Biocldmica et Biophysica Acta, 1135(1992)343-348
343
© 1992ElsevierScience PublishersB.V. All rightsreserved016%4889/92/$05.00
BBAMCR 13187
Regulation of prostaglandin E 2 binding to a murine macrophage
cell line, P388D. by insulin M a r k G . R a e ", D i n o R o t o n d o b a n d A s i m K. D u t t a - R o y b Dicision of Plmrmacofo~o U n i t ' ~ . of Aberdeen, Aberdeen (UK) and b Dicision of Biochemical Scte~ces, Rowett Research Institute, Aberdeen (UK)
(Received 17 December 1991)
Keywords:. Pro~glandin E2 receptor,Insulin;P388DI cell line; cyclicAMP; cyclicAMP phosphodiesterase Preincubation of murine macrophage-like P388DI cells with physiological amounts of insulin resulted in an increase in prostaglandin E 2 binding m these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE 2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0_~0+__0.02 to 0.63 _+0.03 fmo!/106 cells for the high affinity receptor binding sites, and from 2A + 0.31 to 5.0 + 0.41 fmol/106 cells for the low affinity receptor binding sites) rather than m an increase in the affinity uf the binding sites. The insulin-stimulation of PGE 2 binding appeared m be assuciated with a lowering of the cAMP level in these cells, treatment of cells with insulin lowered the cAMP level by increasing the cAMP phesphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressiveaction of this prostanoid, as PGE 2 is known to suppress many steps in the immune response, including interleukin-I expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiologicalconcentrations may enhance the immunnsuppressiveaction of PGE2.
lnlreduction Monocytes/macrophages are essential regulatory cells in most immunological responses [1]. Interleukin 1 (IL-1), a polypeptide, produced by various cells, especially monocytes and macrophages during inflammation and infection, is involved in many acute-phase responses such as fever, hnmunoactivation and ~ucose homeostasis [2,3]. IL-I is directly involved in the activa* tion of helper T lymphocyte.s, and the stimulation of other cell types including monocytes/macrophages to produce secondary mediators, such as prostaglandin E 2 (PGE 0 [2-4]. IL-1 can produce insulin-dependent diabetes by reducing the insulin content of islet cells, as well as by inln'biting insulin secretion through prostaglandin synthesis [3-5]. Several studies have demonstrated that IL-1 is a potent modulator of pancreatic /3-cell function and can potentiate or inh~it glucose-induced insulin secretion, depending on the concentration and time of exposure to IL-1 [6-10]. One of the mechanisms by which monocytes/macrophages are thought to exert their regulatory function is through
~ n c e : A.K. Durra-Roy,Divisionof Biochemical Sciences, RoweRResearchInstitute,Aberdeen, AB2 9SB, Scotland, UIL
the production of PGE 2 [11,12]. PGE 2 inhibits various steps of immune responses, including IL-1 expression, possibly by raising intracellular cAMP level,~ through the activation of receptor-linked adenylate cyclase [1317]. The cAMP phosphodiesterase inhibitors are shown to suppress the production of IL-1, as well as TNFa, by immune cells [13,15,18]. This suppressive effect appears to be mediated by the accumulation of cAMP [13-16]. Inhibition of IL-1 production in monocytes by PGE 2 is thought to be an event fundamental to the initiation and regulation of immune responses. But very little information is available on the regulation of these activities of immune cells by circulating hormones, such as insulin and autacuids, that occur naturally in the circulation. Insulin has been shown to modulate the activity, of prostaglandin receptors in human platelets and erythrocytes [19-21]. The positive cooperation between the receptors for P G E I / P G I 2 and insulin in both human platelets and erythrocytes is important for the normal function of these cells [22]. Although there appears to be a relationship between IL-1 and insulin action and secretion [6-10], little information is available on the regulation of PGE 2 receptor activity of immune cells by insulin. In this paper we report that the incubation of murine macrophage cells, P388Di,
344 with insufin at physiological concentrations increases the binding of PGE 2 to its receptors and increases cyclic AM, q~ formation. The insulin-induced stimulation of PGE 2 binding may help to enhance the immunos u p p r ~ i v e effect of this p:~ostanoid. Materials and Methods
Materials P388D I cells were obtained from I C N / H o w Laboratories, UK. [5,6,8,11(n)-3H]Prostaglandin E z (Spee. act. 56-60 Ci/mmot) was obtained from Amersham, UK, human insulin (Actrapid) was from Novo Industries, Denmark. All other reagents used were of analytical gredes.
vat~ng concentrations of unlabelled PGE 2 (0-1 v M ) for 20 min at 4°C, both in the absence and presence of insulin (1.4 nM). rotal binding was determined from the calculated specific activity of the radioligand obtained by d~uting [3H]PGE2 with known concentrations of unlabelled prostag!andin. The dissociation constants ( K d) and the number of binding sites (n) were obtained from non-linear regression analysis of the equil~rium binding, using a computer programme (Enzfitter, Biosoft, Oxford).
Determination o f cyclic AMP content
Ce/ls P388D I cells were grewn as a continuous suspension culture in Dulbeccu's modified Eagle's medium with 10% horse serum at 37'C, 5% C O , , and 100% humidity in 175 cruZ/800 ml plastic culture flasks [23]. P388D I ceils were initially harvested into sterile vials and centrifuged at 300 × g for 5 min. The cells were then washed twice with 10 ml of RPMI 1040 and resnspended in RPMI 1640 to give a final concentration 2-107 cells/ml. Cell count was estimated using a haemocytometer and the viability of the cells was roufinely between 90-95%, as judged by the trypan blue exclusion method.
Estimation of the cAMP content in P388Dj cells (2-106/ml) was determined by preincubating cells with insulin (0-2 riM) in a total volume of 250 /~1. After incubation for 30 min 10/~M IBMX was added for 5 min before the addition of either RPMI 1640 (controls) or PGE 2 (1 /zM) and incubations continued for a further 20 rain. Reactions were terminated by the addition of 372/tl of ice-cold ethanol (65% final concentration). The tubes were then centrifuged at 17000 × g for 5 min. and the supernatants were transferred to tubes for storage for no more than 48 h before analysis of cAMP. The cAMP content of the supernatants was determined by evaporating aliquots of the ethanol and measuring the cAMP present using a cAMP radioimmunoassay kit (Amersham). Neither PGE 2 nor insulin affected the standard curve for cAMP.
[ 3H]PGEz binding assay
Cyclic AMP pho~hodiesterase assay
Radiolabelled PGE 2 binding to P388D I cells was performed as described previously [23] with slight modifications [24]. Washed cells (2 × 106) were incubated with 3 nM p H ] P G E 2 at 4°C for 20 min in a total volume of 0.2 ml. After incubation, cells were vacuum filtered through G F / C glass fibre (Whatman) filters, presoaked with phosphate buffered-saline, (pH 7.8). The filters were then washed with 10 ml of ice-cold phosphate-buffered saline, then dried and radioactivity was measured by seintillatinn counting. Nouspecifie binding was determined by adding excess unlabeUed PGE 2 ( l / t M ) to the above mixture and specific binding was calculated by subtracting the nonspecific binding from the total binding. The effect of insulin on PGE 2 binding was estimated by preincubating the cells (2 × 106) with insulin (0-10 riM) for different times at room temperature (22°C) before the addition of radiolabelled PGE 2.
Cyclic A M P phosphodiesterase activity was measured as described previously [26]. To determine the effect of insulin on cAMP phosphodiesterase activity in P388D I cells, the cells were incubated with insulin at various concentrati3ns (0-5 nM). The cell membranes and cytosolic fractiow were prepared after washing the cells. Cells were boraogenised in 50 mM Tris-HC! (pH 7.4) and centrifuged at 1 7 0 0 0 × g for 15 min at 4°C. The supernatant was retained and recentrifuged at 1 7 0 0 0 x g for 15 min at 4°C and the resulting supernatant was used as the soluble fraction. The pellet was rasuspended in the original volume of buffer and recentrifuged at 1 7 0 0 0 × g for 15 rain at 4°C. The resultant supernatant was discarded and the pellet was resnspended in 0.25 × the original volume of homogenisation buffer and this was used as the particul a t e / m e m b r a n e fraction. The membrane (120 Itg) and soluble fractious (89/~g) were separately incubated in 20 m M Tris-HC! buffer (pH 7.4) in a total volume of 200/zl for 20 rain at 22°C. [~251]cAMP ( 0 . 2 / t M ) was added to the reaction mixture to determine the phosphodiesterase activity of the membrane and soluble fractious of insulin-treated and untreated cells. The mixture was incubated for 3 min at 2 2 ~ and the reaction was terminated by adding 0.2 ml of 0.2 M
Analysis o f equilibfimn binding of [3H]PGEz to P388DI cells in the presence or absence of insulin The effect of preincnhation of P388Dj cells with insulin on the interaction of PGE 2 with these cells was analysed by the method of Scatcbard [25]. Typically, 2-106 cells were incubated with 3 nM [3H]PGE2 and
345
20G
1.5
to0
1.0
o
!
t
t
t
!
. ._..a
0
1
2
3
4
5
.~Sudn conc (riM)
Fig. I. Effect of preinc,_,_h~_~.innwith increasing concentrations of insulin on [3HIPG[~2 binding to P388DI ceils. Cells (2×106) were preincubated in the presence of insulin (0-5 nM) for 30 rain at 22°C, followedby a funficr incnbarion with 3 nM [3H]PGE2 for 20 min at 4°C. The binding of pH]PGE z to the cells was determined, as dt.~arihed in l',hteriafs and Methods. Values indicate total binding (0). specific binding (0) and non-specific binding (e), of% specific binding in the absence of insulin. Results shown here are the mean +_S.D. of three experiments, each in triplicate.
Z n S O 4 followed by 0.2 ml o f 0.2 M Ba(OH) 2 at 30 s intervals. After vigorous stirring for 1 min, the precipitate was separated by centrifugation at 8000 × g for 4 min. The a m o u n t o f cyclic A M P hydrolysed was calculated from the rate of decrease in radioactivity in the supematant. Results Incubation of P388D t cells with [3H]PGE 2 resulted in a rapid and r e v e r s ~ l e binding of the P G E 2. F_.quil~rium of binding was attained within 20 rain of incubation at 4°C (data not shown). Nonspecific binding was estimated to be around 2 5 - 3 5 % of total binding w h e n 3 n M [3H]PGE2 was t : ~ l in the presence of 1 /zM unlabelled ligand. Addition o f increasing concentrations of insulin to the binding assay mixture, 20 min prior to the addition o f radiolabeiled P G E 2 affected P G E 2 binding (Fig. 1). A t 1A n M concentration, insatin increased P G E 2 binding by approx. 2-fold, compared to control, whereas the presence of higher concentrations (above 1.5 n M or m o r e ) of insulin diminished the stimulatory effects on prostaglandin binding. The stimulatory effect of insulin on P G E 2 binding to P388D t cells could b e observed within 5 min incubation with insulin (data not shown). Substitution of insulin by adrenecorticotropin ( A C F H , 4300 Da), a protein o f similar molecular weight, at various concentrations did not effect the binding of [3H]PGE2 (0.33 -4-_ 0.02 f n m l / 1 0 6 cells) w h e n compared to controls (0.35 + 0.04 finol/106 cells). Scatchard analysis o f [3H]PGE2 binding to these cells in the absence and presence of insulin ( I A nM) showed the presence of one high-affinity-low capacity and the o t h e r low-affinity-high capacity receptor population (Fig. 2). The high affinity receptors showed a
O.OL!
!
!
!
t
!
|
o
5
lO
15
20
25
30
Fig. 2. Scatchard plot of [3H]PGE2 binding to P388Dt cel:s in the absence and presence of insulin (1.4 nM). Cells (2x106) were preincubated in the absence (o) and presence (e) of inulin (1.4,1M) for 30 rain at 22°C, followed by a further incubation with 3 nM [3HlPGE2 plus 0-1 gM unlabeficd PGE2 fox 20 rain at 4'~. Total binding (fmol/106 cells) was determined fox each point by dividing the dpm by the calculated specific zctiviw (dpm/fmol) obtained by diluting 3 nM [3HIPGE2 with known concentrations of unlabelled PGE 2. The binding of PGE 2 to the cells was determined as described in Materials and Methods. Each point represents the mean of three experiments, each with triplicate determinations. dissociation constant ( K d ) of 2.72 + 0.14 n M with a binding capacity (n t) oft0.30 + 0.02 f m o l / 1 0 6 cells. The low affinity receptors had a dissociatien constant (Ko2) of 40 + 9.1 n M and a binding capacity (n 2) o f 2.4 + 0.31 f m o l / 1 0 6 cells. In the case of insulin-treated cells, both the high (n t) and low (n 2) binding capacities for P G E 2 increased to 0 . 6 3 + 0 . 0 3 and 5 . 0 + 0 , 4 1 f m o l / 1 0 6 cells, respectively ( P < 0 . 0 0 1 , Student's ttest). However, the dissociation constants of the receptors on insulin-treated cells remained essentially unaltered when compared with the control values (Table I). According to Klotz and Hunston [27], total binding ( R ) cart be expressed as R=
niKt[PGE2]
n2K2[PGE2]
1+ K,[PGE,-----~ + 1 +Kz[PGEz] I II
where R is total P G E 2 binding, n t is the capacity of the high-affinity binding sites, K t the association con. TABLE I Summary of Scatchard analysis of [ 3H/PGE2 binding to P388DI cells both in the absence and presence of insulin (1.4 riM) (mcan +_S.D., n~3)
a gd * the dissociation constants (nM) and n, the number of binding sites (fmol/106 cells). Treatment Control Insulin
High affinity binding sites
Low affinitybinding sites
Kd I a
~d 2 a
nt
n2
2.72+_0.14 0",,30+_0,02 " ~ 9 . 1 "~±0.31 2.65+0.11 0.63+0.03* 48+_2.3 5.0±0.41 *
• P < 0.001, relative to control (Student's t-test).
346 stant of the high-affinity sites, t/2 the capacity of the low-affinity sites, and K2, the association constant of the low-affinity sites. Components I and El of the equation show the "true' specific and non-specific contn'butions of the total b i n d i n g respectively. By substituting the values of nt, K t = 1/Kdt n 2, and K 2 = l/Kdz in the above equation, the specific and nonspecific binding of tritium-labelled P G E 2 to these cells in the absence and presence o f various concentrations of insulin was found to be within 15% of the values determined by experimental procedures. Cyclic A M P levels in iusulin-treated and untreated P388Dt cells were measured both in the absence and presence of P G E 2 (1 /zM). Fig. 3 shows the levels of c A M P in these cells u n d e r different experimental conditions. The incubation of cells with insulin alone at 1.4 or 2 n M for 30 rain reduced c A M P levels by 12.0% and 18.1%, respectively, compared to control, i n contrast, P G E 2 (1 p M ) stimulated c A M P synthesis by 22.9% in insulin-treated cells (1.4 nM) when compared with that of cells incubated with P G E 2 alone. The maximum production o f c A M P by P G E 2 (1 p M ) in the imulintreated cell was observed with ml insulin concentration of 1.4 riM. To test whether the insulin-induced decrease in c A M P levels in the cells was due to the activation of c A M P p h o s p h o d i e s t e r a s e , the c A M P p h o s p h o diesterase activity in P388Dt cells in the absence and presence of insulin ( 0 - 2 riM) was measured. Fig. 4 shows the effect of insulin on c A M P pbosphodiesterase activity. Insulin at 1.0 n M stimulated the degradation of c A M P from basal level, 40.5 +_. 1.32% to 44.1 _+ 1.03% in the soluble fraction ( P < 0.01, student t-test)
A
4t 1=
40 39
0
o
~
1 2 insert conc (#,t)
Fig. 4. Effect of incubation of P388Dt cells with insulin on phosphodie,erase activity. P388DI cells were homogenised in 50 mM Trls/HCi (pH 7.4) and soluble and particulate/membrane fractions were prepared. The soluble (89/zg (A)) and membrane fractions (120 pg (B)) were incubated with [1251]cAMP(0.2 pM) for 3 rain at 22~C and the amount of cyclic AMP h)'drolysed was calculated from the rate of decrease in radio~livity~n the supematanL Values represent the mean _+S.D. n =4.
and from 32.6 +_ 0.96% to 35.6 + 1.3% ( P < 0.01, Stud e n t ' s t-test) in the particulate fractious. Whereas, at a higher concentration o f insulin (2 riM) phusphodiesterase activity was not affected.
e
g
46
~40
Discussion
2 5 0 t-
0
1.4
2
0
1.4
2
iosuen conc (nM)
Fig. 3. Effect of incubation of P388D1 cells with insullin on the increase of cAMP levels in the absence and presence of PGE 2. P388DI cells were incubated with various concentrations of insidin (0-2 riM}for 30 rain at 2TC. IBMX (10 lgM) was then added for 10 rain and either RPMI 1640LA)or i/zM PGE2 (B) was added before incubations were continued for a further 20 min. Incubations were terminated and cAMP determined by radioimmano~say, as descrt'bed in Materials and Methods. Values represent the mean_+S.D. n=4.
The physiological concentration o f insulin varies between 0.03 n M and 1.5 n M [28]. T h e data obtained in this study show that insulin at physiological concentrations increased the binding of P G E 2 to murine macrophage-like cells, P388D t. This suggests that insulin may enhance the immunosuppressive action o f P G E 2. luldbifion o f IL-I production in m o n o c y t e s / macrophages by P G E 2 through raising c A M P levels via adenylate cyelase activation, is regarded as an event that is fundamental to the regulation of the i m m u n e response [13]. W e have shown recently that IL-I inhibits P G E 2 binding to P388D t cells by a cyclic A M P independent process a n d thereby IL-1 may minimise the immunusuppressive action of this prostanoid [29]. This may have. important impficatious in understanding
347 the complex interactions between PGE 2 and IL-I which can regulate most immune response. Insulin increased beth the low and high affinity binding sites of PGE 2 receptors in P388Dj cells without changing their affinities. The availability and the affinity of receptors which determine, at least in part, the physiological effects of hormones, are regulated by various factors, such as second messenger systems, G proteins and phosphorylation of the receptor. In many cell types regulation of a particular receptor system by a distinct second receptor system is often observed ~.30]. Our results show that insulin directly modifies the prostaglandin receptor number in P388D~ cells. This is not necessarily dependent on new protein synthesis as it seems very unlikely that the synthesis of new protein occurred in the 5-20 min incubation time with insulin at 4°C. The stimulatory effect of insulin on the increased binding of PGE 2 was possibly due to the increased availability of spare receptors in the cell membrane. Interaction between receptors for insulin and prostaglandins in human erythrocytes and platelets has been found to be bidirectional and does not involve new protein synthesis [22]. The exact mechanism(s) by which insulin increases the prostaglandin receptor number in blood cells is not yet known. PGE 2 has been shown to down-regulate its own receptor in P388D~ cells through cAMP formation, by activating re~eptor-linked adenylate cyclase [31,32]. Paradoxically, accumulation of cAMP allows these cells to escape the inhibitory effects of PGE 2 and other agonists, through beterologous desensitisation [17,31, 32]. However, it is unclear whether the lowering of cAMP levels by insulin in the cells stimulates PGE 2 binding. The decrease in cAMP levels in cells incubated with insulin (0.7 to 2.0 nM) most likely allows the cells to bind more PGE 2 than controls (Fig. 4). The lowering of cAMP levels may permit the cells to upregulate the PGE 2 receptor number. Insulin has also been shown to decrease intracellnlar cAMP levels in liver, adipose tissue, and other target organs [33,34]. The binding of PGE 2 to cells is reported to be eseeptial for the inhibition of IL-I production and activity [13,14], and the enhanced binding may facilitate the immunosuppressive action of this prostanoid. PGE 2 inhibits many steps in the immune response, including IL-1 production [13,16,35,36]. This inhibition mainly occurs through stimulation of cAMP synthesis by activation of adenylate cyclase [13-15]. The interaction of prostaglandin with its receptors on the cell membrane has been shown to be a prerequisite for the activation of adenylate cyclase [24]. The enhanced binding of PGE 2 to P388D t cells is accompanied by an increased cAMP synthesis (Fig. 3). The increased binding of PGE 2 appears to be insulin-specific, as a protein of similar molecular weight (ACTH) did not affect PGE 2 binding to the cells. In P388D i cells, insulin was
found to decrease the cyclic AMP level, presumably by activating cAMP phosphodieste~-ase in a concentration-dependent manner (Fig. 4). Degradation of cAMP was maximum when the cells were treated with 1.0 nM of insulin. Despite the lowering of cAMP levels in P388Dj cells by insulin, the incubation of insulintreated cells with PGE 2 increased cAMP levels by 25% when compared with untreated cells. This suggests that increased synthesis of cAMP in insulin-treated cells by PGE 2 was due to enhanced prostanoid binding. In human platelets insulin increases P G E t / P G I 2 binding without affecting cAMP levels [21]. The enhanced prostaglandin binding to platelets results in an increase in cAMP synthesis and thus amplifies the anti-aggregatory actions of the prostanoid [22]. Our data suggest that insulin may be capable of regulating immune cell functions via modulation of prostaglandin binding. As diabetes mellitus involves an absolute or relative insulin deficiency, then this disease may predispose to an inadequate immune response. Acknowledgement A preliminary account of this work has been published as an abstract in the Biochemical Society Transactions 19, 358S, 1991. References 1 Johnston,R.B. Jr. (1988)N. Eng.J. Med. 318, 747-749. 2 Dinarello,C.A. (1984) Rev. Infect. Dis. 6, 51-95. 3 Dinarello,C.A. (1988)FASEBJ. 2,108-115. 4 Bendtz~n,K., Mandrup-Poulson,T., Nerup,J., Neilsen,J.ELand Dioarello, C.A. (1986)Science 232, 1545-1547. 5 Shimizu,H., Uehara, Y., Shimomu,-a,Y., Negishi, M. and Fuktasu, A. (1990)Biochem.Biophys.Res.Comman.173,1280-1286. 6 McDaniel, M.L., Hughes,J.H., Wolf, B.A., Easom, R.A. and Turk, J.W. (1988) Diabetes37, 1311-1318. 7 Hamaguchi,K. and Leiter, E.H. (1990) Diabetes~9, 415-425. 8 Welsh, N., BendU'.en, K. and Sandier, S. (1991) Diabetes 40, 290-294. 9 Garcia-Welsh,A., Schneiderman,J.S. and Baly,D.L (1990)FEllS Len. 269,421--424. 10 Velich, M.R. (1990)Am. J. Physiol.258, Ri070-RI077. 1! Fisher, R.I. and Bostick-Burton, F. (1982) J. lmmunol. 129, 1770-1772. 12 Holdstocg,G., Chastenay, B.F. and Krawitt, E.L (1982)Gastroenterology82, 206-210. 13 Knudsen, P.J., Dinarello. C.A. and Strom, T.B. (1986) J. lmmtmol. 137, 3189-3191. 14 Renz, H., Schimdt, A., Nain, M. and Gemsa, D. (1988) J. lmmm~oL141 2388-2390. 15 Brandwein,S.R. (1986)J, Biol. Chem.261,8624-8626. 16 Monick, M, Glasier, J. and Hannighake, G.W.(1987)Am. Rex,. Resgir. Dis. 135,72-77. 17 Coffey,R.G., Alberts,V.A. and Wealdand, L.L.(1990)J. Leukocyte. Biol. 48, 557-564. 18 Endres. S., Fulle. H.-J.. Sinha, B., Stoll, D., Dinarello, C.A., Gerzer, R., and Weber, PC. (1991) Immunology7~ 56-60. 19 Duna-Roy.A.K. and Sinha, A.K.(1985)Biochim.Bioph~s.Acta. 812, 671-678.
348 20 Ray, T.K., Dutta-Roy., A.IL and Sinha, A.K. (1986) Biochim. Biophys. Acta. 856, 421-427. 21 Kahn, N.N. arid Slaha, A.K. (1990) J. BIO!.Chem. 265, 4976-4981. 22 Durra-Roy. A.I,'~ Kahn. N.N. and Sioha. A.K. (199!) Life .qci. 49. 1129-1139. 23 Femandez-Borlan. 17,. and Suzuki, T. (!985) J° lmmuanL 133. 2662-2667. 24 Dutta-Roy, A.K. and Sinha. A.K. (1987) J. Biol. Chem. 262, 12685-12691. 25 Scatchard G. (1949) Ann. N.Y. Acad. Sci. 51,660-672. 26 Kahn, N.N. and Sinha, A.K. (1989) Biochim. Biophys. Acta. 984. i13-118. 27 Klot~ F.M. and Hunston. D . L (1971) Bh)chemistry 10, 30653069. 28 Harrison's Priociplcs of Medicine, 9th edn. (1980) (Isselbacher, K.J, Adm"n.s, P~D, Braunwald, E., Petersdorf, R.G. and Wilson, J.D. eds.), P.A-4. pp. AI-A7. McGraw-Hill, New York.
29 Roe, MoG. Ro;ondn. D., Milton, A.S. and Dutta-R~', A J L (1992) Biochim. Bioffl~,~ Acta 1i38, 75-79. 30 Po~lte. G. and Crook¢. ST. (1986) (eds.) Mechanisms of Receptor Regulation. Plenum Pre~,s. NY. 31 Shamma. M . S . . Fe~andez-Bortan. R. and Suzuki, 1". (1988) P~ndios 36. 329-341. 32 Femand~_-Bortan. R. and Suzuki. T. (1984) J. ImmunoL 133. 2655-26~1. 3? Bulhcer, R.W. Baird. C.E. and Sutherland, E.W. (1969)J. Biol. Chem. ?.44, 1705-1708. 34 Jefferson. L S . F.xto~ i H . Butcher. R.W. $mherland. E.W. and Park. t~_IL (1968) J. Biol. Chem. 243.1031-1033. 35 Knudsen. PJ. and Strcra. T.B, (1985) Br. J. RheumatoL 24. 65-68. 36 Thomson, P.A.. Jeli~ek. D.F. and Lipskey. P.E. (!984) J. lmmurtoL 133. 2446-2453.
343
© 1992ElsevierScience PublishersB.V. All rightsreserved016%4889/92/$05.00
BBAMCR 13187
Regulation of prostaglandin E 2 binding to a murine macrophage
cell line, P388D. by insulin M a r k G . R a e ", D i n o R o t o n d o b a n d A s i m K. D u t t a - R o y b Dicision of Plmrmacofo~o U n i t ' ~ . of Aberdeen, Aberdeen (UK) and b Dicision of Biochemical Scte~ces, Rowett Research Institute, Aberdeen (UK)
(Received 17 December 1991)
Keywords:. Pro~glandin E2 receptor,Insulin;P388DI cell line; cyclicAMP; cyclicAMP phosphodiesterase Preincubation of murine macrophage-like P388DI cells with physiological amounts of insulin resulted in an increase in prostaglandin E 2 binding m these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE 2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0_~0+__0.02 to 0.63 _+0.03 fmo!/106 cells for the high affinity receptor binding sites, and from 2A + 0.31 to 5.0 + 0.41 fmol/106 cells for the low affinity receptor binding sites) rather than m an increase in the affinity uf the binding sites. The insulin-stimulation of PGE 2 binding appeared m be assuciated with a lowering of the cAMP level in these cells, treatment of cells with insulin lowered the cAMP level by increasing the cAMP phesphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressiveaction of this prostanoid, as PGE 2 is known to suppress many steps in the immune response, including interleukin-I expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiologicalconcentrations may enhance the immunnsuppressiveaction of PGE2.
lnlreduction Monocytes/macrophages are essential regulatory cells in most immunological responses [1]. Interleukin 1 (IL-1), a polypeptide, produced by various cells, especially monocytes and macrophages during inflammation and infection, is involved in many acute-phase responses such as fever, hnmunoactivation and ~ucose homeostasis [2,3]. IL-I is directly involved in the activa* tion of helper T lymphocyte.s, and the stimulation of other cell types including monocytes/macrophages to produce secondary mediators, such as prostaglandin E 2 (PGE 0 [2-4]. IL-1 can produce insulin-dependent diabetes by reducing the insulin content of islet cells, as well as by inln'biting insulin secretion through prostaglandin synthesis [3-5]. Several studies have demonstrated that IL-1 is a potent modulator of pancreatic /3-cell function and can potentiate or inh~it glucose-induced insulin secretion, depending on the concentration and time of exposure to IL-1 [6-10]. One of the mechanisms by which monocytes/macrophages are thought to exert their regulatory function is through
~ n c e : A.K. Durra-Roy,Divisionof Biochemical Sciences, RoweRResearchInstitute,Aberdeen, AB2 9SB, Scotland, UIL
the production of PGE 2 [11,12]. PGE 2 inhibits various steps of immune responses, including IL-1 expression, possibly by raising intracellular cAMP level,~ through the activation of receptor-linked adenylate cyclase [1317]. The cAMP phosphodiesterase inhibitors are shown to suppress the production of IL-1, as well as TNFa, by immune cells [13,15,18]. This suppressive effect appears to be mediated by the accumulation of cAMP [13-16]. Inhibition of IL-1 production in monocytes by PGE 2 is thought to be an event fundamental to the initiation and regulation of immune responses. But very little information is available on the regulation of these activities of immune cells by circulating hormones, such as insulin and autacuids, that occur naturally in the circulation. Insulin has been shown to modulate the activity, of prostaglandin receptors in human platelets and erythrocytes [19-21]. The positive cooperation between the receptors for P G E I / P G I 2 and insulin in both human platelets and erythrocytes is important for the normal function of these cells [22]. Although there appears to be a relationship between IL-1 and insulin action and secretion [6-10], little information is available on the regulation of PGE 2 receptor activity of immune cells by insulin. In this paper we report that the incubation of murine macrophage cells, P388Di,
344 with insufin at physiological concentrations increases the binding of PGE 2 to its receptors and increases cyclic AM, q~ formation. The insulin-induced stimulation of PGE 2 binding may help to enhance the immunos u p p r ~ i v e effect of this p:~ostanoid. Materials and Methods
Materials P388D I cells were obtained from I C N / H o w Laboratories, UK. [5,6,8,11(n)-3H]Prostaglandin E z (Spee. act. 56-60 Ci/mmot) was obtained from Amersham, UK, human insulin (Actrapid) was from Novo Industries, Denmark. All other reagents used were of analytical gredes.
vat~ng concentrations of unlabelled PGE 2 (0-1 v M ) for 20 min at 4°C, both in the absence and presence of insulin (1.4 nM). rotal binding was determined from the calculated specific activity of the radioligand obtained by d~uting [3H]PGE2 with known concentrations of unlabelled prostag!andin. The dissociation constants ( K d) and the number of binding sites (n) were obtained from non-linear regression analysis of the equil~rium binding, using a computer programme (Enzfitter, Biosoft, Oxford).
Determination o f cyclic AMP content
Ce/ls P388D I cells were grewn as a continuous suspension culture in Dulbeccu's modified Eagle's medium with 10% horse serum at 37'C, 5% C O , , and 100% humidity in 175 cruZ/800 ml plastic culture flasks [23]. P388D I ceils were initially harvested into sterile vials and centrifuged at 300 × g for 5 min. The cells were then washed twice with 10 ml of RPMI 1040 and resnspended in RPMI 1640 to give a final concentration 2-107 cells/ml. Cell count was estimated using a haemocytometer and the viability of the cells was roufinely between 90-95%, as judged by the trypan blue exclusion method.
Estimation of the cAMP content in P388Dj cells (2-106/ml) was determined by preincubating cells with insulin (0-2 riM) in a total volume of 250 /~1. After incubation for 30 min 10/~M IBMX was added for 5 min before the addition of either RPMI 1640 (controls) or PGE 2 (1 /zM) and incubations continued for a further 20 rain. Reactions were terminated by the addition of 372/tl of ice-cold ethanol (65% final concentration). The tubes were then centrifuged at 17000 × g for 5 min. and the supernatants were transferred to tubes for storage for no more than 48 h before analysis of cAMP. The cAMP content of the supernatants was determined by evaporating aliquots of the ethanol and measuring the cAMP present using a cAMP radioimmunoassay kit (Amersham). Neither PGE 2 nor insulin affected the standard curve for cAMP.
[ 3H]PGEz binding assay
Cyclic AMP pho~hodiesterase assay
Radiolabelled PGE 2 binding to P388D I cells was performed as described previously [23] with slight modifications [24]. Washed cells (2 × 106) were incubated with 3 nM p H ] P G E 2 at 4°C for 20 min in a total volume of 0.2 ml. After incubation, cells were vacuum filtered through G F / C glass fibre (Whatman) filters, presoaked with phosphate buffered-saline, (pH 7.8). The filters were then washed with 10 ml of ice-cold phosphate-buffered saline, then dried and radioactivity was measured by seintillatinn counting. Nouspecifie binding was determined by adding excess unlabeUed PGE 2 ( l / t M ) to the above mixture and specific binding was calculated by subtracting the nonspecific binding from the total binding. The effect of insulin on PGE 2 binding was estimated by preincubating the cells (2 × 106) with insulin (0-10 riM) for different times at room temperature (22°C) before the addition of radiolabelled PGE 2.
Cyclic A M P phosphodiesterase activity was measured as described previously [26]. To determine the effect of insulin on cAMP phosphodiesterase activity in P388D I cells, the cells were incubated with insulin at various concentrati3ns (0-5 nM). The cell membranes and cytosolic fractiow were prepared after washing the cells. Cells were boraogenised in 50 mM Tris-HC! (pH 7.4) and centrifuged at 1 7 0 0 0 × g for 15 min at 4°C. The supernatant was retained and recentrifuged at 1 7 0 0 0 x g for 15 min at 4°C and the resulting supernatant was used as the soluble fraction. The pellet was rasuspended in the original volume of buffer and recentrifuged at 1 7 0 0 0 × g for 15 rain at 4°C. The resultant supernatant was discarded and the pellet was resnspended in 0.25 × the original volume of homogenisation buffer and this was used as the particul a t e / m e m b r a n e fraction. The membrane (120 Itg) and soluble fractious (89/~g) were separately incubated in 20 m M Tris-HC! buffer (pH 7.4) in a total volume of 200/zl for 20 rain at 22°C. [~251]cAMP ( 0 . 2 / t M ) was added to the reaction mixture to determine the phosphodiesterase activity of the membrane and soluble fractious of insulin-treated and untreated cells. The mixture was incubated for 3 min at 2 2 ~ and the reaction was terminated by adding 0.2 ml of 0.2 M
Analysis o f equilibfimn binding of [3H]PGEz to P388DI cells in the presence or absence of insulin The effect of preincnhation of P388Dj cells with insulin on the interaction of PGE 2 with these cells was analysed by the method of Scatcbard [25]. Typically, 2-106 cells were incubated with 3 nM [3H]PGE2 and
345
20G
1.5
to0
1.0
o
!
t
t
t
!
. ._..a
0
1
2
3
4
5
.~Sudn conc (riM)
Fig. I. Effect of preinc,_,_h~_~.innwith increasing concentrations of insulin on [3HIPG[~2 binding to P388DI ceils. Cells (2×106) were preincubated in the presence of insulin (0-5 nM) for 30 rain at 22°C, followedby a funficr incnbarion with 3 nM [3H]PGE2 for 20 min at 4°C. The binding of pH]PGE z to the cells was determined, as dt.~arihed in l',hteriafs and Methods. Values indicate total binding (0). specific binding (0) and non-specific binding (e), of% specific binding in the absence of insulin. Results shown here are the mean +_S.D. of three experiments, each in triplicate.
Z n S O 4 followed by 0.2 ml o f 0.2 M Ba(OH) 2 at 30 s intervals. After vigorous stirring for 1 min, the precipitate was separated by centrifugation at 8000 × g for 4 min. The a m o u n t o f cyclic A M P hydrolysed was calculated from the rate of decrease in radioactivity in the supematant. Results Incubation of P388D t cells with [3H]PGE 2 resulted in a rapid and r e v e r s ~ l e binding of the P G E 2. F_.quil~rium of binding was attained within 20 rain of incubation at 4°C (data not shown). Nonspecific binding was estimated to be around 2 5 - 3 5 % of total binding w h e n 3 n M [3H]PGE2 was t : ~ l in the presence of 1 /zM unlabelled ligand. Addition o f increasing concentrations of insulin to the binding assay mixture, 20 min prior to the addition o f radiolabeiled P G E 2 affected P G E 2 binding (Fig. 1). A t 1A n M concentration, insatin increased P G E 2 binding by approx. 2-fold, compared to control, whereas the presence of higher concentrations (above 1.5 n M or m o r e ) of insulin diminished the stimulatory effects on prostaglandin binding. The stimulatory effect of insulin on P G E 2 binding to P388D t cells could b e observed within 5 min incubation with insulin (data not shown). Substitution of insulin by adrenecorticotropin ( A C F H , 4300 Da), a protein o f similar molecular weight, at various concentrations did not effect the binding of [3H]PGE2 (0.33 -4-_ 0.02 f n m l / 1 0 6 cells) w h e n compared to controls (0.35 + 0.04 finol/106 cells). Scatchard analysis o f [3H]PGE2 binding to these cells in the absence and presence of insulin ( I A nM) showed the presence of one high-affinity-low capacity and the o t h e r low-affinity-high capacity receptor population (Fig. 2). The high affinity receptors showed a
O.OL!
!
!
!
t
!
|
o
5
lO
15
20
25
30
Fig. 2. Scatchard plot of [3H]PGE2 binding to P388Dt cel:s in the absence and presence of insulin (1.4 nM). Cells (2x106) were preincubated in the absence (o) and presence (e) of inulin (1.4,1M) for 30 rain at 22°C, followed by a further incubation with 3 nM [3HlPGE2 plus 0-1 gM unlabeficd PGE2 fox 20 rain at 4'~. Total binding (fmol/106 cells) was determined fox each point by dividing the dpm by the calculated specific zctiviw (dpm/fmol) obtained by diluting 3 nM [3HIPGE2 with known concentrations of unlabelled PGE 2. The binding of PGE 2 to the cells was determined as described in Materials and Methods. Each point represents the mean of three experiments, each with triplicate determinations. dissociation constant ( K d ) of 2.72 + 0.14 n M with a binding capacity (n t) oft0.30 + 0.02 f m o l / 1 0 6 cells. The low affinity receptors had a dissociatien constant (Ko2) of 40 + 9.1 n M and a binding capacity (n 2) o f 2.4 + 0.31 f m o l / 1 0 6 cells. In the case of insulin-treated cells, both the high (n t) and low (n 2) binding capacities for P G E 2 increased to 0 . 6 3 + 0 . 0 3 and 5 . 0 + 0 , 4 1 f m o l / 1 0 6 cells, respectively ( P < 0 . 0 0 1 , Student's ttest). However, the dissociation constants of the receptors on insulin-treated cells remained essentially unaltered when compared with the control values (Table I). According to Klotz and Hunston [27], total binding ( R ) cart be expressed as R=
niKt[PGE2]
n2K2[PGE2]
1+ K,[PGE,-----~ + 1 +Kz[PGEz] I II
where R is total P G E 2 binding, n t is the capacity of the high-affinity binding sites, K t the association con. TABLE I Summary of Scatchard analysis of [ 3H/PGE2 binding to P388DI cells both in the absence and presence of insulin (1.4 riM) (mcan +_S.D., n~3)
a gd * the dissociation constants (nM) and n, the number of binding sites (fmol/106 cells). Treatment Control Insulin
High affinity binding sites
Low affinitybinding sites
Kd I a
~d 2 a
nt
n2
2.72+_0.14 0",,30+_0,02 " ~ 9 . 1 "~±0.31 2.65+0.11 0.63+0.03* 48+_2.3 5.0±0.41 *
• P < 0.001, relative to control (Student's t-test).
346 stant of the high-affinity sites, t/2 the capacity of the low-affinity sites, and K2, the association constant of the low-affinity sites. Components I and El of the equation show the "true' specific and non-specific contn'butions of the total b i n d i n g respectively. By substituting the values of nt, K t = 1/Kdt n 2, and K 2 = l/Kdz in the above equation, the specific and nonspecific binding of tritium-labelled P G E 2 to these cells in the absence and presence o f various concentrations of insulin was found to be within 15% of the values determined by experimental procedures. Cyclic A M P levels in iusulin-treated and untreated P388Dt cells were measured both in the absence and presence of P G E 2 (1 /zM). Fig. 3 shows the levels of c A M P in these cells u n d e r different experimental conditions. The incubation of cells with insulin alone at 1.4 or 2 n M for 30 rain reduced c A M P levels by 12.0% and 18.1%, respectively, compared to control, i n contrast, P G E 2 (1 p M ) stimulated c A M P synthesis by 22.9% in insulin-treated cells (1.4 nM) when compared with that of cells incubated with P G E 2 alone. The maximum production o f c A M P by P G E 2 (1 p M ) in the imulintreated cell was observed with ml insulin concentration of 1.4 riM. To test whether the insulin-induced decrease in c A M P levels in the cells was due to the activation of c A M P p h o s p h o d i e s t e r a s e , the c A M P p h o s p h o diesterase activity in P388Dt cells in the absence and presence of insulin ( 0 - 2 riM) was measured. Fig. 4 shows the effect of insulin on c A M P pbosphodiesterase activity. Insulin at 1.0 n M stimulated the degradation of c A M P from basal level, 40.5 +_. 1.32% to 44.1 _+ 1.03% in the soluble fraction ( P < 0.01, student t-test)
A
4t 1=
40 39
0
o
~
1 2 insert conc (#,t)
Fig. 4. Effect of incubation of P388Dt cells with insulin on phosphodie,erase activity. P388DI cells were homogenised in 50 mM Trls/HCi (pH 7.4) and soluble and particulate/membrane fractions were prepared. The soluble (89/zg (A)) and membrane fractions (120 pg (B)) were incubated with [1251]cAMP(0.2 pM) for 3 rain at 22~C and the amount of cyclic AMP h)'drolysed was calculated from the rate of decrease in radio~livity~n the supematanL Values represent the mean _+S.D. n =4.
and from 32.6 +_ 0.96% to 35.6 + 1.3% ( P < 0.01, Stud e n t ' s t-test) in the particulate fractious. Whereas, at a higher concentration o f insulin (2 riM) phusphodiesterase activity was not affected.
e
g
46
~40
Discussion
2 5 0 t-
0
1.4
2
0
1.4
2
iosuen conc (nM)
Fig. 3. Effect of incubation of P388D1 cells with insullin on the increase of cAMP levels in the absence and presence of PGE 2. P388DI cells were incubated with various concentrations of insidin (0-2 riM}for 30 rain at 2TC. IBMX (10 lgM) was then added for 10 rain and either RPMI 1640LA)or i/zM PGE2 (B) was added before incubations were continued for a further 20 min. Incubations were terminated and cAMP determined by radioimmano~say, as descrt'bed in Materials and Methods. Values represent the mean_+S.D. n=4.
The physiological concentration o f insulin varies between 0.03 n M and 1.5 n M [28]. T h e data obtained in this study show that insulin at physiological concentrations increased the binding of P G E 2 to murine macrophage-like cells, P388D t. This suggests that insulin may enhance the immunosuppressive action o f P G E 2. luldbifion o f IL-I production in m o n o c y t e s / macrophages by P G E 2 through raising c A M P levels via adenylate cyelase activation, is regarded as an event that is fundamental to the regulation of the i m m u n e response [13]. W e have shown recently that IL-I inhibits P G E 2 binding to P388D t cells by a cyclic A M P independent process a n d thereby IL-1 may minimise the immunusuppressive action of this prostanoid [29]. This may have. important impficatious in understanding
347 the complex interactions between PGE 2 and IL-I which can regulate most immune response. Insulin increased beth the low and high affinity binding sites of PGE 2 receptors in P388Dj cells without changing their affinities. The availability and the affinity of receptors which determine, at least in part, the physiological effects of hormones, are regulated by various factors, such as second messenger systems, G proteins and phosphorylation of the receptor. In many cell types regulation of a particular receptor system by a distinct second receptor system is often observed ~.30]. Our results show that insulin directly modifies the prostaglandin receptor number in P388D~ cells. This is not necessarily dependent on new protein synthesis as it seems very unlikely that the synthesis of new protein occurred in the 5-20 min incubation time with insulin at 4°C. The stimulatory effect of insulin on the increased binding of PGE 2 was possibly due to the increased availability of spare receptors in the cell membrane. Interaction between receptors for insulin and prostaglandins in human erythrocytes and platelets has been found to be bidirectional and does not involve new protein synthesis [22]. The exact mechanism(s) by which insulin increases the prostaglandin receptor number in blood cells is not yet known. PGE 2 has been shown to down-regulate its own receptor in P388D~ cells through cAMP formation, by activating re~eptor-linked adenylate cyclase [31,32]. Paradoxically, accumulation of cAMP allows these cells to escape the inhibitory effects of PGE 2 and other agonists, through beterologous desensitisation [17,31, 32]. However, it is unclear whether the lowering of cAMP levels by insulin in the cells stimulates PGE 2 binding. The decrease in cAMP levels in cells incubated with insulin (0.7 to 2.0 nM) most likely allows the cells to bind more PGE 2 than controls (Fig. 4). The lowering of cAMP levels may permit the cells to upregulate the PGE 2 receptor number. Insulin has also been shown to decrease intracellnlar cAMP levels in liver, adipose tissue, and other target organs [33,34]. The binding of PGE 2 to cells is reported to be eseeptial for the inhibition of IL-I production and activity [13,14], and the enhanced binding may facilitate the immunosuppressive action of this prostanoid. PGE 2 inhibits many steps in the immune response, including IL-1 production [13,16,35,36]. This inhibition mainly occurs through stimulation of cAMP synthesis by activation of adenylate cyclase [13-15]. The interaction of prostaglandin with its receptors on the cell membrane has been shown to be a prerequisite for the activation of adenylate cyclase [24]. The enhanced binding of PGE 2 to P388D t cells is accompanied by an increased cAMP synthesis (Fig. 3). The increased binding of PGE 2 appears to be insulin-specific, as a protein of similar molecular weight (ACTH) did not affect PGE 2 binding to the cells. In P388D i cells, insulin was
found to decrease the cyclic AMP level, presumably by activating cAMP phosphodieste~-ase in a concentration-dependent manner (Fig. 4). Degradation of cAMP was maximum when the cells were treated with 1.0 nM of insulin. Despite the lowering of cAMP levels in P388Dj cells by insulin, the incubation of insulintreated cells with PGE 2 increased cAMP levels by 25% when compared with untreated cells. This suggests that increased synthesis of cAMP in insulin-treated cells by PGE 2 was due to enhanced prostanoid binding. In human platelets insulin increases P G E t / P G I 2 binding without affecting cAMP levels [21]. The enhanced prostaglandin binding to platelets results in an increase in cAMP synthesis and thus amplifies the anti-aggregatory actions of the prostanoid [22]. Our data suggest that insulin may be capable of regulating immune cell functions via modulation of prostaglandin binding. As diabetes mellitus involves an absolute or relative insulin deficiency, then this disease may predispose to an inadequate immune response. Acknowledgement A preliminary account of this work has been published as an abstract in the Biochemical Society Transactions 19, 358S, 1991. References 1 Johnston,R.B. Jr. (1988)N. Eng.J. Med. 318, 747-749. 2 Dinarello,C.A. (1984) Rev. Infect. Dis. 6, 51-95. 3 Dinarello,C.A. (1988)FASEBJ. 2,108-115. 4 Bendtz~n,K., Mandrup-Poulson,T., Nerup,J., Neilsen,J.ELand Dioarello, C.A. (1986)Science 232, 1545-1547. 5 Shimizu,H., Uehara, Y., Shimomu,-a,Y., Negishi, M. and Fuktasu, A. (1990)Biochem.Biophys.Res.Comman.173,1280-1286. 6 McDaniel, M.L., Hughes,J.H., Wolf, B.A., Easom, R.A. and Turk, J.W. (1988) Diabetes37, 1311-1318. 7 Hamaguchi,K. and Leiter, E.H. (1990) Diabetes~9, 415-425. 8 Welsh, N., BendU'.en, K. and Sandier, S. (1991) Diabetes 40, 290-294. 9 Garcia-Welsh,A., Schneiderman,J.S. and Baly,D.L (1990)FEllS Len. 269,421--424. 10 Velich, M.R. (1990)Am. J. Physiol.258, Ri070-RI077. 1! Fisher, R.I. and Bostick-Burton, F. (1982) J. lmmunol. 129, 1770-1772. 12 Holdstocg,G., Chastenay, B.F. and Krawitt, E.L (1982)Gastroenterology82, 206-210. 13 Knudsen, P.J., Dinarello. C.A. and Strom, T.B. (1986) J. lmmtmol. 137, 3189-3191. 14 Renz, H., Schimdt, A., Nain, M. and Gemsa, D. (1988) J. lmmm~oL141 2388-2390. 15 Brandwein,S.R. (1986)J, Biol. Chem.261,8624-8626. 16 Monick, M, Glasier, J. and Hannighake, G.W.(1987)Am. Rex,. Resgir. Dis. 135,72-77. 17 Coffey,R.G., Alberts,V.A. and Wealdand, L.L.(1990)J. Leukocyte. Biol. 48, 557-564. 18 Endres. S., Fulle. H.-J.. Sinha, B., Stoll, D., Dinarello, C.A., Gerzer, R., and Weber, PC. (1991) Immunology7~ 56-60. 19 Duna-Roy.A.K. and Sinha, A.K.(1985)Biochim.Bioph~s.Acta. 812, 671-678.
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