a ge s (B M M ph) grow n for 10 d ays in a liq u id cu ltu re sy s ­ tem in the p resence o f M-CSF can serve as antig en presenting cells ca pa blo o f stim u la t­ ing T c lo n e cells and T hybrido m a cells with specificity for bovine Insulin (Bl) to proliferation a n d /or to IL-2 p ro du ction After activation w ith GM-CSF the m a cro ph a ge s exhibit a dra stically high e r B l-presentation ca pa city S tim ulation o f BM M ph w ith IFN-t is

The serum protein. LPS binding protein (LBP). seems to play an important role in regulating host responses to LPS. Complexes of LPS and LBP form in serum and stimulate monocytes' macrophages or polymorphonuclear leucocytes after binding to

lo ss efficient, a ltho ug h these ce lls express m uch higher levels o f M H C cla ss II m o le cules com p are d w ith G M-C SF-activated BMMph. To in vestigate w hether the d ifferent p re se n ­ tatio n ca p a b ility o f b oth m a cro ph a ge p op ulatio n s is based on a different p ro cessin g ca pa city we in hibite d the activity o f several proteases necessary for d e g ra d a tio n o f the native p ro te in antig en a nd g eneration o f the antigenic pep tide

When serine- a n d /o r

CD 14. Previous studies have described the structure and properties of LBP from human and rabbit serum Since mice are used in expenmental models of endotoxemia or Gram-negative infections, information is needed about the properties of murine LBP.

thiolp ro tea se s were in hibite d b y chym ostatin. leupeptin. or Cbz-Pho-Ala-CH N2 th e Blpre sen tatio n p ote ntia l o f iFN -r-activated BM M ph increased drastically and a pp ro xim a ted the p ote ntia l o f G M -C SF-stim ulated m acrophages, w hile the latter were n o t in flue n ced by

the

in hibito rs

U pon

Inhibition

of m e talloproleases

by

p h o sp h o ra m ld o n

Bl-

pre sen tatio n fun ctio n was w oakly o nhancod in b oth m a crophage populatio n s, w h ile In­

Munne LBP was purified by ion-exchange and HPLC chromatography and its NH2terminal sequence (TNPGLVTRIT) was very similar to human and rabbit LBP (80 to 90% amino acid identity). Murine LBP resembled LBP from other species by promoting

h ibition o f a cid ic p roteases w ith pepstatin A had no influence o n B l-presentation. F u r­ therm ore. w h en wo p ulsod BM M ph w ith Bl In the presence o f ch loroq u ine o r NH4CI, su b sta nco s sh ow n to in crease lysosom al pH

IFN-r-activated m a cro ph a ge s beca m e

m ore efficient presen ting cells, w hile GM -C SF-activated m acrophages w ere n ot a ffected The w e ake r antig en pre sen ting capacity o f IFN-r-treated BM M ph seem s to be th e c o n ­

the binding of LPS to monocytes and by enhancing the sensitivity of monocytes to LPS at least 100 fold. In mice as in rabbits and humans. LBP was found to be an acute-phase protein. Further studies in vivo in mice using anti-C D l4 or anti-LBP

sequ en ce o f a successive degra da tio n o f Bl a nd d estruction o f antig en ic dete rm in a nts b y thlol /serin e proteases. On the other hand, since reduction o f disulfide b o n d s was

reagents will help to understand the role of LBP in response to LPS challenges

sh ow n to p lay a role in the p ro cessin g of insulin, we m easured the levels o f the in tra ­ cellular th io ls gluta thio n e and cystoino

BM M ph stim ulated w ith G M CSF have a s ig ­

nifican tly high e r cysteine and g lutathione co nte nt than IFN-T-actrvated BM M ph

This

h ig h level o f thiols and the w eak p ro te o lytic cle avage by thiol-/serine p ro te a ses m ay be re sp on sib le for the high ly efficient B l-presentation capacity o f G M -C SF-pulsed BM M ph

IN H IB IT O R Y EFFECT OF IRON OVERLOAD ON THE B AC T E R IC ID A L

LPS AND CYTO KINH F.FFliCTS ON TNF a mKNA SYNTHESIS IN RAT KUPFFKR CULLS R GAUSLING M GRfiW h. HC KSTLKR. R HOI I MANN. K GUYI-KO and K DECKF.R

A C T IV IT Y OF MURINE MACROPHAGES IN V IT R O

Biochemisches Institut, Hermann Herder Straße 7, D-7800 Freiburg. Germany Kupffcr cells (KC) arc thought to be the main producers of TNF « protein within die liver upon stimulation with LPS.

G AUTHIER Y . . a n d

ISOARD P.

rim T N I-a release was shown to he inhibited rapidly by PGUi and

dexamethasone However, the regulation o f TNF-a m-KNA synthesis and the influence o f peptide mediators remained undetermined. Rat liver KC in primary culture were stimulated with LPS tlOOng'ml), murine interferon y (INI- y, 50 U/ml). dexamethasone (l>iM ). K ili, (Ip M ). munne IN I--nr (500 U/ml), II IB (100 ll/m l).

U n it é

de M ic r o b io lo g ie

S a n té d e s A rm é e s -

-

B .P .

C e n tr e d e R e c h e rc h e s du S e r v ic e 87 -

3 8 7 0 2 LA TRONCHE C e d e x

-

de

F ra n c e

munne 11-6 (150 U/ml) aod dBcAMP (Im M ) for different periods of time. Total RNA from l.5-2x 1(7 KC was isolated and Northern blotting was employed using biotinylated rihoprohes. Simultaneously. * aliquots were reverse-transcribed and semiquantitative PCR was performed (RT-PCR). Hie identity of the RT-PCR product was established once by Southern blotting. KC stimulated with LPS/INI- y showed an appearance for TNF-a-mRNA artcr 30 min with n maximum between Ih and 2h both in Northern blotting and RT PCR; after 7h the signal had vanished

The

c u lt iv a t io n

m a c ro p h a g e s

Interestingly, the early occurring RNA was 50-100 bases longer than the one detectable at a later stage

such

(approx. 1,44kh). Northern blot analysis o f KiK.-trcatcd KC and also dBcAMP. a stable derivative of the

am m onium

in

a

a s hum an

of

ir o n - s a tu r a t e d

fe r r ic

r e s id e n t

m e d iu m c o n t a i n i n g

c it r a t e

p e r ito n e a l

v a r io u s

tr a n s fe r r in

le a d s t o

a

m u r in e

s o u rc e s

or

of

ir o n

ia c to fe r r in .

d e c re a s e d

or

p h a g o c y to s is -

intracellular second messenger o f PGK,. showed a striking inhibition o f TNF-ot mRNA synthesis. In contrast. KC treated with I.PS ♦ dexamethasone showed hardly a 50% inhibition o f TNF-a-mRNA synthesis, although supernatants of cultured cells did not contain significant cytotoxic activity. Moreover. TNF a itself was able to inhibit I.PS induced TNF-a-inRNA synthesis at a narrow tunc scale. Even a

a s s o c ia t e d an

b a c te r ic id a l

e n te r o p a th o g e n ic

D e f e r o x a m in e

and

a c tiv it y

s tr a in

hum an

of

of

t h e s e s m a c ro p h a g e s a g a i n s t E.

c o li.

a p o - T r a n s f e r r in

Iro n

c h e la t o r s

have a s im ila r e f f e c t .

24h pretreatment of KC with I.PS inhibited an additional endotoxin-evoked TNF-a-mRNA synthesis Additionally, neither II- IB. 11-6 nor 1NF-y alone had any effect, but a 24h pfctrcatmcnl with INF-y prior

Id e n tic a lly .

to the OOmin I.PS challenge seemed to have a priming effect lor I NF-a mRNA synthesis Reverse

a c tiv a te d

th e

e x tr a c e llu la r

m a c ro p h a g e s

is

b a c t e r ic id a l

a ls o

d e c re a s e d

pow er in

of

th e

PMA sam e

transcription of mRNA aliquots followed by scmiquantitahve PCR gave in corresponding results for the various stimuli, thus supporting the data in both mRNA detection assays Ihc presented stimulation and suppression experiments demonstrate a regulation o f TNF-ot production by KC at the mRNA level The time course of TNI- a-mRNA signals and the inhibition

c o n d itio n s . e ffe c t

of

These ir o n

b a c t e r ic id a l

r e s u lts

o v e r lo a d

su g g e st th a t is

m e c h a n is m s ,

to

ra th e r

h in d e r th a n

th e o b s e rv e d th e

in h ib it o r y

e x p r e s s io n

In c r e a s e

th e

of

som e

fo r m a tio n

of

experiments arc in lull agreement with the data on the release of mature TNF-a obtained with cultured KC In contrast to other mycloie cells. TNF-a itself was able to inhibit its own TNF-a-mRNA synthesis, while other cytokines did not seem to have an effect. Nevertheless, the detection o f TNF-a-mRNA by RT-PCR even in unstimulated KC. the incomplete inhibition of mRNA expression by dexamethasone and

fre e and

r a d ic a ls n itr ite

c o n d itio n s

of

to x ic

to

p r o d u c t io n

th e

c e lls .

S u p e r o x id e

a re

m e a s u re d

a n io n

g e n e r a t io n

u nder d iffe r e n t

c u lt u r e

m a c ro p h a g e s .

the change of the length o f mRNA revealed by Northern blot analysis suggest different regulatory

9

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mechanisms within the fine tuning of TNF-a in resting and stimulated KC!, respectively

S t im u l a t io n

of

M onocytes

w it h

S u r f a c e A n t ig e n E x p r e s s io n

M

and

y c o b a c t e r iu m

I n d u c t io n

of

Jens GERCKEN. Hans-Dieter Flad. and Martin ERNST.

Tu b e r c u l o s is : M o d u l a t io n

of

C e l l -A s s o c ia t e d IL-1ß .

P R O D U C T IO N O F A N O V E L S O L U B L E M E D IA T O R D U R IN G T H E IN T E R A C T IO N O F Th1 -L Y M P H O C Y T E S A N D B O N E M A R R O W -D E R IV E D M A C R O P H A G E S C U L T U R E D IN G M - C S F

Forschungsinstitut Börstel. Institut

für Experimentelle Biologio und Modizin. Parkallee 22, 2061 Börstel, Germany By moans of ttow-cytomotry analysis we investigated tho modulation of monocyte surface mole­

T.

Germ ann. E. Rüde,

Institut für Im m unologie der Joh. G utenberg Universität. W -6500 Mainz.

cules as well as tho cell-associated production of IL-1ß following stimulation with M.tuborculos/s. Human monocytes were isolated from PBMC by countortlow élutriation, subsequently stimulated

The recognition o f M HC m olecules in com bination with processed fragm ents of an antigen by the T cell receptor is responsible for the specificity o f an im m une

with live FITC-labelled M.tuberculosis (H37Rv) and culturod for I 8h. In order to analyze the

response and results in partial activation of specific T cells. In addition, accessory

expression of surface moloculos of colls that have phagocytosod labollod bacteria monocytes were

signals play an im portant role in the activation process of T cells. They enhance

stained with phycoerythrin-labolled monoclonal antibodies. The expression of cell-associated IL-tp

and m odify the T cell receptor signal leading to a stronger and sustained stim ula­

was detoctod by intracellular staining with the IL-1ß-specihc monoclonal antibody FIB-3 aftoi

tion. A ccessory signals can be delivered to T cells either by direct cell contact or

pormoabilizalion of the ceo membrane with n-octyl-ß-D-glycopyranoside.

b y soluble m ediators. M acrophages derived from bone m arrow cells b y culture in G M -CS F-containing m edium are very potent antigen-presenting cells for Th1-

With regard to the modulation of surface molecules we found that tho expression of complement

lym phocytes, although they express only few MHC class II m olecules on their

receptor 1 (CD35) and of tho high affinity Fcr-receptor (CD64) is down-regulated on most myco­

surface. W e c ould dem onstrate that these m acrophages express an as yet un­

bacteria-infected monocytes. Other surface antigens constitutive^ present on monocyles. like

defined m em brane m olecule(s) required for the proliferation of Thl-ly m p h o c y te s

HLA-DR. CD14. ICAM-t (CD54), and tho ß2-intogrins (C D tta/b/c) were expressed in similar amounts on phagocytically active and inactivo monocytes of the mycobacteria-exposed cultures.

stim ulated via their T cell receptor/C D3-nom plex

Such an interaction between

m em b ran e-b ound m olecules is also required for the synthesis of a novel soluble m ediator durin g the m acrophage-Thl-ly m p h o c y te interaction. This m ediator by

In comparison to unstimulaied control monocylo culturos. however, antigen densities of CD14.

itself can then act on Th1-lym phocytes, further augm enting their activation. Its ef­

CD54. and CD1 tc wore upregulated.

fects on Th1-lym phocytes include grow th enhancem ent, stim ulation of IFN-r-

Intracellular staining with monoclonal antibody FIB-3 revealed that IL-tß is produced by both,

synthesis as well as triggering of LFA-1/ICAM -1-dependent cell adhesion. The m olecular w eight o f this factor - about 65 KD on SDS-PAGE - and som e other

phagocytically active and Inactive monocytes. A vanablo amount of tho M.tuborcu/osisinlocxod

biochem ical and functional properties in dicate that it is probab ly identical w ith the

colls appoarod immunofluorescence-negative for IL-113. Monocytes having ingested high numbers

recently describ e d cytokine IL-12. So far we were unable to detect this m ediator

of bactoria woro predominantly immunofluoresconco-nogative for IL-tß.

in supe rna tants o f Th1-lym phocytes o r m acrophages stim ulated separately by

Taken togothor. our results demonstrate that tho modulation of surface molecule expression and

various agents. Furtherm ore, only GM-CSF seem s to in duce a functional state In bone m arrow -derived m acrophages which allows its synthesis upo n coculture with Thi -lym phocytes. M acrophages derived from bone m arrow cells cultured in

that M.tuberculosis-infoctod monocytes induce phenotypic changes and IL-1ß-production in

M-CSF (CSF-1) d o not possess this functional capacity. However, short-term

bystander monocytos eilhor by soluble mediators and/or direct coll to cell contact.

treatm ent (24 h) w ith GM-CSF but not IFN-r is sufficient to convert them in to a

This work was supported by BMFT, grant no. OtKI 8827

functional state allow ing the generation of this novel soluble m ediator

A L P H A -M E TIIYLI’RKDNISOLONE CAN M ODULATE LE CT IN O P H A G O CY T O SIS O F YLA STS BY HUMAN AND MURINK M ACROPI1 A G E S. Jean GIAIMIS1. Yves LOMBARD1. Paul FONTENEAU*, Rachel LEVY*. Madeleine M a k a y a -K u m b a 1, Philippe Po indron 1and Jams L azdjns 2. ‘ U n h rrxitf Louts Pasteur, Departement d'Immunologic, Immunopharmacologie r t Pathologic, BP 24. F 67401, ILLKIRCH Cedes. France arut 2Ciha Gcigy Limited, Research Department. KI25 2.17, CH 4002. BASEL. Switzerland. Non-opsonized Saccharomyces cerevistae can tic phagocytozed by uucropbage* (human or murine), in the case ol mouse peritoneal macrophages, it is believed that this phenomenon is mediated by a 0-glucan receptor. Similar results have been obtained using fresh human blood monocytes. However, using in vitro differentiated cells, it has been claimed that the phenomenon is mediated by mannose receptors. Since corticosteroids arc known to modulate expression of mannose ct 0-glucan receptors on murine phagocytic cells, wc examined the effect o f this substance on human cells. The study was designated as comparative study with iiiunnc macrophages. Freshly isolated and in vitro cultured human blood monocytes obtained by combination o f leukaphercsis and centrifugal clutnation and resident mouse peritoneal macrophages (BAl.B/c mice) in vitro cultured were used for this study. Lectinophagocytosis was evaluated using methods previously described (Giaimis el «/.. J. Immunol. Methods, in press, and this conference). Circulating human monocytes do not express the mannose receptor, therefore, non-opsomc phagocytosis is mediated by 0-glucan receptors. Wlicn these cells are induced in vitro to differentiate, they express the mannose receptor. Culture (7 days) m the presence o f a-mclhylpredmsolonc (104M) inducod phagocytosis o f much higher amounts o f non-opsonized yeast particles than non treated cells. This process could be totally inhibited when during phagocytosis u-inaunan and latnmarine (4 mannose receptors which arc increased, whereas thut mediated by 0-glucan receptors was diminished. Freshly isolated murine peritoneal macrophages expressed both mannose and 0-glucan receptors After 4-day culture in the presence of corticosteroids, lectinophagocytosis was significantly reduced. This reduction was due to an effect on 0-glucan receptor mediated phagocytosis. The mannose receptor mediated phagocytosis was not affected. Thus, our studies have demonstrated that in human as well as in murine cells, both types of receptors (ix. mannose and 0-glucan) are involved in the phagocytic process and that corticosteroids affect the functionnal expression of these receptors.

10

BOTH M ANNOSE ANI) 0-GLUCAN RECEPTO R S ARF. IN VOLVED IN P H A G O C Y T O SIS O F YF A ST S BY MURINK M A CR O PH A GE S. Jean Gia im is 1. Yves L o m b a r d 1. Paul Fonteneau *. Christian M u l le r 1. Rachel L ev y 1, Madeleine M a k a y a -K u m b a 1, Janis L a / dins 2 and Philippe Poindron 1. I University Ixtuis Pasteur, Departement d ’hnmunologie. Immunophannacologie ct Pathologic, BP 24. F 67401, ILLKIRCH Cedex, France atui 2Ciba Geigy Umited. Research Department, KI2S 2.17, CH 4002, BASEL. Switzerland. Macrophages arc known lor their capacity to hind and to ingest opsonized particles (opsonophagocytosis) They are also able to bind and to internalize non-opsonized particles (especially microorganisms such as hactena or yeasts) t»caring polyosidic structures on their surface, through endogenous lectin-like receptors (lectinophagocytosis). Two o f these lectins are mainly involved : the mannose receptor and the 0-glucan receptor. I lic mannose receptors arc trypsm-sensitive and calciumdependent. In a previous paper (Giannis r t a l, J. Immunol. Methods, in press. 1992) we have shown that autoclaved Saccharomyces cerevistae can lie used to study both attachment and internalization steps of lectinophagocytosis. Its wall consists essentially ot a-mannans and 0-glueans. We show here that autoclaved 5. cerevisiae can be conveniently used to study both mannose receptor- and ()-glncan receptormediated lectinophagocytosis. fUe mnrme macrophage cell line MALI >used for this study was establislied iri our laboratory (Lombard el at., J. Leukocyte Biol.. 44. 391-401. 1988) These cells were showu to display mannose receptors on their surface (Muller rt a L.i. Leukocyte Biol.. 43. 165-171, 1988). It was first observed that lectinophagocytosis o f 5. cerevisiae was totally inhibited when MALU cells were pre-incubatcd in presence o f a-mannans (10 mg/ml). Under the same conditions, opsonophagocytosis of S. cerevisiae was not impaired. In addition, laminann. a soluble 0-glucan. (10 mg/ml) inhibited by 50 % lectinophagocytosis when used alone. We hypothesized therefore that both receptors were involved in the phenomenon. Ibis was confirmed by the total inhibition of lectinophagocytosis by a mixture of lannnarme and a-mannans at a dose ot 400 pg/ml each, whereas when used alone, these polysaccharides inhibited only partly the lectinophagocytosis o f £ cerevisiae Treating a-mannans by laminannase. a 0glucanasc. resulted in a preparation which now was unable to totally inhibit lectinophagocytosis. indicating that the commercial preparations ot a-mannans are contaminated with 0-glucans. Treatment ot laminarin with a-manni»sida.se did not modifiy its inhibiting activity. Both receptors behave quite differently. Alter being incubated in presence of a-mannans, washed out, and then treated with laminann, the MAl.U cells recovered very rapidly their ability to bind and internalize yeasts. After laminarin treatment, washing out. and addition o f a-mamians. ihc MALU cells did not recover the ability to bind and internalize yeasts. Taken together with the results o f the kinetic studies o f lectinophagocytosis by either receptors, these observations suggested that the mannose receptors recycle wliereas the 0-glucan receptors cannot. Hie lectinophagocytosis which was shown to persist after treatment o f the cells by trypsin or EGTA was totally abrogated if cells were treated by laminarin before addition ot yeasts, and was only marginally affected by a-numnans. This suggested that 0-glucau receptors were trypsin-resistant and calciiim-uidcpendent.

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induction of IL-1ß is not restricted to monocytes loadod with mycobactoria. This findino suooosts

f u n c t i o n a l b -g l y c a n

r ec ep to r

ex pr es sed

b y

m ic r o g l ia

in

TH E C E N T R A L NERVO US SYSTEM

EXPRESSION A N D SECRETION OF MACROPHAGE SPECIFIC PRODUCTS AFTER IN VITRO INFECTION OF H U M AN MONOCYTES/MACROPHAGES W ITH HIV

J. G ia im is * . V . B o c c h in i $. A . M e n g h in i §. P. P o in d ro n “ & C .D . M u l l e r * " • Dipartciiient (riiiinmnologie.lnMiuinoptunnacologic el Pathologic & ** Laboraloirv . FPA(5Q%) and I L -6 (40%) com pared t o c o n t r o l c e l l s . S i m i l a r r e s u l t s w e re o b ta in e d b y P I-P L C t r e a t m e n t. T h u s. LPSa c t i v a c i o n o f human m o n o c y te s , r e s u l t i n g i n c e l l u l a r p r o c o a g u la n t a c tiv it y and I L - 6 - s y n t h e s is a n d r e le a s e , a p p e a rs t o be p a r t l y m e d ia te d th r o u g h CD14.

Several

lines

of

evidence

support

the

concept

that

bacterial

antigens,

especially

lipopolysaccharidc (LPS). persist in the patients developing reactive arthritis after Salmonella. Shigcllu and Yersinia infections lo r longer lim e than in the patients w ith uncomplicated disease. First, strong and long persisting antibody responses against the causative agents in sera and synovial flu ids «if the patients w ith reactive arthritis suggested the persistence ol corresponding antigens. Then. LPS structures were detected in inflam ed jo in ts , w ith in mononuclear and polymorphonuclear phagocytes, in patients w ith Salmonella-, Shigella- and Yersinia-triggered arthritis. It seems lik e ly that the interaction between host and microbe is abnormal and inefficie n t in H L A B27 positive subjects in whom reactive arthritis develops. In order to evaluate that further, wc have recently fed peripheral blood monocytes o f healthy people w ith live Sulmonellu typhim urium bacteria, and follow ed expression o f certain cell surface antigens during 6-7 days o f intracellular life o f Salmonella. Despite Salmonella bacteria were liv in g in traccllulurly. obvious procession o f bacteria started in few hours leading to expression o f Salmonella antigens on ihc cell surface Expression o f H L A B27 antigen decreased, and so did other M H C class I antigens in H L A B27 positive cells. Expression o f M H C class II antigens DR. DP. D Q seemed to be regulated independently. Expression o f C R 1 und f), -tniegrins were usually upregulatcd in H L A B27 negative cells, but downrcgulated in H L A B27 positive cells. Salmonella bacteria inside the monocytes enhanced the adhesion o f these cells on endothelial cell monolayers in vitro. B y modulating the surface expression «>f integrins bacteria inside the monocytes may enhance their binding u> synoviul endothelium.

B IP H A S IC P R IM IN G O F A L V E O L A R M A C R O P H A G E S F O R E N H A N C E D R E S P IR A T O R Y B U R S T B Y P L A T E L E T - A C T I V A T I N G F A C T O R

B .K lc u s e r . T . S c h lü te r a nd G . G c rc k c n U n iv e rs itä t H a m b u rg . I n s titu t f ü r B io c h e m ie u n d L c b c n s m ittc lc h c m ie . A b t. f ü r B io c h e m ie und M o le k u la rb io lo g ie . M a n in - L u th c r -K in g -P l. 6 . D -2 0 0 0 H a m b u rg 13

T h e b io lo g ic a lly a c tiv e e th e r lip id , p la te le t- a c tiv a tin g f a c to r ( P A F ) , w h ic h is in v o lv e d

in

m any

in f la m m a t o r y

p ro c e s s e s ,

in d u c e s

( e .g .)

c h c m o ta x is ,

d c g ra n u ln tio n . a nd the re le ase o f iu te r lc u k in c s nnd re a c tiv e o x y g e n in te rm e d ia te s ( R O l) . F u rth e rm o re . P A F e xe rts a p rim in g e ffe c t o n som e c e ll types. In c u b a tio n o f p o ly m o rp h o n u c le a r n e u tro p h ils ( P M N ) w it h 0.01 to 1 00 0 n M P A F in cre ase s the re s p ira to ry b u rst w h e n th e c e lls n rc c h a lle n g e d b y a second s tim u lu s . T h e a ctiva te d s ta te o c c u r s im m e d ia t e ly

a ft e r th e a d d itio n o f P A F a n d d is a p p e a rs a fte r

a p p ro x im a te ly 6 0 m in . In o u r e x p e rim e n ts w c s tu d ie d the p rim in g e ffe c t o f P A F on c u ltu r e d b o v in e a lv e o la r m a c ro p h a g e s ( B A M ) . A ft e r in c u b a tio n w it h P A F o v e r a p e r io d f r o m 0 m in to 10 h th e c e lls w e re s tim u la te d b y a s e co n d a g o n is t.

Birgit Knepper. Thomas F. Meyer and Jos P.M. van Putten Max-Planck-Institut tor Biologie, Abt. Infektionsbiologie. Spemannstr.34, 7400 Tübingen, Fed. Republic of Germany

Human professional phagocytes: preferred targets for Neintoria gonorrhoeao 7 Abstract Neisseria gonorrhoeae. the causative agent of gonorrhoea, is frequently found intracellularty in human leucocytes and it has been suggested that these cells are a niche enabling N gonorrhoeae to survive the Inflammatory response during infection. We were interested whether the interaction between N. gonorrhoeae and human leucocytes is modulated by the expression of variable surface exposed opacity associated proteins (Opa). We therefore performed in vitro phagocytosis assays with freshly Isolated human neutrophils and monocytes in the absence of serum and examined the dependence of tho interaction on Opa expression by microscopy. Interestingly, only OpaC. the Opa protein which gives rise to an invasive phenotype to epithelial cells (t) was unable to mediate interaction with human neutrophils, whereas the other ten Opa-protelns that can be produced by the same strain Increased the interaction rate about ten-fold (2). Remarkably, tho association of Neisseria gonorrhoeae with human monocytes was Opa-independent. pointing to a different type of interaction with these cells than with neutrophils. The ingestion and survival of «he gonococd inside loucocytes was examined with human monocytes Infected cells were treated with gentamicin to kill extracellular bacteria and the number of viable Intracellular organisms was monitored by colony counting of overnight grown bacteria released from the monocytes by saponin lysis. Our results show that approximately 20% of the Initially pnagocytosed gonococci survive within monocytes lor at least 6 hours. The mechanism underlying this apparent resistance against phagocytic killing is currently being investigated.

R e s p ira to ry b u rst a c tiv it y w a s e v a lu a te d b y m e a s u rin g the rele ase o f R O l in to the in c u b a tio n m e d iu m . A tim e -d e p e n d e n t b ip h a s ic p r im in g w a s o b s e rv e d : B A M e x h ib ite d an e nhanced re s p ira to ry burst a fte r a p re in c u b a tio n tim e o f 0 to 6 0 m in . In

References (t) S.Maklno. J.P.M.van Putten and T.F Meyer (1991) EMBO J. lf l . 1307-1315 (2) E. M.Kupsch. B.Knepper. I.Heuer. T.Kurokl and T.F.Meyer; In preparation

a d d itio n , a second m a x im u m o f e nh anced R O l release c o u ld be seen a fte r a p rim in g tim e o f 3 .5 to 6 h ou rs. T h e e n h a n ce m e n t o f b o th m a x im u m p eaks v a rie d b e tw e e n 15 a n d

1 0 0 % . d e p e n d in g o n

th e ty p e o f th e se con d a g o n is ts , e .g . P M A .

c o n c a n a v a lin A . o p s o n iz e d z y m o s a n , o r q u a rtz d u s t. T h e lo w e s t e ff e c t iv e c o n c e n tra tio n l o r P A F p rim in g w as 0 . 1 n M . and m a x im u m va lu e s w e re re ached at 100 n M P A F . T h e s p e c ific ity o f d ie e ffe c t w as p ro v e n b y in c u b a tio n w ith ly s o -P A F o r w ith P A F a nd its a n ta g o n is t W E B 2 0 8 6 . L y s o - P A F was in e ffe c tiv e , a nd W E B 2 0 8 6 a b o lis h e d the P A F p r im in g e ffe c t. T h u s , the b ip h a s ic p rim in g o f B A M b y

17

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P A F seem s to be u re ce pto r-m e d ia te d process.

TUMOR CRLLS E L IC IT THE RESPIRATORY DURST IN CYTOTOXIC, ACTIVATED

DEVELOPMENTAL REGULATION OF VITAM IN D, METABOLISM AND VITAM IN

BONE MARROW -

D , RECEPTO R EXPR ESS IO N D U R IN G D IF F E R E N T IA T IO N MONOCYTES

DERIVED MACROPHAGES (BMDM)

S te p h a n K ö r n e r

OF H U M A N

Marina Kreutz1, Helm ut Reichel7, Stofan W. Krause' and Reinhard Andreesen’

J o n o l Pharm a GmbH, K c ic n e n b u r g s t r . 2 5 ,

’ Klinik für Innere Medizin I, Universität Regensburg and 'M edizinische Klinik. Sektion Nephrologie. U niversität Heidelberg, Germany

D -8 4 0 0 R e g e n s b u rg

The in v itro m aturation of blood m onocytes IMO) can serve as a model for the in A c tiv a te d

m a c ro p h a g e s

a c tiv itie s s p e c ie s

u g a in s t

(ROS)

a re

c y to to x ic ity , w it h

MDP,

w a lls .

in v o l v e d

s u p e r o x id e

( 02* )

c h e m o lu m in e s c e n c e . to

o lic it

h o u rs

th e

w h e re a s

tre a te d in

c y to to x ic R O I.

s t im u la t e d is

RNI

a re

r e s p ir a to r y

v e ry

and

( r e a c tiv o

a nd c a n

th e

p o te n t

p ro d u c e d

n it r o g e n

la r g e

a fte r

p r o v id e

c u r r e n t ly

by

a m o u n ts

a

by

I n c u h a t lo n

b u rs t

i.e .

of

m u c ro p h a g e s an

T he n o n t o x i c

p o w e rfu l

to o l

u n d e r in v e s t ig a tio n

in

th e

m atured M AC showed no 24.25(O H )7D3-synthesis. Furthermore, w e examined the expression o f the vitam in 0 receptor (VDR). MO c o n stitutively show ed a

a c ts fro m of

MO into M AC differentiation in v itro is associated w ith (i) an enhanced capacity

and

to synthesize 1.25(OH )7D3, (iiJ a loss o f 2 4 .25

in

kD a p ro te in ) is a very d iffe re n tial m odulator o f the effects o f lipopolysaccharide (LP S) on

o r ig in a t e d

w h ic h

a c c e le r a t io n

in

of

lo s s

m urine macrophages. In te rle u kin I production by macrophages in response to stim ulation

of

w ith LPS was drastically reduced, w hile cytotoxicity (tu m o r necrosis factor p ro du ction ) was

g ro w th .a

a n tig e n ic ity

greatly enhanced, when the 39 kD a protein was complexcd w ith LPS.

and

of

2 4h

c u lt u r e d

c e lls L020 u n it m e a s u re d b y u s in g a c c o r d in g

to

The in c r e a s e

th e of

m e th o d

of

PM s u p e r n a t a n t s th e

T.Mosmnnn e t

s u p e rn a ta n t

on

interaction o f bone m arrow-derived, m urine macrophages w ith l-PS and w ith complexes o f

T N F - jc e n s itlv e

c o lo r im e t r ic

MTT

LPS and the 39 kD a protein. Macrophages were allowed to react w ith LPS o r LPS-protcin

assay

al

c y t o t o x ic it y

complexes fo r 30 min. Thereafter, they were washed thoroughly anti the presence o f LPS

w it h

its

c o n c e n t r a t io n

on the macrophage surface was follow ed in a m odified E L IS A over a 24 hour period.

lia s b o o n o b s e rv e d In

c o m p a ris o n

w it h

When IT S alone was adm inistered, it could barely be detected on the macrophage surface c o n tro l

a nd o f PM o b ta in e d f r o m 24021 1 2 p g /m l>

s u p e rn a ta n t

70 3.

c u lls ,

r f iijp s t

fr o m

(iii) a decrease in the expression of VDR mRNA. Since 1,25IO H |7D:i w as show n

( J o r a o l* ) .

L a b o r a t o r y fo r C y t o p h y s i o l o g y . b o p a r r m e n / N o d i c a l S c h o o l , it.y

of

CT 0 6 0 3 0 , USA.

d e fe n s e

s y s te m s ,

in

a g a in s t

an

cancer

a d o p t iv e

w as

tra n s fe r

m o d e l u s in g s c i d m ic e b e a r in g x e n o g r a f t s o f t h e r e s p e c t i v e a u t o lo g o u s tu m o r c e l l s .

Several observations suggest tha t fusion o f tu m o r cells w ith n o rm a l monocytes o r

DEFENSE N .G .

C o n n e c t i c u t M e d ic a l S c h o o l,

s t u d ie d , In s titu t fu r H ygiene. L cop o ld -F ra n zcn s-U n ivcrsitä t Innsb ru ck. A u s tria

IN

M ukhel i i .

re s e d

P l a s t i c a d h e r e n t m a c ro p h a g e s , d e r i v e d f r o m le u c o p h o -

b lo o d

m o n o n u c le a r

c e lls

fro m

th e

r e s p e c tiv e

p a tie n ts ,

w e re g ro w n i n l a r g e p l a s t i c f l a s k s i n A IM V m e d ium (G IB C 0 ) c o n t a i n i n g 100

U /m l

I n te r le u k in - 2

(fo r

th e

fir s t

3

days)

.

s o ld m ic e ( c . B - 1 7 i c r j )

and

1000

0 /m l

clear, how such a fusion process c o uld be in itia te d in v ivo . In v itro it is possible to in duce o f M-CSF ( G e n e t ic s i n s t i t u t e , C a m b rid g e fusion o f monocytes, leading to the generation o f m u ltin u c lc a tc d gian t cells, by

w e re

g ra fte d

in cub a tion w ith a supernatant o f C on A -s tim u la tc d m o n on uclea r cells. W e tested i f such

c e lls

a co nd itio ne d m e d ium could also induce fusion between monocytes and tu m o r cells.

D e p e n d in g

M onocytes isolated fro m p e rip h e ra l bloo d o f healthy don ors and cells o f various tu m o r

tu m o r c e l l s

lines were m em brane-labeled w ith two d iffe re n t dyes. M onocytes and tu m o r cells were

w it h

s u b c u t a n e o u s ly

( m e la n o m a upon

th e

lin e

th e

a u t o lo g o u s

a p p r o x im a t e l y

and

e x p e r im e n t a l

w e re i n f u s e d ,

on day one o n ly ,

w it h

JH -K

ma)

th e

re n a l

d e s ig n ,

m ic e

in t r a v e n o u s ly a n d /o r

c u ltu r e d

m a c ro p h a g e s

on d a y se v e n o n ly ,

1-2

m illio n

cancer

lin e

g ra fte d

tu m o r JR -R ) .

w it n

th e

in tr a p e r ito n e a lly ,

( 1 0 7 - 1 0 8 /in fu s io n ) ,

o r th re e

t im e s w e e k ly .

m ixed and incubated w ith a p p ro p ria te concentrations o f the co nd itio ne d m edium . T he H a rk e d fo rm a tio n o f m u ltin uclea ted g ia n t cells was m o n ito re d w ith an inverted m icroscope

tu m o r s

e quipped fo r d ual w ave-length fluorescence. F o r some o f the tu m o r c e ll lines (e.g., U 937)

g ro w th s

no evidence o f fusion w ith monocytes was fou n d. In o th e r experim ents (e.g.. w ith T A L L

th e

o r S k-M e l 37) g ia n t cells stained w ith both flu o ro ch ro m cs were observed, in d ica tin g tha t

tu m o r

as

w e ll

w e re

r e g re s s io n s

( p a r t ia l

as

to

seen

c u ltu r e d

p a r tia l w ith

b o th

m a c ro p h a g e s .

a n t i- tu m o r

e ffe c t

u n lik e ly

have o c c u rre d

to

to

c o m p le t e

s in g le

th a t

m u ltip le

of

th e

of

p r o te c t io n

and

A n a ly s e s

s u g g e s te d

c o m p le t e )

th e

tu m o r

e s t a b lis h e d fro m

tu m o r

in fu s io n s

m e c h a n is m

of

r e g r e s s io n s

as a r e s u lt o f d ir e c t c y t o l y t i c

of th e

w e re

a c tiv it y

these cells co nta ine d mem branes o f both ce ll types and thus w ere derived fro m fusion Of

th e

m a c ro p h a g e s .

between n o rm a l m onocytes and cells o f c e rtain tu m o r lines u nd er co nd itio ns tha t could

a c tin g

also occur in vivo.

a n o n -T c e l l d e fe n s e

24

In s te a d ,

th e

m ode

of

a c tio n

a p p e a re d

to

h a v e b e e n m e d ia t e d b y a c y t o k i n e ( 8 ) e l a b o r a t e d b y t h e m a c ro p h a g e s , e ith e r d ir e c t ly

on th e

tu m o r c e l l s

s y s te m o f t h e

o r a c tin g

by r e c r u it in g

a n im a l s . Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/18/2019 1:34:47 PM

o f monocytes w ith tu m o r cells. These results indicate, tha t it is possible to in duce fusion

ELEVATED

8

INTERLEUKIN

LEVEL'S

IN

BRONCHOALVEOLAR LAVAGE FLU ID

OF

PATIENTS WITH ACQUIRED EMPHYSEMA. T h . M u lo v .

m

. W te b e l.

T h o r a x k lln ik ,

GENE IN NORMAL AND AUTOIMMUNE MICE.

G. K llt s c h e ,

A r a a ll e n s t r .

5.

RFLP-ANALYSIS OF THE MACROPHAGE-COLONY STIMULATING FACTOR (CSF-1)

V.

S c h u lz a nd W. E b e r t

6900 H o id o lb e r g - R o h r b a c h .

Müller. M . . Jarck, P . , Hecht, M., Hartung, H., Paul, D., Lohmann-

FRG

Matthes, C ig a r e tte w h ic h

s m o k in g

can

p ro d u c e 8

In te r le u k in lu n g s

em physem a.

flu id s

To in

IL 8

E lis a

of

K it

v a lu e s

w e re

e x -s m o k e r s . d if fe r e n c e s t a t is t ic a l m a jo r

r o le

th e

lu n g

fa c to r

th e

m e a s u re d

in

W ilc o x o n b e tw e e n

s m o k e rs

th e

fo r

It

is

p a t h o g e n e s is

c o u ld

a c q u ir e d

th e re m ust e x is t e le v a t e d

It

a nd

em physem a,

a d d itio n a l

IL 8 le v e ls

in

g ro u p vs

lu n g s and

fa c to rs

th e

in

th a t

IL

t h e ir

it

to

b e s id e s s m o k in g

m e d ia n

p < 0 .0 4

8 c o u ld

vs

re a c h p la y

p r e s u m a b ly

be

of

the

reasons

for

an

increased

macrophage

SLE prone mice already at an age of 4

to

normal mice,

serum levels of CSF-1, One

we and others have

weeks

esp.

found

in

where an increased amount of CSF-1 mRNA could be in NZB/W mice.

elevated

in diseased MRL-lpr and BXSB

possible site for this production of CSF-1 is

mice.

the

kidney,

detected,

e.g.

Since structural abnormalities of the CSF-1

gene

might we

be responsible for the dysregulation of the CSF-1

assessed

the CSF-1 gene for a

polymorphism.

Using

restriction

PST 1 and BAM HI,

cleavage

site

in the CSF-l gene,

(BALB/c,

C3H/HeN) and lupus prone mice (NZB/W,

system,

fragment

enzymes

length

possessing

we digested DNA

w it h in

a

n o te d , to

and BXSB) and performed Southern blots.

from

one

normal

MRL-lpr, MRL-+/+

The results

demonstrate

no differences in the RFLP analysis indicating that in SLE

due

mice the CSF-1 gene itself appears to be normal.

th e

on

a m a jo r r i s k

le a d in g

comparison

th e

not

is

Investigating compartment

be

p a t ie n t s

g ro u p

a c tiv a tio n

has

to

te s t).

h ig h e s t

d id

omphysoma

w it h

emphysema

W ilc o x o n

t h a t s m o k in g

a lt h o u g h

fo u n d

w ith

emphysema

c o n c lu d e d

w e re

Q u a n t i k in o

n o n s m o k e rs .

th e

oe d e m o n s tr a te d

was

assessed

e x -s m o k e r s

of

n e u t r o p h ils

8

in

la v a g e

c o m p a ris o n u s in g

(p < 0 .0 0 0 1 .

(p < 0 .0 2

W i t h in

in

and Enunendörf fer, A. Fraunhofer Institute, Dept,

th o u g h t fo u n d

c o n c e n t r a t io n s

IL

8 w e re

IL

is

M.-L.

of Immunobiology, 3000 Hannover 61.

of

n e u t r o p h ils

w a lls

p a tlo n ts

non -o m p hyso m a s m o k e rs

s ig n ific a n c e . in

of

on w h ic h

d is e a s e s

of

m e d ia t o r s

b r o n c h lo a lv e o la r

U S A ).

emphysema

te s t).

r e c r u it m e n t o f

t is s u e .

BALP

le v e ls

8

IL

(AM)

p a t h o g e n e s is

a lv e o la r

emphysema

In c .

th e s e

th e

a c tiv ity

of

p u lm o n a ry

in

w it h o u t

In

w it h

th e

Among in

of

h y p o t h o s io .

o th e r

m a c ro p h a g e s

P M N - e la s ta s e ,

S y s te m s

lo w e s t

s a r c o id o s is .

of

s u p e rn a ta n ts

(R&D

th e

r o le

c h e m o t a c tlc

p a tie n ts

th o s e

a

d e s t r u c t io n

th is

fr o m

a lv e o la r

c y to k in e s .

r e le a s e

th e

h ig h e r

to

R e m a rk a b ly ,

it s

th e

p ro v o

s ig n ific a n tly co m p a re d

of p la y

fo r

s u ffe r in g

a c tiv a te

c o u ld

c e ll- f r e e

(BALF)

p a tie n ts

to

to

to

v a r ie t y

B e s id e s

r e s p o n s ib le

m e a su re d

w it h

a

8 le a d s

IL

be

know n

(II. 8 )

em physem a.

(PMN). to

is

the CSF-l gene proraotor,

and

the

prone

Further studies

on the stability of the CSF-1

degradation of the CSF-1/ CSF-1 receptor

mRNA

complex

will

th a t th e

t h e BALF o f emphysema p a t i e n t s .

probably reveal the explanation for the increased serum levels of this growth factor in SLE prone mice.

UROK.INASE/PLASMINOGEN ACTIVATOR INHIBITOR-TYPE 1 COMPLEX BINDS TO THE

THE

PURIFIED a,-MACROGLOBULIN RECEPTOR.

KILLING

INTRACELLULAR RESISTANCE BY

OF

MYCOBACTERIUM FORTUITUM

TO

MONOCYTE-DERIVED MACROPHAGES

Evidence that cellular degradation In monocyte* o f urokinase receptor-bound complex Is mediated by the Uj-macroglobtilln receptor.

Nzlromaaanga

P . . P.M.

Shah

and W.

Stille

Infektiologle

Nvkjacr A *. Jensen PH". Petersen CM*. Muller B". Mocstrup SK". Holtct TL1. Etzcrodt M*. Thogcrsen H C \ Munch M*Andrcascn PA*. Glicmann J" Institute of Medical Biochemistry". Dept, of Clinical Immunology", laboratory for Gene Expression1.

Zentrum

der

Klinikum

Innere

der

J.U.

Theodor-Stern 6000

Medizin Goethe

Kol

Frankfurt

Universität

7

am

Main

70

Dept, of Molecular Biology*. University of Aarhus, DK.-8000. Aarhus C. Denmark Abstract

Wc and others have shown that urokinase receptor (uPAR)-bound complexes between urokinase (uPA) and plasminogen activator inhibitor typc-1 (uPA/PAI-1) are readily internalized and degraded in monocytes. In contrast. uPA or its aminotcrminal fragment (ATF) arc poorly internalized following

The

interaction

fortuitum

and

binding to the glycosyl-phosphatidylinositol anchored uPAR. Monocytes express the «.-macroglobulin

in vitro using

receptor (a,MR. a monocyte differentiation antigen). Wc have recently reponed (FEBS Lxtt. 300, 13-17

were

(1992)) that T -ccll derived intcrlcukin-2 activoted killer cells express uPAR. but not «.MR. and that they do not degrade uPAR-bound uPA/PAI-1 Wc now report that uPA/PAl-1. but not uPA or ATF. binds to the a-choin o f purified human pluccntal two chain a¡MH The magnitude of binding is comparable to that of the established ligands

for

isolated 7 days

cells

to

the method

1640

observed

macrophages

to

after 4

the

Incubation,

the

cells

atypical

mycobacterial

unstimulated human of

and cultured

in RPMI

were

added

between

normal

with

and

tissue

Into

in culture.

lO OOO

extracellular

May.

culture

10% Fetal

transform

days at

Crowle

on

Calf

The

were

Blood

was

surfaces

The

adherent

Irregularly

mycobacteria After

M.

studied

monocytes

plastic Serum.

large.

bacteria/ml.

bacteria

soeoles

macrophages

shaped

were

1 hour

removed

by

washing.

a--macroglobulin/ptotcinase complex and the novel 40-kDa UjM-associated protein (ci.MRAP) The

Binding of uPA/PAI-1 is blocked by excess natural or recombinant or placebo (0.2 ml NaCl 3x/week). Up to now.

the analysis o f the m ortality rate shows that 57% (4/7) o f mice died in ihc placebo treated

group.

87%

(7/8) died

the

M oIFN y

treated

group

highest score o f proteinuria was reached 11. 17 and 21 weeks after ihc beginning

metastasis in an experimental fibrosarcoma metastasis model. 3x10^ CFS1 cells,

of

the

treatment

w ith

These

M o IF N y.

results

were

placebo fu n h e r

and

m Ab

confirm ed

treated

no death

therefore. Investigated the Influence, of endogenous and exogenous TNF on

respectively.

anti-IFN y nor in the M oIFNy-R

whereas

occurred neither in

methylcholanthrcnc-lnduccd fibrosarcoma cells, were Inoculated t.v. Into mice

the mAb

in

tumor cells due to the broad spectrum o f biological activities of TNF. We.

a nti-IF N y

by

the

groups.

or

The

M o IF N y -R .

im munohistoebernistry

and pulmonary metastascs were counted on day 11. The animals received either a single i.p. Injection of recombinant human TNF (rhTNF). recombinant mouse TNF (rmTNF). or mTNF-nculrallying monoclonal antibodies (Vlq) 5 hours prior to

analysis

o f the

kidneys (hat

showed

a low er

tumor injection. Both groups of TNF pretreated animals showed significant

m Ab

enhancement of metastascs tn the lung, while neutralization of endogenous TNF

Most o f the mice treated with M oIFNy died

led to a decrease. Dose responses of rhTNF and rmTNF demonstrated a stronger

o f anti-dsDNA

metastasis-enhancing effect by rmTNF compared to rhTNF. Treatment with

incidence o f glom erulonephritis

mice treated with M oIFNy-R or mAb anti-IFNy. The treatments w ith a nti-IF N y significan tly

delayed the emergence o f anti-dsDNA

autoantibodics

without having developpcd

autoantibodics. These results confirm

in

M oIFNy-R or

high tilers

the accelerating role o f IFNy

in this pathology and show the capacity o f the MoIFNy-R in in hibitin g the disease

rmTNF or V lq on day 5 after tumor implantation resulted In no change of the metastatic potential Indicating an early effect of TNF No direct effect of rmTNF. rhTNF or V lq on the proliferation rate of tumor cells was seen in vtlro . Histological analysis of mtcrometastascs In the lung on day 5 revealed more metastatic

foci

and

mctabollcally

more active

tumor cells

after rmTNF

pretreatment. Experiments to analyze the mechanisms of this metastasisenhancing effect of TNF are In progress.

M ICR OG LIAL C E L L C LO N ES PRESENT ANTIGEN IN VITRO

MODULATION OK MONOKINK PRODUCTION BY THKMSKLVBS OR BY XKNOHIOTICS M.A. Parant* 1

Pnola Puglia. Francesca G ra nu cci. L in d a C a lm s and P iq Ih R ic c ia rd i C nstaanoli, C N R . C e nte r o f C yto p h a m m c o lo g y . U n iv e rs ity o f M ila n o , M ila n o . I T A L Y .

University Paris 6, 15 ruó de 1•Ecole-de-Médecino, 75270 Paris 6 France.

T o study the fu n c tio n a l properties o f m ic ro g lia l c e lls w e have generated m ic ro g lia l ce ll c lo n e s fro m e m b ry o n ic B a lb /c b ra in c u ltu re s (F. 13) b y im m o r ta liz a tio n w ith re co m b in a n t re tro viru ses. In p re vio u s studies w e have d em onstrated Ihnt m ic ro g lia l clo n e s respond to p h y s io lo g ic a l s ig n a ls such IF N -y and tha t, lik e p rim a ry m ic ro g lia l

Circulating iovel rial

of monokines

following administration of

component can bo modulated by

a bacte­

various agents, including cytokinoo,

in an intricate network. Thua, pretreatment of mice with rMuTNF or rHuTNF

c e lls, they can be in du ce d I«» tra nscribe c y to kin e s genes such as 1 L -1. I L -6 m id T N F - a . In a d d itio n n itric o x id e synthase a c tiv ity has been dem onstrated in the same clones. In

caused

th is re p ort w c have ana lyze d the a b ility o f these m ic ro g lia l clo n e s to express MHC" class II m o le cu le s and present a n tig en . IF N -y acts s y n c rg is tic a lly w ith G M -C F S to

and IL-6. In vitro and ex vivo

enhance the expression o f M H C class II m o le cules. T h e a ctiva te d m ic ro g lia l c e lls w e re tested f o r a n tig e n p re s e n ta tio n o f S p c rn tw h a lc m y o g lo b in to a T h I c e ll

a marked enhancement in the yiold

tiating effect of both

TNF. IL-1 a or B

dopronood the

of LPS-induced serum TNF, IL-1

assays have also shown tbo strong

TNF response

poten­

displayed differential offocts. They upon subsequent LPS

challenge without

h y b rid o m a . 1 3 .2 6 .8 -H 6 .1. re c o g n iz in g the 1 32-147 m y o g lo b in p e p tide in a H -2 ^ re stricte d co n te xt. F ifty thousand T c e ll h y b rid s /w e ll w e re cu ltu re d w ith the same

affecting

n um be r o f A B C s u sing the n a tive a n tig en doses in d ica te d in fig u re I. A ft e r 24 hr» l(X) |t l a liq uo ts o f supernnntnnt were transferred to fresh m ic ro titc r plates together w ith five

phages to LPS (mRNA or protein) was not influenced in the proaence of IL-

Ih o u sa n d /w cll I I . -2 dependent H T -2 ce ils. ^ H -T h y m id in e in c o rp o ra tio n o f H T - 2 was measured 24 hou rs later. A s sh ow n m ic ro g lia l c e lls were a ble to process and present the s p e c ific antig en upon a c tiv a tio n w ith IF N - y «nd G M -C S F . T h e a b ility o f resident m ic ro g lia l c e lls to process and present a n tig e n s in v itro raises the q u e stio n o f the p otential A P C fu n ctio n s o f endogenous m ic ro g lia l ce lls in vivo .

serum IL-6 titer,

but in vitro

the response

of mouse macro­

1. Both IL-1 and IL-6 were used in assays performed in mico that had been adrenalectomized. Whereas the inhibitory offeet of IL-6 on TNF production was maintained in these animals, pretroatment with IL-1 exerted a priming effect for an effect of

enhanced cytokine response to

IL-6 appeared to bo

two contradictory effecte of nerum corticosterone may mice, and the be

related to

observed after

LPS. Therefore, whoreaa the

a direct one on

TNF-producing cello, tho

IL-1 were indirect.

The enhanced lovol

of

account for depression of TNF relnaoo in normal

stimulating effect in tho abaence other mediators.

The

of adronaln appeared to

potentiation of

pretroatment with muramyl

reaponooo

dipeptides (MDP) was

to LPS shown to

depond on a direct action on macrophages loading to expression of mRNA of the three cytokines the

and to a strong increase

LPS challenge. Tho level of protein

MDPs was rather low in the absence of any

cytokine did not seem

in mRNA accumulation after

roloased after stimulation with

of a socond signal and tho production

responsible for the priming

offeet of the

26

Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/18/2019 1:34:47 PM

synthetic compounds.

PREVENTION OF CIRCULATORY SHOCK BY THE MONOCYTE/MACROPHAGE ACTIVATOR MTP-PE IN A PIG-MODEL OF SEPTICEMIA

T H E C Y T O S T A T IC A C T IV IT Y A C Q U IR E D BY H UM AN M O N O C Y T E S D U R IN G T H E IR D IF F E R E N T IA T IO N IN T O M A C R O P H A G E S IS INDEPENDENT O F NO AND TNFa

Passlick,B.*, Izbicki,J.R.*, Ostertag,P. *, Löffler,Th.* and Ziegler-Heitbrock,H.W.L.'

Je an -F ran çois P e tit. Phan B ich L ie n and G eneviève L e m a ire , U R A 1116. B â tim e n t 4 3 2 . U n iv e rs ité P aris-S ud , 914 05 -O rsa y C h an tal M a rtin a c h c and M a n ue l L op ez, IN S E R M U 76. C N T S . 53 Bd D id e ro t. 7 5 0 1 2 -P a ris .

*Dept. of Surgery and 'institute of Immunology, University of Munich, Munich, Germany Septicemia is still a major problem in patients with polytrauma or

W hen d iffe re n tia te d

major surgery, leading to septic shock, organ failure and death. AB

Prevention of septic shock in patients at risk would be tempting

in to m acrophages in te flo n bags in the presence o f

serum , hum an m o n ocyte s a cqu ire a c y to s ta tic a c tiv ity aga in st tu m o r c e lls

(A n dree sen et a i., J. Im m u n o l.

M ethods.

2 95 -3 04 .

way to face this problem. We therefore conducted a trial with the

d u rin g

monocyte activator muramyl-tripeptide-phosphatidylethanolamine

é lu tria tio n at day 0 (D o ) o r m o n on uclea r c e lls fro m

seven

d ays

u nd er

these

c o n d itio n s

e ith e r

obta ine d by é lu tria tio n at D 7 . C y to s ta tic a c tiv ity

1983).

We

m o n o cyte s w h ic h

was m o n ito re d

b y c o c u ltu rc

o f D 7 m acrophages

and tu m o r c e lls at an e ffc c to r/ta rg e t ra tio

Haterial and Methods: Liposome encapsulated MTP-PE (50 pg/kg; n=5)

presence

th y m id in e .

Wc

or liposomes alone (n=5) were given i.v. 24 h before sepsis was

tritia te d

used

m a s to c y to m a

induced by LPS (S.abortus, 4pg/kg/h). Animals were anaethesized

TNFa, lin e s

and mechanically ventilated and monitored over an 8 h period.

the w as

(re s id u a l

c o m p le t e ly

The e x h ib it

animals and control animals were found with respect to septic

even

leuco- and thrombocytopenia, hemo-concentratlon and urinary

5 00

P ifi r a f o r t t f . E . T r i t a r e lll * .

B io lo g ía

C e llu la r e S u p e rio re

a nd di

U.

T e s la *.

Minen ¡

M.

E m a to lo g ia - O n c o lo g ia *

S a n ita *,

V ía le

R egina

K lena

299,

00161

ROMA,

as e ffe c to r o f

phagocytes*

IT A L Y

P u r p o s e ; N itr ic m ediated

o xide

c yto sta sis

synthesized

by

(NO®)

was recently

a n d /o r

e nzim auc

id e n tifie d

c y to to x y c ity .

o xid a tio n

of

NO®.

a

L -a rg in in c ,

is

( N 0 2 * ) form ation. Wc studied the a b ility o f H IV monocytes to produce N O c and M e th o d s :

Peripheral

produ ction

of

in volves

ihc

D M PO )

w hich

and

by

form a tion

is

saturated

nitrone

com pound

a of

rap idly

a

(spin the

G riess

s p littin g expected from

not

to

give

n itrite

a

DM PO

by

adduct

a

was able

in

fro m

p ro life ra tio n

v it r o

by

the

m u rin e

one of

the

three

d if f e r e n t ia t e d

m o n ocytes o r

fro m

P815

s e n s itiv e

to c e ll

hum an

m o n o n u c le a r c e lls

c o n d itio n e d

tw o c la s s ic a l

m e d ia

;

the

c y to s ta tic

a c tiv it y

is

in h ib ito rs o f N O -syn tha sc, N - m o n o m e th y l-L -

the

a c tiv it y

a c tiv ity

is

also

a ga in st

presence o f a n ti- T N F a

TNFa,

inde p en d en t

tw o

of

T N F a re sista nt

TNFa. c e ll

M a c ro p h a g e s

lin e s .

a ntib od ie s able to n e u tra liz e

w c s t ill observed an unchanged a c tiv ity

M o re o v e r, m o re

than

against the three

The

technique

to

form in

more

the

cell

D M PO

dismutasc.

w ith

o x id a tiv e

the

to stim ulate

can

th u s

c y to s ta tic

c o n c lu d e

tha t

m e c h a n is m s

bases arc c u rre n tly

hum an

m o n o cyte s

in d e p e n d e n t

of

d iffe re n tia te d

NO

and

in

v it r o

TN Fa. T h e ir

u nder in v e s tig a tio n .

Abstract CONCOMITTANT INCREASE OF ALVEOLAR MACROPHAGE RECEPTORS AND OF SOLUBLE RECEPTORS IN EXOGENOUS ALLERGIC ALVEOLITIS, SARCOIDOSIS AND LUNG FIBROSIS A. P fo rte 1. A. S c h ie B le rl, S.U. Kress1, A. Brunner1. B. 8ehr3, P. Gais4 , H.W.L Z iegle r-H eitb rock* j Medical C lin ic , Medical Department2 LMU, Munich1 I n s titu te o f Immunology LHU Munich* Central H ospital GautingJ GSF Munich

A ll the d iffe r e n t types o f a lv e o litis are characterized by an increased c e llu la r i t y in bronchoalveolar lavage due to an increase o f T-lymphocytes and alveo­ la r macrophages (AM). The AM are activa te d and we can demonstrate increased le v e ls o f IL-2R (CD25) s ta in in g in immunocyjochemistry. Cytometry reveals th s t in a lle r g ic a lv e o litis 21 % o f AM are IL-2R compared to 2 % in c o n tro ls. At the same time soluble serum IL-2R are increased by fa c to r 2 w ith a p o s itiv e c o rre la tio n fo r IL-2R* AM and ssIL-2R. A comparable pattern is seen in sar­ coidosis and in lung f ib r o s is . Furthermore, increased expression o f CD14 in AM a lv e o litis pa tie n ts wss also associated w ith increased serum le v e ls of sCD14 and ICAM-1. Response to therapy was associated w ith a decrease o f both AM associated and soluble serum receptors. The data suggest th a t soluble serum receptors msy be derived from AM and th s t serum parameters can be employed to study the status o f AM in various forms o f i n t e r s t it ia l lung disease.

the

NO2 ' release (150 %).

was observed

L -a rg in m e

Nevertheless, gave

radicals

and

superoxidc

adducts suggest

DM PO

days.

rcageni. concentration

of

0-4 T h is

N O 2 * was q u a n tifie d

o f 64 +_ 7%

in h ib ito r

observed

of

ra d ica l in

(5 .5 -d im c th y l-1-p y rro lin c -N -o x id c .

adduct increase)

m o d ified

constants o f the

those

so lu tio n

at p M

fo r

technique.

short-live d

adducts).

An in h ib itio n

s tc rc o s p c c ific

interm ediate

cultured

spin-trap p in g

trapping

(100-200%

was

an

free

gp 120 protein to activate blood

were

by

using

production

(P B M )

studied

radicals

and acetyl salicylate.

NO *

monocytes

c o n clu sio n s: Gp 120

radicals

d iffe re n t

of

is capable

s h o rt-liv e d

N02‘

was

add itio n

n itro x id c

adduct

blood

radicals

supernatants

h ypcrfinc

the

c y to s ta tic

U /m l

m o le c u la r

H U M A N B L O O D M O N O C Y T E S A C T IV A T IO N BY G P 120 H IV E N V E L O P E P R O T E IN . E V ID E N C E FO R N IT R IC O X ID E R E L E A S E .

a rg in in c ,

in

d e v e lo p

preventing septic shock and death in an experimental model.

The

a rre s te d

and

o f th y m id in e < 10% ).

m a c ro p h a g e to

c y to s ta tic

Wc

Conclusions; Monocyte activation by MTP-PE is effective in

PBM

lym p h o m a . The

w h e th e r o b ta in e d

to T N F a ,

c ry th ro lc u k c m ia

ta rge ts, in clu d in g the U 9 3 7 c e ll line.

output.

Results

in s e n s itiv e

K562

the

a rg in in c and c u - n itr o - L - a r g im n e .

significantlly improved. No differences between MTP-PE pretreated

culture

in

in s e n s itiv e

the arterial pressure is stable and blood oxygenation is

persistent

h is tio c y tic

in c o rp o ra tio n

d e te cte d

out of 5 control animals . The treated animals develop no fever,

1st i l u to

lin e s

hum an

o f 4 in

T he m acrophage c y to s ta tic a c tiv ity is not due to N O : n itrite s cannot be

shock and death: None of the MTP-PE protreated pigs died, but 5

D.

tu m o r c e ll the

U 937

m acro ph a ge s,

Results; MTP-PE is effective in preventing LPS-induced septic

tw o and

by

m acrophages w ere

(MTP-PE) in an experimental pig model.

of

c u ltiv a te d

o b ta in e d

form ation

h yp c rfin c

b ubbling

of

adduct

w ith

indom cthacin

w ith

N -n itro -L -

p ath w ay. of

s p littin g

NO®

The

D M PO -O H

g3S

h y p c rfin c

constants in

a N 2* s p littin g

constants supcrim posablc to that o f DM PO -O H. The reaction leading to DM PO -O H form ation

from

O ur data

indicate

NO® is unclear. that

gp

120

stim ulate

L -a rg in in c

o xida tive

pathway

in

PBM.

27

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N O ° / N 0 2 * release may contribute to ce llu lar and tissutal damage o f A ID S disease

FUNCTIONAL

AND

RECEPTOR CHARACTERIZATION

OF

MOUSE

PERITONEAL

TRYPANOSOMA

CRUZI

N a t h a lie

F re e

In

th is

b a c t e r ia l

of

s tu d y ,

we d e s c r i b e ,

a n a ly s is

of

b o th

The

d e t e r m in e d

u s in g

d is c o n t in u o u s

a

p ro c e d u re

im p ro v e d ty p o s .

m em b ra ne a n t ig e n s

same f r a c t i o n s b a c te r ia l assays

ty p ic a l

th e

lin e a g e .

m a c r o p h a g ic

fr a c tio n s

th e s e c e i l s

in

th e

t im e ,

a

c r ite r ia c e ll

and

a nd

fir s t Then,

a ls o

a h ig h

ra te

M o r p h o lo g ic a l

MACROPHAGE

and

c o u ld

e x p la in

some

of

B r u s s e ls ,

FRACTIONS

in f e c t io n

of A ll

c e ll

r a t io

p e rc e n ta g e

T h is

th e

v a r io u s

c e ll

per

sh ow e d

th a t

d if fe r e n c e

a n d 2 . 4G2 m o n o c lo n a l

o f Fc r e c e p t o r - m e d ia t e d and

e n z y m a t ic

fr a c tio n s c r u z i.

g r a d ie n t.

th e

c o lls

p a r a s ite .

th e

h

h ig h e r

of

in f e c t io n

th e

le v e l

is

These

d if fe r e n c e s

b a c t e r ia l

p h a g o c y to s is

a re

th e th e

im m a tu re m a tu r e

m a c ro p h a g e s . and

fu n c tio n a l

im m a tu re

d iv e r s ity

of

Immune r e s p o n s e .

ANTIGEN-PRESENTING AND ACCESSORY FUNCTION OF MONOCYTES AFTER PHAGOCYTOSIS OF

o b s e rv e d

a c tiv a te d to

th e

m u lt ip lic a t io n . s ta te

of

o b s e rv e d

24.

in

le s s

fro m

th e

In d e e d , th o

w it h

we

-

on

P e r c o ll p a r a s ite

In f e c t io n ,

have

o b s e rv e d

F ir s t,

th e

im m a tu re

m a c ro p h a g e s

to

be e x p la in e d

th e

th e

m a tu r e

w ith

th o

Im m a tu re m a c ro p h a g e s a r e th e

of

at

w ith

a

in fe c tio n fr a c tio n s .

In te rfe ro n -y

le s s

p a r a s ite

a th o

m a tu r e

( e s p e c ia lly

in

th e

a m a s tig o te s

le v e l

a nd m o re s e n s i t i v e

th o c ru z i

th e

in f e c t io n

o b s e rv e d

to T.

m u ltip lic a tio n

id e n t if ie d :

im m a tu re t o

a ls o

r e fle c tin g

th e

Is o la te d

A p r o g r e s s iv e e v o l u t i o n

w e re

p e r ito n e a l

s e n s it iv e

s t u d ie d

48 a n d 72 h o f

and in

b y a lo w

T h ts c o u ld

th e c e lls

1070

r e s id e n t

w e re in c u b a t e d

and - se co n d ,

m a c ro p h a g e s .

a c tiv a t io n

F re e

L e n n ik ,

h e t e r o g o n o it y , o n e ca n a sk

we h a v e

w o re c o u n t e d .

le v e l.

th a t

c e lls

M e d ic in e ,

a n d t h e mean n u m b e r o f

in f e c t io n

in f e c t io n )

to

th e s e

A fte r c o lls

Two c e l l g r o u p s w e re

u n d e rs c o re

of

1.

m a c ro p h a g e s c h a r a c t e r i z e d 72

of

R o u to d o

o f m ouse th is

c o u ld b e m o re o r

fr a c tio n s

in f e c t e d

in

808,

7 m a c ro p h a g e f r a c t i o n s

o f 3 to

in fe c te d

Due t o

T h e re fo re ,

th e c e l l

of

V ra y .

F a c u lty

B e lg iu m .

w e ll know n.

c e ll T.

r o u tin e .

c r ite r ia

a n d p e r fo r m e d b y b e tw e e n

U n iv e r s it y

f u n c t i o n a l h e t e r o g e n e it y

p a r a s ite

to

B e rn a rd

Im m u n o lo g y ,

o u r r e s u lt s

a p p a rte n a n c e

a n t ig e n s

if

a nd

of

m a c ro p h a g e s i s

in

a n a ly s is

M l/ 7 0

was

g r a d ie n t.

a ll

w e re

e n z y m a t ic

fr a c tio n s

The

flo w -

a n t ig e n s

T h e same f r a c t i o n s

P e r c o ll

b y F 4 /8 0 ,

d iffe r e n c e s

m a c ro p h a g e

fir s t

w e re r e a l i z e d

On t h e w h o le ,

e x p re s s e d

o b s e rv e d

de

e x p r e s s e d b y h ig h d e n s i t y m a c ro p h a g e s . T he

d is p l a y e d

c o n f ir m e d

p r e d o m in a n t ly

o f th e

h a r v e s t in g o f

d e te c te d

ro u te

P la s m a n

L a b o ra to ry

B r u s s e ls -

m em brane

f lo w - c y to m e tr ic

p h a g o c y to s is .

m a c ro p h a g e

The

th e The

BOB,

M e d ic in e ,

im mune s e ru m ) p h a g o c y t o s i s o f

c o n t in u o u s

w e re m o s t ly

th e

m o r p h o lo g ic a l d e n s it y

of

B e lg iu m .

fr a c tio n s .

P e r c o ll g r a d ie n ts

p e r ito n e a l

a n tib o d ie s

by

s p e c if ic

-

fo r

an

F a c u lty

B r u s s e ls ,

v a r io u s

( o p s o n iz e d o r n o t w i t h

c h a r a c t e r iz e d

assays.

of

1 07 0 B r u s s e ls

m ouse r e s i d e n t p e r i t o n e a l c e l l a ls o

Im m u n o lo g y ,

U n iv o r s ity

L e n n ik ,

N a t h a lie

P la s m a n a nd B e r n a r d V r a y .

L a b o ra to ry

c y to m e tr ic

INFECTIO N OF DIFFERENT

ISOLATED ON PERCOLL GRADIENT.

MACROPHAGES.

p e r m is s iv e e n tra n c e

a nd

by th e d if f e r e n t

m a tu r ity

a f u n c t io n a l h e te r o g e n e ity

a lr e a d y

o u r o t h e r e x p e r im e n t s

(s e e p o s te r s e s s io n ) .

M O L E C U L A R P A T H O G E N E S IS OF M O N O C Y T E S /M A C R O P H A C E S H I V - 1 IN F E C T IO N

BACTERIA. J.

Pryima'. J. Baran'. and H.-D. Flad*. ’Institute of Paediatrics. Copernicus School ot Medicine. Wiolicka

265. 30-663 Cracow. Poland: ’ Forschungsinstitut Borstel. D-2061 Borstei. Germany

H. R a o u l^ , R. l x N aour^l. A. Mabondzo^ ). F. B o u s s in ^ and D. D o r m o n t^ ) .___ (1). Laboratoire de Neuropathologie expérimentale et Neurovirologic. CRSSA/DSV/DPTE, CE A , Fontenay-aux-Roscs. France. (2). Institut Pasteur. Paris. France.

Monocytos/macrophagos (MO) are involved both in the induction and effector mechanisms ot the immune response, although it is not clear whether both functions can be performed by tho same cell at the same time. Thorelore, we examined the antgenpresenting (APC) and accessory function (AC) of monocytes after phagocytosis of bacteria. Ponphorai blood monocyios isolatod from mononuclear cells by counterflow elutnation wero incubatod with a suspension of bacteria (S.aureus. E.coK P. aeruginosa. S.entonMis) in the presence of fresh normal human serum under conditions whore at least 80% ot monocytes engulfed microorganisms These cells were either pulsed with aniigen (purified derivative of tuberculin - PPD, tetanus toxoid - IT) and usod as APC for autologous T lymphocytes, or their AC function was examined in pokowoed mitogon (PWM)activated cultures of T or T and B ceils it has boon found that phagocytosis of bactona by MO reduces their ability to trigger antigen- and mitogendnven proliferation. In contrast, when added to PWM-activated culturos of T and B lymphocytes. MO containing microorganisms enhanced B coll differentiation mto immunoglobulin-secreting cells. The reduced proliforative response of T lymphocytes was not due to a change of the kinetics ot the response or triggo ring of 'suppressor* mechanisms. Furthermore, antigen processing was not much offoctod afior phago cytoss of bacteria, since antigen-pulsed and paraformaWohyde-fixed cells containing bacteria were comparable to control colls as APC. in oontrasi as a result of phagocytosis of bacteria, a reduced ex­ pression of adhesion molecules (CD11. CD54), FcRI (CD64). CD14 and a vanablo reduction of HLA-DQ and HLA-DP molecules was observed. In conclusion, phagocytosis ot bacteria by MO affects their APC and AC functions presumably due to changes in the expression of molocu'os ossontial for MO-T cell inter­ actions.

O b je c tiv e s : Because c lin ica l and biological features indicate that a dysrégulation o f both cytokine pattern, and m icrobicidal a ctivity occurs in the monocytcs/macrophages o f HIV -1 infected patients, the regulation o f TNFa synthesis as w ell as MnSOD gene expression during macrophage H IV infection, has been investigated. A s previously reported, H IV - l/L A I infected macrophages did not produce any b iologically detectable TNFa w ith in the few hours follow ing lentiviral infection, whereas a high expression o f the M nSOD and TNFo genes was observed 2 and 4 hours after the infection. In this view, wc attempt to determine 1) the consequence on the regulation o f TNFo synthesis as w ell as M nSOD gene expression using monocytotropic H I V - 1 strains (IU V -1 /D A S and H IV -l/P A R ) 2) the infectious events in volved in these phenomena, testing the effects o f heat inactivated virus (L A I. D A S . PAR), and recombinant GP160 (rGP160) on the macrophages. M e th o d s : Human peripheral blood monocytes (P B M ) were obtained fro m healthy HIV -1 seronegative donors using the centrifugal élutriation technique. They were cultured by adherence in RPM I 1640 supplemented w ith 10% fetal c a lf scrum fo r 7 days fo r diffe re n tiatio n into macrophages. Cells were then infected w ith H IV -l/D A S or H IV -l/P A R (0,001 T C lD 50 /ccll). or treated w ith heat inactivated HIV-1 strains (56°C, 30 mn) o r K3P160 (lOng/lON'ells). Culture supernatants were harvested every 2 hours during the firs t 10 hours, fo r a TNFo biological activity test using Actinom ycinc D-trcated L-929 cells. Concomitantly, expression levels o f the MnSOD and TNFa genes were determined by Northern blots analysis. R e s u lts : C o ntrarily to the results obtained w ith H I V - l/L A I. biological active TNFo was detectable during the early phase o f in vitro infection o f human macrophage cultures w ith the 2 monocytotropic strains tested. The results were the same when the different H IV -1 strains used were inactivated. No TNFo release was detectable in the supernatants o f macrophage rGP160 stimulated. A t the molecular level, between 2 and 4 hours after infection, an increase o f TNFa and MnSOD m R N A levels was observed compared to negative control, the virus being cither inactivated o r not. W hile these expressions decreased after 4 hours, and reached control values 10 hours p.i.w ith H IV -1 /L A I, no decrease was observed u ntil 10 hours using H IV -l/D A S and PAR strains. Treatment with 1G P I6O docs not change the expression level o f these 2 genes in the macrophages compared to control cultures. C o n c lu s io n : The detection o f b iologically active TNFo and the increase o f TNFo and MnSOD gene expression in the macrophage cultures is apparently related to the cellular tropism o f the viral strains used. These results may reflect an enhancement o f the infection. The results obtained with all the inactivated viral strains indicate that the phenomena observed are related to the viral entry. Therefore, the binding o f the rGP160 docs not seems to be sufficient to induce T N Fa or M nS O D expression. Experim ents to test the vira l specificity and to assess the mechanisms involved in the macrophagic response observed are now in progress, and results w ill be presented during the meeting.

28

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(Supportod by grant KBN 2040/4/91 of the Polish Research Committee)

THE TO X IC IT Y OF O X ID IZE D LO W DENSITY LIPOPROTEIN TOWARDS

PROSTAGLANDIN AND LEUKOTRIENE SYNTHESIS IN A MACROPHAGE HYBRID CELL LINE (H4-7)

MACROPHAGES IN VITRO

J Riese and V Kaever Institute of Molecular Pharmacology. Medical School. D-3000 Hannover 61, FRG

Reid. V.C.. Mitchinson, M.J.

Studies on the regulation of macrophage eicosanoid synthesis are often

Department o f Pathology. Division o f Genetic and C ellular Pathology, University o f Cambridge.

hampered by the inability of most m acrophage-like cell lines to secrete measurable

CB2 1QP, England.

amounts of arachidonate 5-lipoxigenase

(5-LX ) products. In contrast to

the

commonly used cell lines RAW264 7. PU5-1.8. or P388D1 the newly established The characteristic lipid-laden foam cells that predominate in the intima in the early stages o f human

mouse macrophage hybrid cell line H4-7 [1 ] was able to produce prostaglandins atherosclerosis arc known to be macrophages. I he more advanced lesions have a necrotic centre,

(PG) as well as leukotrienes (LT) upon calcium ionophore stimulation and this appears to be due principally to macrophage necrosis.

As shown by Western blotting (specific antisera were kindly provided by There is now considerable evidence that lipoprotein oxidation occurs w ith in the developing atherosclerotic lesion. Macrophages can oxidize low density lipoprotein (L D L ) in vitro. The properties o f oxidized L D L have aroused a lo t o f interest in the study o f atherosclerosis. One property which is o f particular interest is its toxicity towards various cell types found in the

Merck Frosst. Canada) H 4-7 cells contain 5-LX and 5-LX

activating protein

(FLAP). Addition of ionomycin to intact H4-7 cells led to a concentration- (optimum at 2.5 pg/m l) and tim e-

(maximal after 5 min) dependent translocation and

activation of 5 -L X and subsequent LTC4 synthesis (determined by ELISA). LTC4

atherosclerotic lesion in the artery w all. These have included endothelial cells and smooth muscle

production was inhibited by the specific 5-LX translocation inhibitor MK886 (Merck

cells.

Frosst. Canada).

Since, however, it appears that the necrosis in the advanced stages o f the disease is due to

We have used H4-7 cells as model system for macrophage in vitro activation.

macrophages dying we decided to investigate whether macrophage-mediated lipoprotein oxidation

Addition of lipopolysaccharide (from Salmonella abortus equi) and recombinant

was toxic for the macrophages. We looked at artificial lipoprotein preparations containing the main

murine interferorigamma led to an enhancement of nitric oxide (measured as NOg“

cholcsteryl esters found w ithin L D L and known to be oxidized in vivo. We also looked at oxidized

by Griess reagent) and tumor necrosis factor (measured by bioassay) synthesis, in

L D L itself. Both were toxic to mouse peritoneal niaciophagex in vitro ana this toxicity was reduced

this m vitro system we are currently investigating changes m PG and LT production

by the addition o f antioxidant. The results might explain the im portant event o f the onset o f

during m acrophage activation

necrosis in the developing human lesion.

1

MACRO PHAG E A C TIVA TIO N BY M Y C O P LA S M A ARTHRITIDIS-DERIVED SUPER ANTIG EN (M AS ) NEEDS MORE TH A N l-E a OF MOUSE M H C -C LA SS II

Higuchi M, Higashi N. Taki H. Osawa T J Immunol 1990. 144 1425-1432

THE UPTAKE OF BORRELIA BURGDORFERI A PROMISING MODEL FOR MEMBRANE PROCESSING DURING PHAGOCYTOSIS

L Rm k1. W Nicklas2. M K oe ster1. M R euter1 and H K irchner1 'Institute o l Im m unology and Translusion M e d ian e . University o l Lübeck. M edical School, Lübeck. FRG. and :'G erm an C ancer Research Center. Heidelberg. FRG

MG RITTIG». T HAUPL*. UE SCHAIBLEc, M MODOtLEL'. MM SIMON', GR BURMESTER* From the •Department of Anatomy I and the •‘Institute of Clinical Immunology and Rheumatology. Department of Medicine III. University of Erlangen-Nürnberg, Erlangen. Germany, and the r MaxPlanck-Institutfür Immunbiologie, Freiburg, Germany.

Mycoplasm a arthritidis produces a potent T cell mitogen, which belongs to the previously described group ot superantigens M ycoplasm a arthntidis-denved superantigen (MAS) nas a s p e a fio ty (or distmci V ß-regions ol the T cell receptor (TCR) like every

We performed an olectron microscopal study on tho uptake of the spirochete Borrelia burgdorferi by human and murine phagocytes from different lineages. The spirochetes have a diameter of 0.2S pm

superantigen In contrast to other superantigens MAS also has a specificity for the MHC

but a length of up to 30 pm. As a consequence, these organisms are at least twice as long as most

class-11 subtype and appears to be capable o f activating monocytes even in the absence

phagocytes. Tho uptake of such matorial may present a particular problem - Our study revealed that

of T cells. Only munne ce lls expressing an intact l-Ea molecule respond to MAS In this

the spirochetes were preferentially internalized via coiling phagocytosis. A sheath-like cell protrusion

study we show that not all l-E a positive cells respond to MAS Raw 264 7 and J 774.2

wrapped around a part of a spirochete in multiple turns. This led to a cylindrical coil-like complox

murine m onocytic cell lines respond to MAS as do

murine bone m arrow-derived

which subsequently was internalized. The clefts between the pseudopod rotations wero continuous

macrophages. Responses w ere m easured by IL -i and TN F-u production. On the other

with the extracellular space until the adjacent membranes of the coiled pseudopod fused and

hand P 388 d i . W ohi 3b and M t m unne m onocytic cell lines were not stim ulated by MAS All of these cell lines were definitely l-E a positive and able to produce IL-1 and TN F-« after induction with other stim ulants

It w as shown by other investigators that l-Ea

dissolved. In contrast. In conventional phagocytosis a part of a spirochete was dragged into the phagocyte where vacuoles developed along its lino. The clefts between the invaginated cell membranes also were continuous with the extracellular space until the membrane fusion. Ultrastructural studios have shown that intormodiato stages in endosome formation are connected to

transfected fibroblasts respond to MAS. However, the L 929 m unne fibroblast cell line the coll surface via narrow necks. Regarding the uptake of B. burgdorferi cells, invaginations

stimulation From these data, we conclude that there must be a receptor for MAS different from l-E a. Maybe for this reason MAS. in contrast to other superantigens, directly activates monocytes

stretched along the entire length of the spirochete sections internalized. These clefts were occupying a considerable area of tho coll membrane. Duo to tho multiple wrapping, these membrane areas were more extonsivo during coiling phagocytosis as compared to conventional phagocytosis. - In summary, membrane events which are not usually encountered during the uptake of other matorial are displayed to a large extent during tho phagocytosis of B. burgdorferi. In addition, other unusual features are regularly observed which have been only reportod as occasional events so far. The phagocytosis of B. burgdorferi may serve as a promising model tor membrane processing during phagocytosis.

29

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which w as strongly I-E « positive after treatm ent with IFN-y did not react to MAS

INTERFERO N IN D U C TIO N BY HIV-INFECTED MONOCYTES/M ACROPHAGES.

DIFFE R E N TIA L IN D U C T IO N OF M A C R O P H A G E E AR LY GENES, c -fo s . K C . JE A N D T N F -A L P H A BY L IP O A R A 8 IN O M A N N A N FRO M V IR U LE N T A N D A V IR U LE N T S T R A IN S OF M Y C O B A C T E R IU M TUB E R C U LO SIS

K.

Rokos and G. Pauli. A ID S-Zentrum dcs Bundesgesundhcitaamtes, Robert Koch-Instuu..

Nordufer 20, D-1000 Berlin 65. Germany. R o ach T IA , C b a ite rje e D. B la c k w e ll J M ; D e p t. M e d ic in e . U n iv e rs ity o f C a m b rid g e . A d d e n b ro o k e ’ s H o s p ita l. E ngland

Increasing evidence point» to n crucial role o f cell* o f the monocyte/macrophagc (M D M )

L ip o a ra b in o m a n n a n and 14 healthy volunteers on consecutive days (D l.3.5.7.10) post trauma were studied

nisms in mononuclear phagocytes and to mediate vasodilatation, for example in endotoxin shock Important amounts o f R N I arc secreted by resident and inflammatory munne macro­

Protein release was measured via RIA (IL-10) respectively ncuiropliil-glucoromdase release bioassay tIL-K),

phages after induction o f NO-synthasc (N O S ) in v itro Most powerful inductors are LPS. IFN-

mKNA expression follow mg cytolyw* was determined over northern blotting and radioactive hybridisation with

y and the combination thereof Induction o f NOS can be counteracted by different cytokines

the ’ 2P-labclcd corresponding cDN'A's The ictults foi Ihc calculation of live protein production of the

and some pharmacological agents a) IL-4 and TG F-fl dramatically inhibit release o f R N I by

individual MO. cxpiesscd as synthesis factor 10 1 - ID. flL-X). nre depicted m ihc tabic ( X*SEM, • p-0 05 vs

LPS and IFN-y-stimulated murine peritoneal macrophages

b) Dexamethasonc directly

control t( >1 suppresses induction o f NOS by LPS and IFN-y and synergizes w ith c) 2.4-diamino,6-hy__________ D1________ D3________ D5

___ D7________ DIO_______ C_________ droxypynmidine (D A H P ), an inhibitor o f dc novo-biosynthesis o f tctrahydrobiopterm (B H4),

flL -l______

V4*»0 7

3,X-»0 9

2 6*«0.7

f.r.O 4

X8*2 2

5 5»Q8 an obligate cofactor o f NOS d) D A H P itse lf appears to inhibit NOS. since release o f R NI can­

Wo saw an almost identical puttem of synthesis for both monokines dining Ihc time of observation, with a

not be completely restored by scpiaptcrin. an analogue o f B H 4 . Accordingly, inhibition o f NOS

considerable impairment until D5 and recovery thereafter While for IL-8 in the majority of patients a dear

by D A H P protects mice in vivo from lethal endotoxin shock (P < 0 05) W c conclude that

concurrence between the mRKA signal intensity and the quantity of piotcm release was found, only in 3 out of

NOS is an im portant target fo r regulation by biological immunomodulators (LPS. IFN-y, IL-4.

14 patients this was also live case for IL-10 From these data we conclude that the launching mechanisms fot

T G F -fl) as well as by glucocorticoids o r antagonist o f cofactor-biosynthesis which might be

the dc-novo synthesis for both monokine* under stress differ greatly, with II.-X being clearly regulated on tlic

exploited for treatment o f experimental endotoxin shock

transcriptional level while Ihc dowiircgnlalion of IL-10, initially post trauma, occurs most likely on the

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povltmnvcriptiniial level

THE ROLE OF MONONUCLEAR PHAGOCYTES IN THE PATHOGENESIS ATHEROSCLEROSIS G Schm itz E Kovacs. G Rolho Institute for C linical Chem istry and Laboratory Medicine. University of Regensburg

OF

In atherosclerosis m ononuclear p hagocytes play a key role in the destruction of the vessel wall through endothelial adherence and invasion o f circulating monocytes induction of sm ooth muscle cell proliferation through the secretion of growth factors, and transform a­ tion into lipid-loaded foam cells or activated cells It is not clear, however, whether pheno­ typic or functional alterations of peripherally circulating monocytes and their subpopula­ tions may be an independent factor in the pathogenesis o f atherosclerosis as suggested by the occurrence of atherosclerosis In only a subset of patients w ith hypercholesterole­ m ia and in contrast m acrophage accum ulation in the spleen and RES In the genetic plasm a HDL deficiency of Tangier disease Therefore, in this study the expression of acti­ vation and differentiation markers (CD14, C 016, CD4. CD64. HLA-DR). membrane bound com plem ent regulatory proteins (C D i1 b , CD18, CD25, CD35, CD46. CD5S CD59) and adhesion molecules (CD11a CD48 CD58) was studied on monocytes from freshly drawn blood of patients with hypercholesterolem ia and Tangier disease The hypercholesteroiem ic patients were divided into two groups according to the presence (C H D +) or lack of (CHD") coronary heart disease A significant decrease of differentiation, activation m arkers and m embrane bound com plem ent com ponents was detectable on monocytes from C H D 4 and Tangier patients suggesting either a downreguiation o f surface receptors or the selective depletion of mature monocytes This hypothesis was further supported by the reduction of the C D1447CD164' subpopulation in CHD+ pationts To study the effects of lipoproteins on circulating m onocytes, mononuclear phagocytes from healthy controls and a Tangier patient were cultured for 1-7 days in the presence of LDL, its modified form s (acLDL. oxLDL). HDL2 and HDL3 LDL and its m odified form s as w ell as HDL subclasses induced higher expression of the complem ent regulatory proteins and adhesion m olecules during the culture o f monocytes from controls In Tangier diseaso in contrast the m arkers were down regulated by HDL subclasses A reduced oxidative burst was furtherm ore detected in 1 day H D L3 cultured Tangier monocytes In prolonged culture of 7 days, monocytes showed a retarded expression of HLA-DR, CD14 and CD4 in the presence of the LDL derived fractions, but an increase of the C D 14^/C D 164

NATURAL SOLUBLE HUMAN CD14 (sCI)14) PREVENTS BINDING OF LPS TO MONOCYTES ANI) THEIR ACTIVATION C . Schütt, C . Krüger, T . Schilling, U . Grunwald. Dept. Med. Immunology, Ernst-Moritz-Arndt-Umversity. Greifsw ald. Germany. Binding o f the L P S:L B P complex to monocytes via CD14 causes them to be activated and release large amounts o f cytotoxic factors including T N F or oxygen radicals. This activation is thought to be the major pathway leading to endotoxin shock; therefore inhibition o f cell activation might provide an useful theory. Purified native, spontaneous released soluble C D I4 is a functionally active receptor. The specifily of endotoxin binding by s C D !4 could be demonstrated using blocking and non blocking anti C D 1 4 antibodies. s C D l4 inhibits the binding o f F IT C -L P S to both human and bovine monocytes. In comparison to human CD14 the binding affinity o f bovine CD14 to LBP-LPS-complexes is lower. Human CD14 suppresses the LPS-induciblc oxidative burst response o f normal monocytes. Its suppressive capacity works even in the case o f C D 14-negative monocytes o f PNH patients which were highly responsible to LPS activation in vitro. The potential of s C D !4 as a new therapeutic for septic shock is being evaluated.

subpopulation in the case of HDL subfractions in controls In contrast. 7 day cultured Tangier monocytes reacted with an enorm ous expression of complem ent regulatory pro­ teins and adhesion molecules in the case of HDL2 . HDI.3 and oxLDL suggesting an al­ tered signal transduction of m onocytes in Tangior diseaso On the basis of our results we conclude that the hotorogeneity and prim ing of circulating monocytes is an independent in dicator for the atherogenic risk during hypercholesterolem ia o r splenom egaly in Tangier disease and should bo of value for the developm ent and evaluation o f now thorapeutical approaches

THE RELEVANCE OF SUPPORTING ANI) NEUTRALIZING COM ­

INFLUENCE OF PAF AND LTB4 ON THE RELEASE OF CYTOKINES AND 8UPEROXIDE ANION BY

POUNDS IN BODY FLUIDS FOR CELLULAR ACTIVATION BY

DIFFERENTIATED HL-60-CELLS AND ALVEOLAR MACROPHAGES

ENDOTOXINS C . Schütt, C . Krüger, F . Steller, T . Schilling, S. Witt, K . Vaczi. U . Grunwald

I. LUid. A. Seidel Kcmiorschungszentrum Karlsruhe. Insillu t fdr Genellk und Toxtkologlc. Postfach 3640. D-7500 Karlsruhe 1. Germany

Dept. M ed. Immunology, Ernst-Moritz-Amdl-Univcrsity. Greifswald, Ger­ many

In experimental toxicology one single cell type Is usually exposed In vitro to the noxious agent and

The measurement o f endotoxin concentrations in patients blood is not of

then, for example, the release of cytokines or superoxide anion ts measusred. In vivo granulocytes and

prognostic relevance for their outcome. However, serum proteins like H D L . LPS-binding proteins (LBP. Scpun) or sC D I4 can influence the cellular activation via C D 1 4 or other receptors and its further consequences. The binding o f L P S to C D 1 4 is mediated by purified LB P . this function o f LBP

macrophages may be subject to the Influence of lipid mediators which are released by other components of the tissue and which could possibly modulate the response of the test cells. The atm of our study was to determine whether potent lipid mediators such as PAF or LTB4 could induce the production of some cytokines and superoxldc by themselves and whether they can modulate the stimulus (LPS- or Zymosan) Induced release of these mediators.

is not species restricted. There seems to be a different affinity o f C D I4 to

III.-GO cells were differentiated to granulocytes IHL-60-G) by 1.3% DMSO or macrophages (HL-60-M) by

endotoxins in different species as well as between membrane bound and

Calcltnol (10 6M) and cultivated on membranes directly In gas phase II was found that the cells can

soluble C D 14 in the human system. The soluble form is functionally active.

secrete the cytokines Into the culture medium betow the membranes LPS (10 pg/ml) is n potent Inducer

It seems likely that the relationship between LBP and s C D I4 in different species reflects their different endotoxin sensitivity. Endotoxins and different cytokines regulate the production o f s C D l4 and LBP. Soluble CD14 is able to interfere with activation o f CD l4-negativc cells by LPS too. Using a lot

for the release of TNF. 1L-6 and 11.-8 by HL-60-G as well as HL-60-M. PAF alone 11 ng/mll does not cause any secretion of these cytokines. It has no effect at all on the response of both cell types towards LPS stimulation. Bovine alveolar macrophages (BAM) obtained by post-mortem lavage were also kept in gas phase in this case, the secretion of a TNF-llke activity had to be determined by the cytotoxicity of the medium to WEH1 cells. As with HI.-60 cells. BAM secrete TNF when stimulated with 1.PS. Neither

o f anti C D l4 m A b we could demonstrate, that there is a variation in epitope

PAF nor LTB4 (20 11MI showed any effect when applied as single substance. In analogy to the findings

expression on human C D I4 during monocyte/macrophage differentiation or

reported above, their combination with LPS did not change the stimulus-induced TNF production PAF

granulocyte activation which raised many new questions. These results indicate, that a multiparameter monitoring looking for H D L -. LBP-. and s C D 14-conceniraiions can realize a more detailed view o f patients situation.

was also not able to stimulate any superoxldc anion release, neither In gas phase culture nor in normal culture conditions. Summing up. the results suggest that PAF seems not to play an important role In the regulation of the response of macrophages and granulocytes to stimuli. By itself it has no influence on the release of

32

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some important promllammatory mediators by macrophages and gninulocytcs

ACCESSORY CELLS DERIVED FROM MONOCYTES RESEMBLE

PYRROLIDINE-DITHIOCARBAMATE (PDTC) INHIBITS BOTH NP. B MOBILIZATION

LANGERHANS CELIAS IN PHENOTYPE AND PRIMARY MLR

AND TNF PRODUCTION IN HUMAN MONOCYTES. T.Sternsdorf■ M. Strobel. A.

Fulko Steinbach and Bernhard Thiele* Institut fill- Virologie, FT? Berlin and •Chttrittf, III’ Berlin

Wedel.

R.

Institute

Schreck,

for

P.A.

Bauerle.

Immunology.

H.W.L.

University

Ziegler-Heitbrock.

of

Munich,

MPI

for

Biochemistry. Martinsried

II bus been shown c u rlie r (hut human peripheral blood monocytes can be

Lipopolyaaccharide (LPS) stimulation of human Mono Mac 6 cells

diffcrentiatiaicd into accessory cells ( I ) and that an alternative way o f differentiation

will result in s rapid and transient transcription of the TNF gene

leads to a 1xrngerhans cell phenotype, which we lately described first (2).

with a maximum at 2 ha. Proceeding this transcriptional activation

f o r this purpose, we isolated monocytes via H coll-paque and selective adherence to plastic dishes. As shown by Gicinsa95% and via b ility >98%. functional activity has been measured by non­ specific esterase and phagocytosis o f Candida albicans. Surface markers were

there is a mobilization of the nuclear factor NF* B as detected by binding of a p50 homodimer and a p50/p65 hetarotetramer to a -605 motif

in

the

TNF

promoter.

transcriptional activation

In

order

to

demonstrate

that

the

is mediated by NP* B , we have blocked

NF* B mobilization with PDTC (300 yM at -1 h). Such treatment will

investigated using flow cytonictiv

not affect binding of nuclear factors to SP-1, CRE or c/EBP motifs The analysis o f surface markers characterizes the cells as C D 1 + and 0 D 1 4 ♦ or .

in depending on the stage o f differentiation. As C P I is an originally lymphocyte marker, and C D 14 picks only a subpopulation o f these cells, we tried lo define further criteria fo r m orphological characterization o f m onocytic accessory cells. As a result, we provide evidence that, by using m annosyl-B SA -FITC . lectins like the mannose receptor arc highlv sufficient homogenous markers fo r all populations o f inonocylie cclls.

the human

nuclear

TNF

extracts

promoter, is

while

reduced

to

binding

activity

constitutive

of

levels.

NF* B in

This

PDTC

treatment effectively prevented the rise in transcript levels and it almost completely blocked synthesis of TNF protein. A similar blockade of TNF transcript levels was observed in PCR analysis of PDTC traeted blood monocytes. The data support the idea that NF* B is central to expression of the human TNF gene in monocytes and

Dense cultures o f human peripheral blood monocytes often show a p ro life ra tiv e

they suggest that substances like PDTC may be useful

a c tiv ity , but it has to be underlined that p ro life ra tio n is not required fo r the

detrimental effects of TNF.

to control

generation o f this phenotype. Our recent studies on the lym phocyte s tim ulatin g capacity in a m ixed lym phocyte reaction have fu rlh e i demonstrated that the m onocytc-dcrivcd cells resemble l^angcrhans cells in phenotype function

1) H M N * jw . A .C B iu -C a p d o || | C. R .K .I I G lc s e k i anvJ J .H Pcicr*. fu r . J.t W / .H o cker .Gelder fokin.ThicIc *n J Stcinbarti, hnm. /-fit . . 4 0 U/ml).

conditions,

as

even

The inducibility o f CD14 and LBP expression was analysed by P CR and

extracts. Such constitutive NF* B can be detected in several cell

demonstrated

under

by

gel

serum shift

free

and

analysis

LPS of

free

nuclear

protein analysis. We found that the cells were able to express C D I4 - as well

lines of the myelomonocytic lineage, but not in Molt 4 T-cells or

as LBP-m R N A constitutively. Whereas the level o f C D 14 m RN A did not

K562 stem cells.

change after stimulation with L P S and/or IL6 the level o f LBP-m R N A in­

enhanced with differentiation, but the constitutive NF*B

creased after stimulation with IL6. It was not possible to detect membrane

increased in the more mature cell lines,

bound C D 14 on the cell surface by F A C S analysis or soluble C D 14 in the

after

culture supernatant by E L IS A technique. LBP concentrations were measured

constitutive

by a newly developed functional assay using FACS-analysis o f L P S-F IT C binding to C D 14. IL6 was able lo induce LBP release into the supernatant, whereas L P S did not influence the cells. The IL6 inducing capacity could be enhanced by simultaneous addition o f LPS.

treatment

several

NF*B

tissue

The amount

of LPS-induced

with

vitamin

D3.

was

detected

in

macrophages

A

NF* B appears

to be was not

like Mono Mac 6. even variable

primary

independent

expression

blood on

monocytes the

stage

of and of

differentiation. The data show that cells of the monocyte system under

conditions

constitutive NF*B

that

exclude

major

external

signals

do

show

without an apparent correlation to the stage of

differentiation.

33

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Schütt

B. Passlick. P.A. Bauerle.

Institute for Immunology, University of

o f s u r f a c e m a r k e r s ON ALVEOLAR MACROPHAGES (AM) FROM PATIENTS WITH HIV-INFECTION OR SARCOIDOSIS AS DFTECTFP BY FLOW CYTOMETRY

MACROPHAGE DIFFERENTIATION IN M ACROPHAGE CO LONY STIMULATING

M. Subklowc. K. Wassermann. E. Schell Frederick

Kiyoshi Takahashi. M akolo Naito. Yuki Morioka. Shu|l Um eda. and Leonard D. Shullz.

expression

I/III Mmlizinlacho Kllnik. Haus 11, Joseph Siel/.mann Sir. 9.

S econd Departm ent of Pathology. Kum am oto U niversity School of Medicine. Kumamoto.

D-bOOO Koln 41, Germany

Japan, and the Jackson Laboratory. Bar Harbor. Maine, USA

the cjoal of thin tit tidy In to detect and quantify surface antigens on human alveolar macrophages by flow cylometry. Expression of surface molecules has been shown to be associated with the functional capacity of these cells. The primary antibodies were selected to measure antigen p r e a enrmg capacity CHI A DP, DO, DR), monocyte like cells (Leu M3, Leu M5, My7, My9> and tlie 8tat>* of cel) activation

(Transferrin R, leu 15, 27E10,

RM 3/1, IL-2R, TCAM 1). FITC-laboled P(ab’)2 antt-Ig served as a second antibody.

FACTOR-DEFICIENT MICE FOR THE OSTEO PETRO TIC (op ) MUTATION.

A CD 2 .

3,

19 cocktail

was used

to gate out

lymphocytes. The results are expressed as the median linear fluorescent intensity of the tent antibody divided by that of the Isotype control. This quotient indicates the intensity of specific antigen expression and corrects for variation in cell autofIuorescence between patients. To date we have analysed •'ells from 30 HIV infected patients, 10 patients with newly diagnosed sarcoidosis and 13 normal controls. The results chow significantly increased intensity of Leu M3. Leu Mb. Leu lb and HLA-DP expression In both patient groups. In contrast, there is ‘¡ignlf leant, ly highor expression of Transferrin R in sarcoidosis as compared to controls but no increase in AM from HIV-patlents.

In mice hom ozygous for o p mutation (o p /o p mice), this mutation results in a defect in the coding region of M-CSF gene, leading to a total absence of functional activity of m acrophage colony stim ulating factor (M-CSF). In the m utant mice, serious deficiencies of monocytes in peripheral blood, m onocyte-derived macrophages, splenic marginal zone macrophages, and osteoclasts w ere observed. In m any tissues, num bers of m acrophages variably decreased, particularly in the ovaries and uterus. However, num erical reduction of m acrophages was not prom inent in the spleen and brain. These o p m acrophages were small, round, and im mature. Ultrastructurally. they showed poor developm ent of intracellular organelles, particularly lysosom al com partm ents, and few cytoplasm ic projections. In the o p /o p mice, serum G M -C SF level was higher than in the normal littermates

In co-culture o f norm al m ouse bone marrow cells on a fibroblast cell

line established from an o p/o p m ouse, they stopped differentiating into macrophages at the stage of m onocytes. However, less than 5% of im m ature macrophages were detected, corresponding to the o p m acrophages

in culture, a major cytokine secreted by

the o p fibroblast cell line was dem onstrated to be GM-CSF. Com pared to m acrophages developed in cultures with GM-CSF, M-CSF, o r IL-3 respectively. G M -C SF-derived m acrophages closely resem ble the o p macrophages.

All these results suggest that GM-

CSF mainly supports the differentiation of m acrophages in o p 'o p mice and that the op m acrophages are M -CS F-independent m acrophages derived not from monocytes.

THE ROLE OF MEDIATORS IN THE PATHOGENESIS OF SILICOSIS. I. STIMULATION OF CELL

L IP ID A P A R T IA L STRUCTURES:

PROLIFERATION

PRO DUCTIO N. A .J.U lm e r1. W.Feist«. T .K irika c2. P.Kirikae2, H .H e in e'. S.Ku»umoto>.

OF

HUMAN

PNEUMOCYTES

TYPE

II

(LINE

A-549)

INDUCED

BY

IN H IB IT IO N OP LPS-1NDUCED M O N O K IN E

T.K usam a', H.Bradc2, U .Schade2, E.Th.Rictschcl2, and H .-D .F la d '. 'D ept.of Immunol.and

SUPERNATANTS OF QUARTZ DUST EXPOSED HUMAN MACROPHAGES

Cell B io l., and 2Dept.of Imnuinochem.and liioch cm .M icrob io l., Forschungsinstitut Bonsiel, Borstel,

U r s u la T h ie l. B . J u n g , N .H .S e e m a y e r . H e lg a Id e l

Medical Institute of Environmental Hygiene at the University of Düsseldorf. Gurlittstr.53, D-

Germany,

JD cp t.o f Chem istry,

Osaka U niversity,

Osaka,

Japan.

4Daichi

Pharmaceutical Co. L td .. Tokyo. Japan. 1-ipopoly.saccharide (LPS) from the cell wall o f gram-negative bacteria is responsible for a

4000 Düsseldorf. F.R.G.

variety o f pathophysiologic events (c.g. fever, septic shock) during infection. These events arc predominantly mediated by monokines (IL -1 , IL -6 , and TN F) produced by the infected host.

Silicosis is a pulm onary disease caused by quartz containing dusts resulting in lung fibrosis.

LPS consists o f a hydrophilic polysaccharide and a covalently linked hydrophobic lip id part,

In pathogenesis o f silicosis macrophages play a pivotal role

termed lipid A Lipid A represents the endotoxic principle o f LPS, i.e. the biological effects o f

Human marophages were obtained by cultivation and differentiation of human monocytes

I.PS arc reproduced

from the peripheral blood. Macrophages were incubated for 24 hours w ith Dbrentrupor

corresponding partial structures o f LPS. such as Escherichia co li-type lipid A (compound 506

crystal q ua rn flour (grinding no. 12. particle size smaller than 5pm) at a non-toxic

or 1.A-I5-PP) and precursor la (compound 406 o r I.A-14-PP) has provided the experimental

by free lip id A

The successful chemical synthesis o f lipid A and

basis to investigate the bioactivc regions o f LPS with regard to structure-activity relationships.

concentration Thereafter supernatants woro collected. In earlier studies we found, that supernatants of quartz dust troated human macrophages in

A structure-dependent hierarchy o f LPS and LPS partial structures with respect to their IL - I- . IL -6*. and TNF-inducing capacity was demonstrated, showing that the hcxaacyl type lip id A

culture lead to remarkablo cell proliferation of human lung fibroblasts (Wi-38).

I.A -I5 -P P is able to induce monokine production by human monocyte«, while the tctraacyl

By addition o f a "competence factor" (plateled derived growth factor) stim ulating effect of the cell proliferation factor was further enhanced

type"

The

factor

w hich

was

The synthetic L A -I4 -P P and its

phosphonxycihyl analogue PE-4 became o f particular interest since our studies show that

These results suggest that the cell proliferation factor belongs to growth factors of the "progression

compound L A -I4 -P P exhibited no inducing capacity.

provisionally designated

as

"fibroblast

LA-14-PP is able to block LPS-induccd monokine production by human monocytes in a specific manner In order to achieve a better understanding concerning the molecular basis o f

proliferation factor" is characterized by a relative therm ostability (56°C, 1 hour).

this inhibition, wc have investigated whether LA-14-PP and other synthetic lipid A partial

Now we investigated the effect o f supernatants on human pneumocytes type II cells (line A-

structures (L A -I5 -P P , LA-22-PP. I.A-23-PP. LA-24-PP, and PE-4) arc able to influence the

549). After addition of the supernatants initia lly spindle shaped pneumocytes were detected,

binding o f LPS to monocytcsrinacrophages. When comparing the inhibition o f binding o f

follow ed by epithelial-like cells when cell proliferation progressed Induction of spindle-shaped pneumocytos typo II cells could also be seen after addition of

,25I-LPS to the inhibition o f biological activity wc found that both.

LA-14-PI* as well as

PF.-4, arc the best inhibitors o f l-PS-binding to human monocytes as well as preventors o f monokine production tested. Taken together, our results provide strong evidence fo r the

observed

concept that inhibition o f

LPS -cdl interaction by LA-14-PP and other lipid A partial

structures is based on a competitive inhibition o f a specific LPS-binding protein o f the

Results indicate that supernatants contain at loast two mediators: the tumor necrosis factor

responder ce ll.

alpha and a proliferation factor.

endotoxin-induccd reactions o f an infected host.

34

In this way these compounds may be able to prevent the fatal

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pure tum or necrosis factor alpha (TNFa) Howover, in this case no cell proliferation was

SOLUBLE CYTOKINE RECEPTORS AND THE TREATMENT OF INFLAMMATION. David L Urdai

A C T IO N O F P IC H IL A N O N N IT R IT E P R O D U C T IO N B Y A I.O N O -T E R M C U L T U R E D R E S ID E N T M A C R O P H A G E L IN E

Imm unex Corporation, 51 University Street, Seattle. W ashington

98101 Cytokines are proteins responsible for the regulation of the hem atopoietic

N. V allot ' , l. F. B oudard C . L’abancr 9 0 *) to MMC

supernatant mediated cytostasis could be fully reversed (colorimetric MTT-cytoioxidty assay). Ught and electron

whereas TNF-a resistant HL60 cells (promyelocytes) showed a low susceptibility (10-50%). After differentiation

microscopic examination showed the characteristic features of apoptosis of U937 cells after incubation with either

induction of HL60 cells into a monocytic direction with 1.25(OH),D, these cells became intermediately sensitive

monocytes or rhTNF-a. No signs of necrosis could be observed. The rhTNF-a induced apoptosis (10* U/mL) as

(50-90%), although these cells remain TNF-a resistant. In contrast KG I cells (myelohlastic. TNF-a resistant)

measured by flow cytometry (hypotonic proptdium iodide solution) paralleled the functional decrease in cellular

were fully resistant to MMC and at E/T > 1were induced to a higher proliferation rate. Leukemic blast cells from

viability (MTT-assay) of 20* ± 3 after 24 hours up to a maximum of SO* 2 4 after 48 hours. Actinomydnc-D

patients with AML showed a similar differential susceptibility to MMC depending on their myeloid or monocytoid

(0.025-2.5 nM) and Cydoheximide (0.2-20 nM) showed a dose dependent synergistic actvity with TNF-a. whereas

origin according to the FAB-classification of A M L M l celb (myeloblasts) were induced to proliferation. M2/M4

EDTA (0.1-1 mM) did not show any inhibitory activity of TNF-a induced apoptosis. In contrast, quantification

cells (mycloid/myclo-raonocytic) showed no or a low susceptibility to non-activatcd MMC whereas M5 cells (mono-

of apoptosis by flow cytometry of monocyte mediated cytotoxicity was lower as compared to ihc percentage of

cyllc) were intermediately to htghly susceptible. After simultaneous incubation with IFN- Ylcukcmic celb showed

monocyte mediated cytotoxicity as measured functionally in ihc MTT-assay. Moreover, morphologically monocytes

an increased susceptibility. These differences tn susceptibility to MMC paralleled differences in responses to TNF-

showed a highly phagocytosing activity with apoptotic bodies in phagolysosomes which may have led to underes­

a, IFN-Y and IL-3/GM-CSF (CSFs). MI-M4 celb were promoted to proliferate in the presence of CSFs whereas

timation of flow cytometric quantification of apoptosis. In conclusion, these data provide evidence that apoptosis

M5 celb did not show any response to CSFs. Furthermore. TNF-a served as growth (actor for MI celb and some

is the major mode of TNF-a dependent monocyte mediated leukemic cell death. Furthermore, we adapted a

M2 samples. In the presence of IFN-Y . TNF-a showed a highly cytolyiic effect on M4/M5 celb. In conclusion,

method to quantify cytokine and cellular induced apoptosis of leukemic cells by flowcytomciry.

these data provide evidence that the differential susceptibility of leukemic celb to human monocytes may be related to their lineage specific origin. These differences paralleled a differential response to TNF-a, IFN-Y and CSFs. These data may have implications to select patients for adoptive immunotherapy with cytotoxic monocytes in AM L

ROLE O F M AC R O P H A G E S IN THE R E SISTA N C E A G AIN S T TR YPA N O SO M E S

H IV -IN D U C ED S YN C Y TIA FO RM A TIO N IN H U M A N M O NO C Y TE S/M AC R O P H A G E S: A M IC R O C IN EM ATO G R A PH IC STUDY

P. Vincendeau. S. D a ulouôde, M Letocard. V. N eaud. Lab ora toire de P a rasitologie. Lab ora toire de s in teraction s cellulaires

H. v o n B rie sen 1. U. S an d er2. K .-H . S ea ck2. M . K re u tz3. K. B e cke r1. S. M ü lle r1, H. R ü b sa m e n -W a ig m a n n 1. R. A n d re o se n 3

U n iversité de B ordeaux II. Bat. 3A. 3èm e étage, 33076 B o rde aux Cedex. France. 1) 2)

M a cro p h a g e s p lay a key role in the c o n tro l o f p ro tozoan parasites. In this study, th e ro le s of m acro phages in the re s is ta n c e aga in s t Trypanosom a

3)

G eorg S peyer-H aus. P au l-E h rlich -S tr. 4 2 -4 4 . 6 0 0 0 F ra n k fu rt/M . (G erm any) In s titu t fü r den W is s e n s c h a ftlic h e n Film . N o nn o nstie g 7 2. 3 4 0 0 G ö ttin g e n (G erm any) M e d izin ische K lin ik I, H ä m a to lo g ie /O n ko lo g ie , F ra n z-Jo sef-S tra uß -A llee 1 1 , 8 4 0 0 R egensburg (G erm any!

m u scu li. a n a tu ra l p a ra s ite o f th e m ouse a n d T b ru c e i gam biense, th e c a u s a tiv e a g e n t o f h u m a n sle e p in g s ic k n e s s , w e re in v e s tig a te d . As th e im portance o f the L -arginine : NO m etabolic pa th w a y has bee n describ ed in th e a n tim ic ro b ia l fu n c tio n of a c tiv a te d m a c ro p h a g e s , th e p a rtic ip a tio n of reactive nitrogen oxides w as also investigated . M u rin e p e rito n e a l m a c ro p h a g e s fro m th e 10lh d a y of T m u s c u li in fe c tio n w e re a c tiv a te d (H 2 O 2 p ro d u c tio n , la e x p re s s io n , e n d o c y to s is ). p ro d u c e d n itrite and. u n like re s id e n t m a c ro p h a g e s , did not fa v o r in vitro parasite m ultiplication This cytostatic effect w a s supp resse d by addition of NG m onom ethyl-L-arg inine (N M M A ). an inhibitor o f NO synthase. In vitro resident peritone al m acrophages activated w ith iFN-y + LPS exerted a cytostatic effect o n T. m usculi w hich w a s blocked b y NMMA. T reatm e nt w ith authentic NO gas inhibited th e proliferation o f T. m usculi C o m pa red to th e parasite m ultiplicatio n o b se rved in vitro in presence of K upffer ce lls from norm al m ice, a trypa nosta tic effect w as also observed In c o ­ cu ltu re s o f tryp a n o so m e s w ith K upffer cells fro m T. m u s c u li- or BC G -infected mice. H u m an b lo o d m o n o c y te s w e re c u ltu re d in v itro fo r 7 d a y s b e fo re a d d itio n o f 7. b ru c e i gam biense

A try p a n o s ta tic effect w a s observed w hen

To in v e s tig a te th e co nse qu e nce s o f H IV in fe c tio n in p rim a ry hum an m o n o cyte s/m a cro p h a g e s (M O /M A C ) w e have esta b lish ed a c u ltu re sy s te m w h e re M O /M A C w o re g ro w n on h y d ro p h o b ic T e flo n m em branes. A fte r in v itro in fe c tio n w ith th e m o n o c y to tro p ic HIV -1 iso la te D 1 1 7 III the v iru s re p lica te s to h igh titre in M O /M A C . T h e m a xim u m v iru s p ro d u c tio n is reached b e tw e e n d a y 2 0 and 3 0 and re m a in s a t a h igh lo vel o v e r se veral w e e ks. No ce ll d e a th is o b se ve rve d b u t the in d u c tio n o f m u ltin u clo a te d g ia n t ce lls. S y n c y tia c o n ta in in g 5 0 to 1 0 0 n u cle i w ith a d ia m e to r o f 2 0 0 to 4 0 0 / / m are fo u n d in H IV -in fe c te d M O /M A C cu ltu re s. T h e aim o f th is s tu d y w a s to fo llo w th e p ro cess o f g ia n t ce ll fo rm a tio n b y m icro c in o m a to g ra p h ic m e th o d s. T h erefo re . c u ltu re s w e re m o n ito re d m ic ro s c o p ic a lly b y tim e lapse c in e m a to g ra p h y w ith 3 5 m m film s. A speed o f s h o o tin g o f 1 fram e per secon d to 3 0 fra m e s per hou r resu lted in vis u a liz a tio n o f c e ll m o ve m e n t at an 2 5 fold to 3 ,0 0 0 fo ld a ccele ra ted speed. The e v a lu a tio n o f th e processe d film revealed three m o rp h o lo g ic a lly and k in e tic a lly d is tin c t M O /M A C p o p u la tio n s : large ro u nd -sh ap e d, sessil ty p e s w ith o n ly s lo w m o tio n (typ e A ), large m o v in g ty p e s w ith va ria ble sh ap e w h ic h c o v e r lo ng d ista n ce s (typ o B). and sm all a g ile ty p e s w h ic h are v e ry a c tiv e ly and ra p id e ly m o vin g th ro u g h o u t th e c u ltu ro s (typ e C). In n o n -in fe cte d c u ltu re s w e v e ry ra re ly fo u n d sp o n ta n e o u s fu s io n p ro cesse s b e tw o o n M O /M A C , and in som e cases w e co uld d e m o n s tra te e n d o m ito s is . Hence, o n ly fe w ce lls ( < 1 5 % ) w ith m ore tha n o ne n ucle u s w e re fo u n d . In H IV -Infe cted M O /M A C c u ltu re s n u m ero u s ce ll fu s io n s w e re d e m o n stra te d b e tw e e n th e sessil M A C ty p e (A ) and the agile M A C ty p e (C). F re q u e n tly, th is p ro ce ss to o k place p o la rly a t o n e site o f the ty p e A M A C . D u rin g a la ter phase fu s io n s also b e tw e e n t w o e x is tin g m u ltin u c le a te d s y n c y tia w e re seen. The e n tire p ro ce ss fro m sin g le -n u cle u s s ta g e to a m u ltin u clo a te d g ia n t ce ll c o u ld be p e rce ive d .

m onocytes w e re activated by supernatants o f lym pho cyte cultures.

activate m onocytes in vitro are in progress t o cla rify the m echanism of hum an m acrophage trypa nosta tic activity.

36

T h is film d o cu m e n ts In v itro th e c y to p a th o lo g ic a l fo rm a tio n o f H IV -in d u ce d g ia n t M O /M A C w h ic h can be fo u n d in v iv o in m icro g lia l n od ules o f A ID S p a tie n ts ' b ra in . The Georg-Speyer-Haus is supported by the Bundesministerium für Gesundheit and the Hessische Ministerium für Wissenschaft und Kunst. Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/18/2019 1:34:47 PM

U se o f n e u tra liz in g a n tib o d ie s d ire c te d to c y to k in e s to in hibit the p ro p e rtie s of lym pho cyte supe rna tants and u s e o f recom bina nt c ytokine s to

A CTIVA TIO N OF IKON RESPONSIVE ELEMENT B IN D IN G PROTEIN (IR E -B P ) BV 7 ,8 -D ll IYDRONEOPTERIN IN H UM AN MONOCYTIC CELLS Weiss G. Doppler W, H a n tM M W *, W em er-Felm ayer G, Fuchs D , W achter II In s titu te fo r M edical C hem istry and B iochem istry, U n ive rsity o f Innshm ck, A-6020 Innsbruck. A ustria. • ) European M ole cular Biology L aboratory, 0-6 90 0 Heidelberg, Germany. In tra c e llu la r Iron m u lu lxjllsni It» regulated at the le vel o f uiRNA s ta b ility and tra n sla tio n o f iron storage m id trnn*|»ort proteins mKNA by the in te ra c tio n o f a sp e cific tra n s -a c tin g cytop la sm n tlc p ro te in . Iron responsive elem ent binding p ro te in (IR E -B P ), w ith n c is -a c tin g RNA m o tif, ca lle d IRE, Id e n tifie d on 5 ’ f e r r it in mKNA and 3’ tra n s fe rrin re ce pto r mKNA. U>w In tra c e llu la r co nce ntratio ns o f low m olecular w eight Iron cause n c liv a tlo n and binding o f IRE-UP to th e IRE w hich results in reduced fe r r it in tra n sla tio n and Increased tra n s fe rrin -re c e p to r mRNA s ta b ility and tra n s la tio n e t vic e versa. We in vestigated the in flue n ce o l neopterin and 7 ,8 -dlhydroncopterin on 1RE/IRK-BP in te ra ctio n s In th is human m onocytic c e il line w ith c h a ra c te ris tic s o f macrophages (T H P -H . C ells w e re cu lte re d In RPMI co ntaining 10 % FCS, supplemented w ith medium. Iron (lOOyeM fin a l co n ce n tra tio n ), d efe rrio xa m in e (2 0 0 > jM ), n eopterin. 7 ,8 -d lh ydron co ptcrin (SOyuM each). 2,6-din m ln o -hyd ro xy-p yrlm id ine (5 mM) o r interferon-gam m a (500 U /m l.) f o r 2*1 hours and c e ll e x tra c ts were prepared by using the d etergent T rito n X-100. In p a ra lle l, a ^P -la b e l led o lig on u cleo tid e, id e n tic a lly w ith th e IRE, was synthesized in v it r o according to tire method o f M illig an e t a l. (NAR, 1S:8783;ID87). C o ll e x tra c ts w e re incubated w ith the -lab elled IR Ii and binding o f th e IRB-BP t o th e IRE was analyzed by m o b ility s h ift assays. As previously shown, e x tra c ts from c e lls p re tre n te d w ith Iron showed low IRE-BP binding a c tiv ity to th e IRE, w h ile a d m in istra tio n o f the Iron c h e la to r d esferrioxam ine resulted in Increase o f IR E /JR E-BP In te ra c tio n . IRE-BP a c tiv ity was also Increased a fte r ndm nustation o f 7.8*d!hydroncoptenn, w h ile It was not Influenced by neopterin. Concom itant a d m inistratio n o f iron and 7 ,8 -d ih ydron co ptcrin also Increased IR E /IR E -B P in te ra c tio n s , thus reversing th e in a c tiv a tin g e ffe c t o f iron on IRE-BP. S tim u la tio n o f ce lls w ith IFN-gommn resulted In fo rm a tion o f

T N F -a-IN D U C E D MHC CLASS II ANTIGEN EXPR ESSIO N IS INHIBITED BY IFN-yON THE MONOCYTIC U937-SUBLINE C119/9. Martin Willheim. Alois Gessl. Andreas Spittler, Zsolt Sz6pfalusi, Hermine Agis. Othmar Forster, and Georg Boltz-Nitulescu Department of General and Experimental Pathology. Neubau AKH. Währinger Gürtel 18-20. 1090 Wien. Austria IFN-y has been shown to increase the expression of class II antigens in a variety of cell types We report the establishing of a subclone of the monocytic cell line U937. termed C119/9. In contrast to U937 cells, on C119/9 only TNF-a can induce HLA-DR and partially -DP molecules The strong effects of TNF-« (% of HLA-DR-bearing cells increases from 5 to 90. mean fluorescence intensity from 6 to 266) can be inhibited almost completely with IFN-y. The effects of TNF-a and IFN-y seems to be restricted to HLA-DR and -DP expression, whereas HLADQ is induced neither by TNF-a nor by IFN-y Preincubation of the cells with iFN-y does not enhance its inhibitory effect, pretreatment with TNF-o up to 24 h shows that addition of IFN-y blocks the TNF-a-triggered HLA-DR expression. Other surface antigens, like MHC class I molecules. FcyRI and FctRII are upregulated by TNF-a and IFN-y. Interestingly, various other cytokines which have been reported to be involved in MHC dass II regulation, such as IL-4. GMCSF. TGF-ß and IFN-a are also able to suppress the TNF-induced MHC dass II expression on C119/9. Furthermore. IL-6. which shares some regulatory effects with IFN-y on monocytic cell lines, also inhibits the effects of TNF-a on MHC class II expression Downregulation of MHC class II molecules by IFN-,- has only been reported in some experiments with B-cells. where it inhibits the action of IL-4 C119/9 cells dearly show monocytic features, e g they can be induced to differentiate upon 1.25-dihydroxyvitamin D3-treatment (induction of CD14 ability to reduce NBT). This aberrant form of U937 cells may be useful to study the regulatory mechanisms of cytokine-induced MHC class II antigen expression.

e xtrem ely high co n ce n tra tio n s o f 7,8-dihydroneoptertn and In a dd itio n a c tiv a tio n o f IRE-BP could be observed. When c e lls were stim u la ted w ith IFN-gamma In th e p rw o n ce of 2.U-dlunilno-hydrr>xy-pyrlmldin©. on in h ib ito r o f 7 ,8 -dlhydronooptcrln synthesis, p te rid ln o fo rm n tlon as w e ll ns IR E /IR E -B P In te ra ctio n were d ra s tic a lly reduced. Wc conclude tha t dihydro n eo pte rin may a c tiv a te IRE-BP by red ucin g cysteine residues, w hich was shown to be o f m ajor Importance fo r Increasing IRE-BP a ffin ity .

Zawatzkv. R. and Nickolaus, P. Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum Heidelberg, and Falk, W. Innere Medizin I, Klinikum, Universität Regensburg IL-4 was initially described as a TH cell factor based on its ability to enhance proliferation in stimulated B lymphocytes. Lateron, it was recognized that IL-4 had important effects on other non-lymphoid cells involved in the regulation of the immune response. In cultures of mouse bone marrow cells, IL-4 was reported to inhibit growth stimulated by IL-3 but to enhance colony formation in response to M-CSF. Moreover, similar to IFN-1T, IL-4 is able to induce tumoricidal activity and antigen-presenting capacity in mouse macrophages. Interestingly, however, IL-4 can also block the secretion as well as some biological effects of IFN-J^ in human cells and recently, an inhibitory effect on the development of the IFNinduced antiviral state in mouse L-929 cells was also attributed to IL-4. In cultures of mouse bone marrow derived macrophage# (BMM), we have therefore studied possible antagonistic effects between IFN-a/ß and IL-4. Culture of bone marrow cells in the presence of M-CSF plus IL-4 completely inhibited the biological effects of the endogenous IFN-ß constitutively secreted by these cells. Macrophages became highly permissive to virus infection and induction of the IFN-responsive enzyme 2*-5' oligoadenylat-synthotase (OASE) was inhibited on RNA and protein level. IL-4 treatment, however, proved ineffective in inhibiting the biological effects of exogenously added IFN-a. These results prompted us to investigate the effect of IL-4 on the expression of endogenous IFN-ß in BMM. Therefore, a PCRbased semiquantitative analysis of IFN-ß mRNA was developed. Using an IFN-ß reference cDNA harboring a deletion within the sequence to be amplified, we detected 5-10 IFN-ß but no IFN-a transcripts per lOOng of total RNA from M-CSF cultured BMM. IL-4 treatment, however, inhibited the appearance of IFN-ß transcripts in BMM. This observation points out to a specific physiological effect of IL-4 on the expression of endogenous IFN-ß in macrophages since virus-induced IFN gene expression was not affected in IL-4 treated BMM.

GANGLIOSIDES SUPPRESS TNP PRODUCTION IN HUMAN MONOCYTES H W.L

Z le g lo r-H c ltb ro c k *.

‘ In s titu te

fo r

E. K ftffe rle ln *.

Immunology.

‘ I n s titu t

M.

f f lr

Ströbel*.

Prophylaxe

C. der

Weber*.

D.

Flieger*.

K re islauferkrankungen.

U n ive rsity o f Munich. 8000 München, Germany Both

normal

and

m alignant

cells

contain

gangllosldes

as

Im portant

cell

membrane c o n stitu e n ts, which, a fte r being shed, may Influence cells o f the Immune system. We have stud ie d the Im pact o f gangllosldes on the expression o f TNP In blood monocytes and In the m onocytic ce ll lin e Mono Mac 6. While under standard cu lture cond itio ns, bovine brain gangllosldes (100 ug/ml) suppressed LPS stim ulated TNF production fiv e fo ld In p e riph e ra l blood mononuclear ce lls (PBMC) and ten fold In Mono Mac 6 ce lls, suppression was more e ffic ie n t under serumfree conditions. P urified gangllosldes GD3. GDla. GM3. GM2, and GM1 (10 ug/ml) were a ll e ffe ctive In reducing

TNF production

In PBMC and

In

Mono Mac 6 by fa cto r

10 to 60.

Gangllosldes appear to a ct a t an e a rly step o f a c tiv a tio n In th a t TNF tra n scrip ts were reduced

and

th e

m o b iliza tio n

of

the

nuclear

fa cto r

NFiB

was

blocked.

Furthermore. In time kin e tic s , gangllosldes were e ffe c tiv e fo r up to 30 min a fte r addition o f LPS. Gangllosldes also blocked TN F-prod uctlo n Induced by staphylococci and by Paf. b ut p h o rb oleste r-ln d uce d O r

production was n ot affected. The data

Indicate th a t gangllosldes can s p e c ific a lly p re ve n t a c tiv a tio n o f the TNP gene at an early step o f sig n a llin g .

37

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ANTAGONISTIC EFFECTS BETWEEN ENDOGENOUS INTERFER0N-ß(IFN-ß) AND INTERLEUKIN 4 (IL-4) IN MOUSE MACROPHAGES

REGULATION OF CD14 IN THE MONO MAC 6 CELL LINE: H.W.L. Zieoler-

THE NOVBL SUBSET OF CD14*/CD16*

Hoitbrock» .

OF TISSUE MACROPHAGES: H.W.L.

w.

Schrauc* .P.

Sternsdorf* . M. Ehlers*,

C.

Immunology.

of

University

University Greifswald.

Wendelgaß« . Schütt*.

J.G.

Munich,

Germany.

*Dep.

M. Haas*.

*Dep. Mol.

Ströbel* ,

T.

‘Institute for

Med. Biol.,

Immunology, Massachusetts

General Hospital. Boston. M A . USA.

Stróbel. Pforte.

W.

Schraut.

Institute

for

F.

BLOOD MONOCYTES EXHIBITS FEATURES Zieoler-HeitbrocK. G. Fingerle. M.

Stelter.

Immunology.

Medicine. University of Munich.

C.

Schütt.

Dept.

Dep. Med.

B.

Surgery.

Passlick. Dep.

A.

Internal

Immunology. University

Greifswald.

The human monocytic cell line Mono Mac 6 expresses substantial

The CD14*/CD16*

cells

in human peripheral

blood

account for

amounts of CD14 as assessed by staining with the My4 antibody (86%

about 10 % of all monocytes in apparently healthy donors. Those

positive

cells, when comparod to the regular (CD14* •/CD16* ) monocytes, show

further

cells).

Treatment

maturation

as

with

evidenced

LPS, by

PGEj

or TPA

enhanced

will

induce

phagocytosis

and

a low expression of the CD14 antigen and a high expresión of the

expression of mCSF-receptor mRNA. LPS and PGE* will enhance cell

CD16 antigen.

surface staining

with

regulated as evident from concomitant changes in transcript levels

density and with

a maximum

a more

than

effect at

twofoldincrease day 2.

in antigen

Furthermore,

both

These differenences appear to be transcriptionally

as assessed by PCR. In 3-color-immunofluorescence, the CD14*/CD16*

reagents will increase soluble CD14 (sCD14) in the supernatant of

cells exhibit twofold higher levels for class II. while CDllb and

Mono

CD33 antigens

Mac

6

by

transcriptionally the constitutive

factor

8-12.

controlled, level

This

since LPS

of CD14

mRNA 6

effect and

appears

to

be

PGEi , both enhanced

to 9-fold.

By contrast,

are

twofold

decreased.

Alveolar

appear to be similar to the CD14*/CD16*

macrophages

cells,

(AM)

since they also

express higher levels of class II and lower levels of CDllb and

treatment with TPA reduces cell surface staining of CD14, it does

CD33 when compared to the regular monocytes.

not induce sCD14 and there is a transient downregulation of CD14

derived macrophages, obtained by 5 d culture of mononucloar cells

Finally,

monocyte-

mRNA. These data show CD14 expression in Mono Mac 6 cells can be

in 10 % human serum, will develop low CD14 and high CD16 and they

differentially affected by different inducers of maturation.

also upregulate class

II and downregulate CDllb and CD33. Taken

together, our data demonstrate features of mature macrophages in

38

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the novel subset of CD14*/CD16* blood monocytes.