A monoclonal triggers

antibody

specific

polymorphonuclear

for immunoglobulin

neutrophil

A receptor

superoxide

release

Li Shen Department

of Microbiology,

Dartmouth

Medical

Schoo4

Abstract: An immunoglobulin M (1gM) monoclonal antibody, My43, specific for IgA Fc receptor (FcaR) on human monocytes, bound to human polymorphonuclear neutrophils (PMNs) and inhibited their ability to bind IgA but not IgG. It was observed that the PMN oxidative burst

was induced by both polymeric IgA and aggregated whereas 1gM was without effect. The IgG-mediated oxidative burst was inhibited by anti-Fc’yRII Fab and IgG,

anti-Fc’yRIII F(ab’)z but not by My43. Conversely, the IgA-mediated oxidative burst was inhibited by My43 but not by anti-Fc’yRII or anti-Fc7RIII. When anti-Fc receptor monoclonal antibodies (mAbs) were used directly as ligands, it was observed that both anti-Fc7RII Fab and anti-FcyRII F(ab’)2 promoted the oxidative burst when cross-linked. Moreover, My43, when cross-linked with F(ab’)2 antimouse 1gM, also triggered the oxidative burst, whereas an 1gM anti-CD15 mAb, PM81, did not stimulate function. This demonstrates that IgA receptors on PMNs are function-triggering molecules and that an antiIgA receptor mAb may be substituted as a ligand.J. Lcukoc. Biol. 51: 373-378; 1992. Key

Words:

Fc receptor

chemiluminescence

Hanover,

New

8] as well IgA rosette

Hampshire

as

Receptors for the Fc portion of immunoglobulin stitute the major class of function-triggering phagocytic cells. FcRs that bind IgG antibodies been extensively characterized and are known tivities such as phagocytosis [2], antibody-dependent cytotoxicity [28], and secretion of microbicidal

(FcRs) conmolecules on (FcyRs) have to mediate accellular products in-

cluding superoxide anion [32]. Through these mechanisms phagocytic cells greatly amplify the protective effects of antibodies. Although FcyRs are important in situations where immunoglobulin G (IgG) antibodies predominate, this is probably not the case at mucosal surfaces. At these sites, which in humans constitute several square meters, the body’s tissues are separated from the environment by a mucous membrane that is protected by mucous secretions containing antibody, chiefly of the IgA isotype [30]. In fact, it is estimated that the body produces more IgA on a daily basis than any other isotype, most of which is transported into secretions [6, 22]. Despite its prevalence, the mode of action of IgA antibody in mucosal protection is incompletely understood. Its chief documented activities are fixation of complement by the alternative pathway [15] and inhibition of bacterial adherence to epithelia [18, 31]. Because significant numbers of phagocytic cells are also associated with mucosal areas, for instance, the alveolar macrophages of the [14], it seemed possible that these cells might amplify protective function of IgA antibody through Fc receptor teraction. Consistent with this idea, receptors specific IgA

(FccrRs)

have

been

detected

on

mucosal

phagocytes

lung the infor [3,

blood cells [7] by methods However, it has been well

such as demon-

strated in studies on Fc’yRs that more sensitive detection and quantitation of receptors could be achieved using a receptorspecific monoclonal antibody (mAb) [9]. This laboratory has produced an mAb specific for IgA receptors on human monocytes [29]. This report describes the binding of the antibody to human polymorphonuclear neutrophils (PMN5), which results in both inhibition of IgA binding and triggering of FcaR function.

MATERIALS PMN PMNs were

AND METHODS

Preparation from peripheral blood freed from mononuclear

of cells

normal adult and red cells

volunteers on a Ficoll

M85 Hypaque gradient [10] and washed in calciumand magnesium-free Hanks’ balanced salt solution (HBSS) at room temperature in preparation for the superoxide production assay. PMNs were usually more than 95% pure, the rest of the cells being lymphocytes and eosinophils.

Superoxide INTRODUCTION

peripheral formation.

Assay

Forty microliters of human myeloma-derived heat-aggregated IgG (HA IgG), polymeric IgA (pIgA), 1gM, or mAb (20 sl) plus 20 jel of F(ab’)2 antimouse Ig were mixed in a luminometer cuvette with 100 el of PMNs at 5 x 106/ml in Ca2and Mg2-free HBSS and 100 1d of 10 M luminol (5-amino-2,3dihydrophthalazine-1,4-dione) (Sigma, St. Louis, MO) in HBSS containing 2.6mM CaCl2 and 2 mM MgSO4’7H2O. The oxidative chemiluminescence an LKB 1251

burst

produced by the PMN-generated of the luminol [4] was monitored using luminometer to give a readout in millivolts

(mV). PMNs were kept at room temperature to 2 h after isolation, during which time superoxide response remained constant.

Fluorescent PMNs complete

their

and used for IgA-mediated

up

Staining

were incubated equilibration

for 2 h at of antibody

4#{176}Cwith binding

mAbs in the

to allow presence

Abbreviations: BSA, bovine serum albumin; FcR, Fc receptor; FITC, fluorescein isothiocyanate; HA, heat aggregated; HBSS, Hanks’ balanced salt solution; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; mAb, monoclonal antibody; OE, ox erythrocytes; P/B/A, PBS, BSA, and sodium azide; PBS, phosphate-buffered saline; pIgA, polymeric IgA; PIPLC, phosphatidylinositol-specific phospholipase C; PMN, polymorphonudear neutrophil; PNH, paroxysmal nocturnal hemoglobinuria; SPOP, 3-(2-piridyldithio)propionic acid N-hydroxy succinimide ester. Reprint requests: Li Shen, Department of Microbiologu Dartmouth Medical School, Hanover, NH 03756. Received June 18, 1991; accepted September 16, 1991.

Journal

of Leukocyte

Biology

Volume

51,

April

1992

373

of 5 x 10 M normal human IgG. The cells were then washed in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 0.1% sodium azide (P/B/A) and incubated at 4#{176}C with fluorescein isothiocyanate (FITC)-labeled F(ab’)s antimouse Ig for 1 h. After further washing in P/B/A the PMNs were fixed in 2% paraformaldehyde and analyzed by flow cytometry using a Cytofluorograph system 50H (Ortho Diagnostic Systems, Westwood, MA). The relative mean numbers of FITC anti-MIg (F(ab’)2 per cell (second antibody per cell) were determined by comparison with a standard curve derived with latex beads (Flow Cytometry Standards Corp., Research Triangle Park, NC) containing per

bead

six different

concentrations

of fluorescein

molecules

NaC1, and

4.5,

treated

with

0.0l

M

dithiothreitol

(30

mm),

through a G.25 Sephadex column (Pharmacia) equilibrated in 0.1 M phosphate, 0.1 M NaCI, pH 7.5. Equimolar amounts of the anti-OE F(ab’)s and IgA or IgG were then mixed and incubated at 18#{176}Cfor 4 h, after which crosslinking was terminated with 2 mM iodoacetamide. Preparations

contained

Antibodies

less

and

than

15%

Antibody

non-cross-linked

1g.

Fragments

The development and properties of IV.3, a mouse IgG2b mAb to Fc’yRII [20], and 3G8, a mouse IgGi mAb to the granulocyte FcyRIII [11], have been reported. The IgG fraction

[19].

pH passed

of ascitic

hybridoma

fluid cells

from was

pristane-primed obtained by

mice precipitation

injected with

with 40%

saturated ammonium sulfate. Ion-exchange high-performance liquid chromatography (HPLC) with a protein-pak 5PW Rosette Inhibition Assay DEAE column (Waters Chromatography Division, Millipore, Milford, MA) was used to purify the antibodies. The PMNs (1 x 106) were incubated wtih 200 jel of mAbs at 20 F(ab)2 fragments of 32.2 and 3G8 were made according to eg/ml for 45 mm at 4#{176}C.The PMNs were then adjusted to the method of Parham [24] by pepsin digestion at pH 3.5 and 2 x 106/ml and 25 el of PMNs was centrifuged together with purified by HPLC gel filtration chromatography with a Bio25 il of a 1% suspension of IgAor IgG-coated bovine 250 column (Bio-Rad). Fab’ fragments were oberythrocytes (Wilfer Labs, Oren, MN) and incubated at 4#{176}C Sil TSK tained by reduction with 1 mM dithiothreitol for 2 h at 18#{176}C, for 90 mm. The cells were then resuspended gently in followed by alkylation with 2 mM iodoacetamide for 1 h at ethidium bromide-acridine orange and the percent of 18#{176}C.The Fab fragment of IV.3 was made as previously rosette-forming cells was determined by counting a minidescribed [20] with minor modifications. Briefly, IV.3 antimum of 200 viable PMNs in duplicate samples using body at a concentration of 2 mg/ml in PBS containing 10 ultraviolet and incident light microscopy. Cells with four or mM cysteine and 2 mM EDTA was incubated with papain more attached erythroyctes were scored as positive. (Sigma) at an enzyme-to-substrate ratio of 1:25 for 3 h at Phospholipase C Digestion 37#{176}C.The reaction was stopped by addition of 20 mM iodoacetamide and the digest was dialyzed against 5 mM phosPMNs at lOx x106 ml in RPMI were incubated with a phate buffer, pH 8.0. The digest was then placed on a protein 1:1000 dilution of phosphatidylinositol-specific phospholipase A-Sepharose 4B column to remove Fc fragments. Other C (PIPLC), a generous gift from Dr. Martin Low (College mAbs were used as hybridoma supernatants. Antibody conof Physicans and Surgeons, Columbia University, New tent was determined using Diffugen radial immunodiffusion York), in the presence of 5 mM EDTA, 2 mM phenlmethylplates (Tago, Burlingame, CA) and supernatants adjusted to sulphonylfluoride, and 5 eg/ml of soybean trypsin inhibitor 20 eg/ml. F(ab’)2 fragments of antimouse Ig were purchased (Sigma) for 30 mm at 37#{176}C. They were then washed in from Organon Teknika (Malvern, PA). RPMI-l0% FCS prior to immunofluorescent staining.

Human

RESU LTS

Ig

Human dimer IgAl paraproteins (Sa IgA and Ca IgA) purified from myeloma sera by gel chromatography Sepharose CL-6B (Pharmacia Fine Chemicals). The corresponding to dimer IgA was cleared of contaminating

were on peak

IgG

con-

on

protein

A-Sepharose

tained less than 0.5 munosorbent assay Birmingham, AL). plasma of a myeloma phy on an Affi-Blue

CL-6B;

these

preparations

eg of IgG/mg IgA by enzyme-linked ml(Southern Biotechnology Associates, Human IgGi was purified from the patient by ion-exchange chromatograGel column (Bio-Rad, Richmond, CA).

Heteroantibodies Conjugates of IgA, IgG, or purified mAb linked to Fab antiox erythrocytes (OE) were made using the bifunctional reagent SPDP (Pharmacia, Uppsala, Sweden) [17]. F(ab’)2 antiOE was prepared from rabbit anti-OE IgG (Cooper Biomedical, Malvern, PA) by pepsin cleavage and passage through protein A-CL-6B. IgA or IgG and F(ab’)2 anti-OE (at 1 to 3 mg/mi) were treated separately with an eighfold molar excess of 3-(2-piridyldithio)propionic acid N-hydroxy succinimide ester (SPDP) for 2 h at 18#{176}C.SPDP-treated anti-OE Fab was dialyzed in PBS, pH 7.2; SPDP-treated IgA, IgG, or mAb was dialyzed in 0.1 M acetate, 0.1 M

374

Journal

of Leukocyte

Biology

Volume

51,

April

1992

In order receptors

to determine for IgA on

whether PMNs, the

the My43 mAb recognized ability of this mAb to inter-

fere with FcaR (IgA receptor) binding was examined (Table 1). PMN IgA rosette formation was completely inhibited by My43, whereas their binding of IgG-coated OE was unaffected. FcyRIII, neither PMNs.

MAbs respectively, did PMN My43 had

3G8 produced forming cells.

IV.3

and 3G8 specific for FcyRII and did not reduce IgA rosette formation; 29, an 1gM mAb specific for CD15 on no effect on PMN IgG rosettes, whereas

a marked reduction These data indicated

in percentage that the

of rosetteMy43 mAb

bound to the PMN FcaR binding site or a closely associated epitope. The relative expression on PMNs of FcaR, FcyRII, FcyRIII, and CD15 was compared by indirect staining and flow cytometric measurement of the mean number of FITClabeled antimouse Ig F(ab’)2 bound per cell (Table 2). These results indicated that FcaRs were expressed in relatively low numbers compared to IgG Fc receptors. The percentage of FcctR-bearing PMNs was also lower than that expressing FcyR; however, a greater proportion of FcaR-positive cells was detected using the My43 mAb than by IgA rosette formation

(Tables

1 and

2).

TABLE

I.

PMN

lgA and IgG Monoclonal

Rosettes Antibodies

and

Their

Rosette-formi mAb

Inhibition

ng cells

by

IgG

46

3G8 IV-3 PMN29

35±

5

40±

8

74±7

43j

10

91±1

were

treated

and

at 4#{176}C with

Methods,

after

± 14 0

89

3G8

3

±

and

antibodies/cell My43

3G8

treatment

to

Staining

My43

Medium

89,157

±

1,215

4,118

±

55

PIPLC

30,452

±

1,176

4,274

±

78

28±10

they

or IgG-coated bovine eiythrocytes. After rosettes was determined by microscopy. of three experiments.

Cell

89±4

medium

which

Different Sensitivity of PMN PIPLC

Second

Medium My43

PMNs

3.

(%)

IgA

in Materials

TABLE

or different were

incubated

90 mm the number The results show

‘PMNs

mAbs

as described IgAof PMNs forming the mean ± SE at 4#{176}C with

incubated

were

als and Methods and P3, followed by FITC (second antibodies/cell) using flow cytometry. and show the mean

hibit

the

with

medium

or

then stained with F(ab)2 anti-mlg. were enumerated The ±

results

are

of

experiments.

SE

triggering

of

two

PIPLC

as described

in

Materi-

3G8, My43, or the nonspecific The FITC F(ab)s anti-mlg by comparison to a standard corrected

PMNs

by

for

IgA

nonspecific

almost

IgG, per cell curve

Ig

to

binding

baseline

levels.

The

expression

of

Fc’yRIII

on

PMNs

was

found

to

be

highly reduced in patients suffering from paroxysmal nocturnal hemoglobmnuria (PNH) and was sensitive to PIPLC, both observations indicating that it is linked to the lipid bilayer of the cell membrane by a phosphatidylinositol glycan linkage [26]. Table 3 compares the sensitivity of FcceR and Fc’yRIII to PIPLC digestion and shows FcaR expression to be unaffected by this enzyme. The functional role of FcctR was then examined. The ability of PMNs to respond to aggregated IgG, polymeric IgAl, or pentameric It was found lesser extent

1gM was measured that IgAl elicited than heat-aggregted

using the a response, human IgGl,

luminol although whereas

assay. to a 1gM

did not trigger this activity of PMN (Fig. 1). The response of PMNs of both IgG and IgA was inhibited in presence of 50 g/ml superoxide dismutase, in agreement with previous observations [4] and indicating that under these conditions luminol primarily detected superoxide anion generation (Fig. 2). In order to test the receptor specificity of PMN responses to IgG and IgA, inhibition studies were performed using anti-FcR mAbs. It was observed that anti-Fc-yRII Fab IV.3 or anti-Fc7RIII F(ab’)2 3G8 both produced partial inhibition of superoxide release triggered by aggregated IgG. Virtually complete inhibition was obtained with a mixture of Fab IV.3 and F(ab’)2 3G8 (Fig. 3). Neither Fab IV.3 nor F(ab)2 3G8 alone induced an oxidative burst in PMNs (data not shown). By contrast, IgA-triggered superoxide release was unaffected by Fab IV.3 and F(ab’)2 3G8, even when the two mAbs were combined (Fig. 4). The inhibitory properties of My43 mAb were then examined (Fig. 5). This mAb did not block slight

the ability enhancement

of IgG to mediate the oxidative was often seen. My43 did,

Antibodies ligand binding

to Fc-yR have been shown in triggering FcR-mediated

to substitute functions,

for ex-

for

ample, whereas

antibody-dependent cell-mediated cytotoxicity, mAbs against nonreceptor antigens have been found to be nontriggering [13]. Therefore, the abilities of anti-Fc’yR mAbs and My43 to promote the PMN oxidative

burst were compared. Both Fab IV.3 and F(ab)2 3G8 stimulated superoxide release when cross-linked with F(ab’)2 antimouse Ig, whereas neither the anti-FcyR fragments nor F(ab)s antimouse Ig alone was stimulatory (Fig. 6). The anti-FcaR 1gM mAb My43 also triggered superoxide release (Fig. 7). The antibody alone produced a slight stimulation that was greatly 1gM. Another CD15

on

PMNs,

alone

or

when

increased 1gM mAb, did

not

by addition of F(ab)2 antimouse PM81, which is directed against stimulate

the

oxidative

burst

either

cross-linked.

DISCUSSION Monoclonal antibodies are important tools for detecting, measuring, and isolating cell surface components and have been invaluable in dissecting the properties of different IgG Fc receptors [9]. Corresponding antibodies specific for IgA receptor have until recently been lacking, and our understanding of FccrR has thus remained less comprehensive. This laboratory recently succeeded in producing the 1gM

801

70

burst; in fact however, in60

TABLE

2.

Relative

Binding Antibodies

mAb

Antigen

My43

3G8 IV-3 PMN

PMNs

29

(CDI6) (CD32)

LNF-III

(CD15)

and Other

Monoclonal 50

to PMNs Second

FcaR Fc7RIII FcyRII

were stained Ig as described

of My43

antibodies/cell

% positive

C

5,700 79,000 34,000

±

6,000 ± 800

93 ± 2 94 ± 1

52,000

±

93

±

700

7,000

75

±

±

40

21

2

with different mAbs followed by FITC F(ab)2 antimouse in Materials and Methods. The FITC F(ab)2 antimlg molecules per cell (second antibodies/cell) were enumerated by comparison to a standard curve using flow cytometry. The results are corrected for nonspecific binding obtained when the cells were stained with the nonspecific P3 IgG and antimouse Thy 1.2 1gM followed by FITC F(ab)2 anti-mlg and show the mean ± SE of three experiments.

30

Fig. 1. PMNs with HAIgG tameric 1gM

x 105) were mixed (#{149}), pIgA (A) or pen(#{149}) to a final concen-

tration

sg/ml

of

40

(5

in

the

of luminol as described in Materials and Methods. The resulting chemiluminescence was measured in a luminometer giving a reading in millivolts. Results are shown as the mean ± SE of three experiments.

S/zen

IgA

20

presence

receptor

specific

10

4

8

12

I6

20

Minutes

mAb

triggers

PMN

375

Fig.

50

4. PMNs

20 jug/mI medium IV.3 40

incubated

(A),

PIgA

with

IgA (pIgA) plus 8 jug/mI

plus

8 jug/mI

plus Fab

20

F(ab)2

3G8 (A), PIgA and a mixture of Fab lV.3 plus F(ab)2 3G8 (0), or 20 jug/mI

MV

were

polymeric (I), pIgA

30

BSA

and

(---),

produce

an

measured

using

their

oxidative

abili.y burst

to was

C

0

as described

luminol

in Materials and Methods. Results the luminometer readings in

show

millivolts

20

mean

and ±

SE

are

expressed

of three

as

the

experiments. 4

I

8 2 Minutes

it

20

l0

..-e--.---.-

0

4

8

-#{149}-

12

FcyRII.

-. 6

62024

4

8

12

16

20

24

MINS with HAIgG plus medium (0) or su(#{149}) pIgA plus medium (A), SOD (A), or BSA (#{149}---S) in the presence of luminol as described in Materials and Methods. The final concentration of IgG and IgA was 20 ug/ml. The resulting response was measured in a luminometer giving a readout in millivolts. Results are expressed as mean ± SE of two experiments. Fig.

2.

peroxide

PMNs

(5

dismutase

x 105)

were

(SOD)

mixed

to 50

ug/ml.

There

environment mAb My43 against the monocyte IgA receptor [29]. data presented here show that this mAb also inhibits binding of IgA to PMNs, indicating that FcsRs of PMNs antigenically identical to those of monocytes.

The the are

3G8 IgG with FcyRIII appeared

IgA-coated to be very

erythrocyte binding, densely expressed on

surface in 10-fold or greater amounts 3G8 did not inhibit PMN recognition polymers in the superoxide generation

the

because PMN

than FcaR. F(ab’)2 of small soluble IgA studies, presumably

because pIgA is a much smaller ligand than an IgA-coated erythrocyte and requires clustering of far fewer FcaRs for successful binding. Interestingly, IgG rosettes were inhibited approximately 66% by the anti-Fc7RIII mAb 3G8, whereas the anti-Fc’yRII mAb IV.3 produced only about 16% inhibition. This could be due in part to the greater expression of FcyRIII

on

the

PMN

surface,

approximately

double

that

of

also

evidence

that

FcyRIII

may

play

a

it would be of greater adaptive advantage high levels of FcyR. This idea that FcR expression may be influenced by levels of different isotypes in the environment surrounding the cell is suggested by observations on PMNs from oral mucosa, an environment with a greater IgA-to-IgG ratio than serum. The cell population contained a higher percentage of IgA-binding cells and a lower percentage of IgG-bmnding cells than peripheral blood PMNs [3]. These data also confirm the findings of Gorter and coworkers [12] that FcaRs mediate activation of the oxidative burst when triggered by particulate IgA complexes and demonstrate that soluble IgA complexes can promote the same function. The PMN oxidative burst in response to IgA was always less than that produced by equivalent amounts of IgG; however, since the cells have many more Fc’yRs than FcaRs, this is not surprising-indeed, one might have expected a much greater difference between IgG and IgA stimulation. It is possible that the heat-aggregated IgG preparation did not contain as high a percentage of agfor

Inhibition of IgA binding by My43 was highly specific; the mAb had no effect on IgG binding. MAb IV.3, which blocks the Fc7RIII binding site, likewise had no effect on IgA binding, whereas mAb 3G8, which binds to Fc-1RIII, reduced IgA rosette formation by approximately 24%. This phenomenon probably resulted from steric interference by

is

principal role in recognition of insoluble IgG complexes [5]. The relative number of Flit-labeled antimouse Ig antibody molecules bound after indirect immunofluorescent staining with My43 and other mAbs suggests that FcaRs are expressed in low numbers on peripheral blood PMNs compared to FcyRII and Fc7RIII. This low FcaR expression might be related to the fact that the ligand for FcaR, IgA, is only a small fraction of total serum immunoglobulins. In contrast, IgG is present in high amounts. Thus PMNs are more likely to encounter IgG-opsonized targets in the serum and

PMNs

to

expres

60

50

40

3. PMNs

Fig.

(5

x 105)

were

inFig.

cubated with HAIgG at 20 ug/ml alone (#{149}), HAIgG in the presence of 8 jug/mI F(ab’)2 3G8 (A), HAIgG with 8 jug/mI Fab IV.3 (A), HAIgG

30

medium My43

with F(ab)2 3G8 plus Fab IV.3 (0), or 20 jug/mI BSA (---), and their ability

to produce

was

measured

described

Results ings

Minutes

376

Journal

of Leukocyte

as the ments.

Biology

in

an

oxidative

using

in Materials

as

Volume

51,

April

1992

(5

x 105) were inHAIgG plus (#{149}), HAIgG plus 2 jug/mI (0), 20 jug/mI PIgA plus 20 jug/mI

My43 (A), or 20 jug/mI ESA (---), and their ability to produce an oxmedium (A), PIgA plus 2 jug/mI idative burst was measured using luminol ad described in Materials and Methods. Results show luminometer readings in millivolts

I0

Methods.

show the luminometer readmillivolts and are expressed mean ± SE of three experi-

PMNs

with

20

burst

luminol and

5.

cubated

0

4

8 Minutes

2

and three

are

expressed experiments.

as mean

±

SE of

gregates only

as

the

a fraction

teraction with The inhibition My43 and the My43

again

Likewise,

polymeric

demonstrate IV.3 and 3G8

tion by receptor.

IgG, indicating Interestingly,

gregated Fab IV.3

IgG was or F(ab’)2

Fc’yRIII

play

gregated

IgA

IgG.

preparation,

and

therefore

a

the interfered

role

in

with

for

PMN

FcaR.

3C

inhibited suggesting

in that

presence of both FcyRII

release

showing

that

after both

binding Fab

1, failed to inhibit to IgG-opsonized IV.3 was capable of Crockett-Torabi

These

for

results

demonstrate

FcaRs

on

PMNs

the

specificity

IgA

of

binding

([I]), PM8I antimouse

(0),

(A),

or

burst

and

SE

and

2 40 (U),

1gM

1gM

oxidameasured

described

in

Methods.

Results

readings

are

of three

(A),

was

luminometer

livolts

40

plus

subsequent as

2

plus

antimouse

production

luminol

with

My43

F(ab)2

Their

(---).

Materials

expressed

in

mil-

as mean

±

experiments.

ag-

IV.3

and

io

the PMN oxidative burst in Staphylococcus aureus Wood, of inhibition. However, the and Fantone [5] that cross-

by

anti-MIgM

PM8I F(ab)2

show

linking of both FcyRII and Fc’yRIII using Fab IV.3 and Fab 3G8, respectively, plus a F(ab)2 second antibody promoted oxidase activity are in accord with the data presented here. A possible reason for these differences may be the use of different and Fc’yRIII mAbs, and further experiments are required to resolve this issue. Superoxide generation occurred upon cross-linking My43 with F(ab)2 anti-IgM on the PMN surface, whereas the anti-CD 15 1gM mAb PM81 did not induce activity when cross-linked. This further demonstrates that the molecule recognized by My43 is a functional trigger and that multiple cross-linking of FcaR by mAb plus second antibody is necessary for the receptors to generate a signal, as has been observed in the Fc’yR-dependent oxidative burst [25]. Alone, My43 induced almost no release of superoxide, despite the fact that as a pentameric 1gM molecule it should have the potential to cross-link up to 10 antigenic sites. It is likely that the steric folding of the 1gM molecule plus the geometry of the FcaR on the cell surface prevents optimal 1gM crosslinking. In addition, the lack of stimulation following treatment with PM8I indicates that the ability to trigger oxidase activity is not a general property of surface molecules but is restricted to those with receptor function. mAb

F(ab)2

using

2 20

treated

jug/ml

tive

either and

wcre

jug/mi

alone

stimula-

PMNs My43

PIga

binds through a separate burst in response to ag-

superoxide

data

of My43

only

that IgA the oxidative partially 3G8,

The

specificity

7.

jug/mi

jug/mi

F(ab)s 3G8 plus F(ab’)u antimouse IgG induced the oxidative burst also suggest that both receptors directly mediate this function. This is contrary to the report of Huizinga et al. [16] demonstrating that another anti FcyRIII mAb, CLB FcR-gran response whereas findings

Fig.

4C

IgG receptors became cross-linked by inthe aggregated IgG to activate NADP oxidase. of IgA-triggered superoxide generation by lack of inhibition of IgG-mediated function by of

the

and

5

20

lb

I

Minutes

functional and

has

triggering. been

The

mAb

instrumental

also

in

binds

cloning

of

monocyte FcaR

FcceRs

cDNA

from

U-937 line [21]. From the cDNA sequence it was deduced that FcaR is a transmembrane protein with two extracellular Ig-like domains. The insensitivity to PIPLC of PMN My43 binding strongly suggests that FcaRs on PMNs are also transmembrane proteins and is in agreement with the observation by Monteiro and co-workers [23] that PIPLC did not affect polymeric IgA binding to PMNs, monocytes, and U-937 cells. The binding of both monocyte and PMN FcceRs by My43 and the identical size of FcaRs isolated from monocytes and PMNs [1, 23] strongly imply that FcaRs on these cells are identical. The ability of FcaRs to trigger the oxidative burst in both monocytes [27] and PMNs [12] demonstrates that these receptors can trigger microbial killing by phagocytes and therefore that FcaRs might greatly augment the protective function of IgA. Conversely, this phenomenon could contribute to the pathology associated with IgA deposition, such as that seen in certain the

monocyte-like

skin

disorders,

trophil

because

release

ious

My43

inhibition

4

enzymes

of

the

toxic

into

deposited

oxygen

IgA

surrounding

would

trigger

neu-

probably varresulting in inflam-

metabolites

and

tissues,

mation.

ACKNOWLEDGMENTS 50

This 19053,

U,

Center

(CA

gift

40 Fig. 6. PMNs were incubated with 4 jug/mI F(ab)2 3G8 (U), F(ab)2 3G8 plus 160 jug/mI F(ab’)2 antimouse IgO (0), 4 jug/mI Fab IV.3 (A), Fab IV.3 plus 160 jug/mI F(ab’)s antimouse IgG (A), 20 ug/mI HAIgG (#{149}), or F(ab’)2 antimouse Ig alone (---), and their production of an oxidative burst was measured using luminol as described in Materials and Methods. Results show luminometer readings in millivolts and are expressed as mean ± SE of four experiments.

ported

work was supported by NIH grants Al 22816, Al and CA 44794. The cytofluorograph was a generous the Fannie Rippel Foundation and is partially supby the Core grant of the Norris Cotton Cancer

of

23108).

30

REFERENCES

0

1. Albrechtsen,

2C

tion

of the

M., IgA

Yeaman,

receptor

G.R., from

and

human

Kerr,

MA.

Characteriza-

polymorphonuclear

Ieuko-

Immunology 64, 201, 1988. 2. Anderson, CL., Shen, L., Eicher, D.M., Wewers, M.D., and Gill, J.K. Phagocytosis mediated by three distinct Fcy receptor classes on human leukocytes. j Exp. Med. 171, 1333, 1990. 3. Barbeau, J., Bourget, J., and Deslaurier, N. RFc alpha bearing cytes.

I0

0

4

8

Minutes

12

16

cells

and

Adv.

Exp.

S/zen

IgA-mediated Med.

IgA

Biol.

receptor

phagocytosis 216A,

563,

specific

in the

mouse

oral

mucosa.

1987.

mAb

triggers

PMN

377

4. Campbell, logy and 6.6,

AK. Cheiniluminescence: Principles and Application Medicine. Chichester, England: E. Horwood,

p.

342,

1988.

5. Crockett-Torabi, mune by

complexes

and

Fantone,J.C.

activate

Fcy

human

of

press 1980. 8. Fanger,

human

G.N., and in the mouse:

im-

145,

MW., of

9. Fanger,

L.,

Proc. SN.,

for

and

IgA

the

3275,

phagocytosis

61, 303, Graziano, cytes cells.

P.S., MT., by

Faber,

ment Mo!.

of

Van

Fanger,

the

is essential

Journal

IgG.

procedure

for

leukocytes Hypaque-Ficoll

Human Proc. NaIL

van der and Daha,

Low,

respiratory

L.A.,

trigger

Daha,

van

and

Gorter, M.R.

Borne, Fc’y

A.E.G. receptor

the

IgG-induced

for

IgA F.,

phagocytosis. j Immunol. B., Titus, J.A., Stephany, of target-specific effector

of Leukocyte

Biology

and

IgAl

Sd.

on

mono-

for

tumor

ME., of comple-

Dol-

Tetteroo, on

PA., human

and neu-

respiratory 142, 2365,

burst 1989.

and

D.A., and cells using

Volume

Segal, D.M. hetero-cross-

51,

j

96,

Mishal,

Z., by

ImmunoL

IgA

Monteiro, distribution,

system: 40, 105,

R.C.,

416,

flow

61,

G.N., bear

cells

a

recep-

serum the

type M

IgA

protein.

1987. R.A.,

cytometry

Methods

anti-Fc

against

Lemonnier,

and

of

301,

and two

Kourilsky,

HLA

class

I

1983.

Anderson, distinct

CL. Human receptors for

Fc

1986.

C.J.,

Schoenborn,

cloning 1990.

M.A.,

of a human

Jackson,

S.,

Gimpel,

Fc receptor

and

Brown,

C!in.

reassessment.

for

TA.

The

Immunol.

Im-

1986.

Kubagawa, regulation

in humans.

j

and Exp.

R.C., and biochemical

Med.

26.

Selvaraj, major and

ME., IgA-

L.,

Kr., (FcRII)

Laryngo!.

PP.,

and

IgA

activity

and

Cooper,

M.D.

nature 597, 1990.

of

Cellular an Fccs

171, of monoclonal IgGi, IgG2a j Immuno!. 131, 2895, 1983. 25. Pfefferkorn, L.C., Guyre, P.M., and Fanger, M.W. Functional comparison of the inductions of NADPH oxidase activity and FcyRI in IFN-’y treated U937 cells. Mo!. ImmunoL 27, 263,

27.

Fc

P., Rosse, receptor

is deficient

in

W.E, Silber, blood has

in paroxysmal

nocturnal

Shen, antibody

L., IgA

Lasser, R., and that reacts with binding

and

Fanger, human

triggers

30.

1989. Tomasi,

31.

Characteristics of an immune nal secretions. j Exp. Med. Williams, R.C., and Gibbons,

TB.,

Tan,

E.M.,

herence gen 32.

R., and Springer, a phosphatidylinositol

T.A.

The anchor

haemoglobinurea.

Na-

ture 333, 565, 1988. Shen, L., and Collins, J. Monocyte superoxide secretion triggered by human IgA. Immunology 68, 491, 1989. Shen, L., Guyre, P.M., and Fanger, MW. Polymorphonuclear leukocyte function triggered through the high affinity receptor for monomeric IgG. j linmunoL 139, 534, 1987.

cyte

fragments.

Koenderman,

Rhino!.

R.J., Abraham, and U-937

receptor

Davis, G.S. Defense Annu. Rev. Respir.

A., Stuurman, Activation

Secretory

antibody

Parham, P. On the fragmentation and IgG2b from BALB/C mice.

29.

RB.,

Kemenade,

Fc-yRII molecules

membrane.

J., and

and

G.

and

1686,

1990.

Acad.

induce a respiratory burst leukocytes. Immunology

Fc-yRI

Mogi,

24.

neutrophil

Sluys, MR.

Looney, monocytes

munopatho!.

P.M. Immunol.

1984.

Quantification

with

1978.

P.C.J., L.A.,

are cytotoxic 3536, 1987.

K.M., von dem D. The 40-kDa

IgG-induced 17. Karpovsky, Production

Guyre,

for

J.C.

serum IgA, secretory 25, 527, 1988.

TW.J.,

trophils

389,

Leijh, Es,

M.W.

G.J.,

Es,

by human Immuno!.

16. Huizinga,

24,

S. aureus

479, 1977. P.S., Biewenga,

A.,

OtoL

human 23.

and

and

LeBouteiller,

cells

28. and

G.M.,Jakab,

15. Hiemstra,

expression

one-step

Unkeless, structure.

poiymorphonuclear

and granulocytes j Immunol. 139,

mechanisms Dz.t. 115,

van

IgA-opsonized

1987. R.F.

14. Green,

3640,

cell

160,

IgG. j Immuno!. 136, 1641, 21. Maliszewski, C.R., March, S., and Shen, L. Expression IgA. j Exp. Med. 172, 1665, 22. Mestecky, J., Russell, M.W.,

1982.

A., Hiemstra, Barselaar,

secretory

and and

77,

Cytofluorographic

R.F.,

A rapid

Methods

USA.

and

L.

ex-

polymorphonuclear

and polymorphonuclear a modification of the

Immuno!.

and

Shen,

USA.

Fc receptors

Y.H.

Wright, S.D., distribution

Gorter, van den

Sci.

monocytes

of receptor 1019, 1983.

by human

Fleit, H.B., Fc receptor 79,

Acad.

20, Graziano,

of mononuclear blood using

j

and

human

correlation

Today 10, 92, 1989. 10. Ferrante, A., and Thong, purification from human

and on

Mo!. Immunol. Shen, L., mediated

and

Nat!.

20.

anti-target

Med.

Exp.

secretion

molecules.

to clearance

containing j

Y.,

nasal

Ann. 19.

Hepatobiliary

15, 893, 1985. Bernier, G.M. Subpopu-

granulocytes

Goldstine,

Cytotoxicity

J.,

Pugh,

IgA.

receptors

MW.,

technique.

Kurono, in

oxidase

ImmunoL

j

Vaerman, J.P. contribution

Immuno!.

peripheral for

and monocytes phagocytosis.

378

NADPH

mechanisms.

Eur. j

IgA. Shen,

receptors

analysis

man, Roos,

insoluble

F.M. DL., Malburny, of plasma IgA

lations

13.

and

neutrophil

receptor-specific

of intravascular 7. Fanger, MW.,

12.

Soluble

aggregates antibodies.

tor

1990.

6. Delacroix, transport

11.

linked 18.

E.,

distinct

3026,

in BioSection

by secretory disposal. &ience

Willis, Ruddy,

and superoxide 1988.

of

function.

Solomon,

receptors generation

My43, cells, j

A.,

and

system common 121, 101, 1965. R.J. Inhibition

immunoglobulin 177, 697, 1972.

HE., Browder, S. Monocional

Cross-linking

MW. myeloid

B., Feister, antibody

a monoclonal inhibits mono-

Immuno!.

inducers

R.A.

to certain of

exter-

bacterial

A: a mechanism

lysozomal

4117,

Prendergast,

A.J., Mohanakumar, to human IgG

by neutrophils.j

143,

ad-

of antiT.,

and

Fc receptors.

enzyme

ImmunoL

release

140,

234,