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The Journal of Immunology, 2002, 169: 3831-3836.
Copyright © 2002 by The American Association of Immunologists

Mac-1 (CD11b/CD18) as Accessory Molecule for Fc{alpha}R (CD89) Binding of IgA1

Annemiek B. van Spriel*,{dagger}, Jeanette H. W. Leusen*, Henriëtte Vilé* and Jan G. J. van de Winkel2,*,{ddagger}

* Immunotherapy Laboratory, Department of Immunology, University Medical Center, {dagger} Medarex Europe, and {ddagger} Genmab, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IgA, the principal ligand for Fc{alpha}RI, exists in serum as monomeric IgA and at mucosal sites as secretory IgA (SIgA). SIgA consists of dimeric IgA linked by joining chain and secretory components. Human polymorphonuclear leukocytes (PMN) and mouse PMN transgenic for human Fc{alpha}RI exhibited spreading and elicited respiratory burst activity upon interaction with either serum or SIgA. However, PMN devoid of the {beta}2 integrin Mac-1 (Mac-1-/-) were unable to bind SIgA, despite expression of Fc{alpha}RI. Consistent with this, serum IgA stimulated Mac-1-/- PMN oxygen radical production, in contrast to SIgA. Binding studies showed the secretory component, by itself, to interact with Mac-1-expressing PMN, but not with Mac-1-/- PMN. These data demonstrate an essential role for Mac-1 in establishing SIgA-Fc{alpha}RI interactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Immunoglobulin A, representing the predominant Ab isotype in humans, is present in the circulation primarily in monomeric form (serum IgA), and in mucosal secretions as dimeric complexes, called secretory IgA (SIgA)3 (1). SIgA exists as dimeric IgA (dIgA) containing an additional joining chain and secretory component (SC), which has been proposed to increase IgA stability. Serum IgA is produced by splenic and bone marrow-derived plasma cells. Mucosal plasma cells in the lamina propria produce joining chain-associated IgA dimers (dIgA), which bind with high affinity to the polymeric IgR (pIgR) present on the basolateral surface of mucosal epithelium (2). Subsequently, dIgA-pIgR complexes are endocytosed and transported to the mucosal lumen. SC, a portion of the pIgR, is cleaved off during this transepithelial transport, leading to local SIgA production (3).

Fc{alpha}RI (CD89) is the IgA receptor, which is uniquely expressed on polymorphonuclear leukocytes (PMN), monocytes, eosinophils, and select macrophages (4, 5). Fc{alpha}RI binds serum and SIgA with low affinity (Ka, ~106 M-1) (6). Fc{alpha}RI expression and function can be up-regulated by fMLP, GM-CSF, IL-8, IL-1{beta}, TNF-{alpha}, and LPS, and down-regulated by TGF-{beta}. The Fc{alpha}RI ligand-binding chain contains a short cytoplasmic tail and its expression and function are dependent on FcR {gamma}-chains, which contain specialized immunoreceptor tyrosine-based activation motifs (7).

Serum IgA can initiate numerous immune effector functions via Fc{alpha}RI, which include phagocytosis, cytotoxicity, respiratory burst activity, release of inflammatory mediators, and Ag presentation (4, 5, 8). Moreover, serum IgA-Fc{alpha}RI interactions on Kupffer cells may provide a "second-line" of defense in mucosal immunity (9).

SIgA is considered crucial for the "first-line" mucosal defense (10, 11, 12), however, its precise in vivo role remains unclear. SIgA has been shown to interact with microorganisms and to block virulence factors, which may prevent microbial invasion. Furthermore, SIgA can trigger phagocyte respiratory burst activity, degranulation, and TNF-{alpha} production (13, 14, 15, 16, 17). Another role suggested for SIgA is neutralization of viruses within infected epithelia (18, 19). Significant correlations have been reported between specific titers of SIgA and resistance to infection (20).

PMN and macrophages are the major phagocytic populations at mucosal sites. Upon microbial invasion these phagocytes are recruited, increase receptor levels, and become activated (or "primed"). Fc and complement receptors are essential for the recognition and elimination of foreign targets. Mac-1 (CR3, CD11b/CD18, or {alpha}m{beta}2) is an important PMN complement receptor that binds multiple ligands, including C3bi, ICAM-1, fibrinogen, and {beta}-glucan. Mac-1 is important for leukocyte adhesion, migration, phagocytosis, cytotoxicity, and chemotaxis (21). Furthermore, Mac-1 has been proposed to act as "signaling partner" for other leukocyte receptors, including IgG receptors (22). Although Mac-1 has been implicated in FcR function, this is the first study demonstrating a crucial role for Mac-1 in FcR ligand binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

dIgA was purified from human serum by HPLC on HR200 Superdex size-exclusion columns (Pharmacia, San Diego, CA). Anti-CD11b mAb M1/70 (rat IgG2b) and 44a (mouse IgG1) were purified from supernatants of American Type Culture Collection hybridomas (Manassas, VA). Anti-CD11b mAb Bear-1 (mIgG1) was obtained from Caltag Laboratories (Burlingame, CA). mAb 14.1 (human IgG1, anti-Fc{alpha}RI) was produced by immunizing mice transgenic (Tg) for human Ig (23) with recombinant soluble Fc{alpha}RI (24) in Ribi adjuvant (Sigma-Aldrich, St. Louis, MO). Splenic lymphocyte suspensions were fused to P3 x 63-Ag8.653 mouse myeloma cells (American Type Culture Collection), and hybridomas were selected by ELISA and FACS on Fc{alpha}RI transfectants (7). mAb 14.1 recognizes Fc{alpha}RI within extracellular domain 2, outside the IgA-binding site. mAb My43 (IgM anti-Fc{alpha}RI) blocks the Fc{alpha}RI ligand-binding domain (25).

Mice

CD11b knockout mice (Mac-1-/-) in the C57BL/6 x 129SV background were kindly provided by Dr. T. N. Mayadas (Harvard Medical School, Boston, MA) (26, 27, 28). To study Mac-1 involvement in human Fc{alpha}RI function, mice were crossed with human Fc{alpha}RI (CD89) Tg FVB/N mice (27), yielding four different genotypes: nontransgenic (Ntg) Mac-1+/-, Ntg Mac-1-/-, Tg Mac-1+/-, and Tg Mac-1-/-. Mac-1-deficiency does not affect PMN FcR expression levels (27, 28).

PMN isolation

Human PMN were isolated from heparinized venous blood of healthy volunteers by Ficoll-Histopaque (Sigma-Aldrich) density gradient centrifugation. PMN purity determined by cytospin preparations exceeded 95%, and cell viability was >98%.

Before murine PMN isolation, mice were injected s.c. with 15 µg polyethylene-glycol G-CSF (kindly provided by Dr. J. Andresen; Amgen, Thousand Oaks, CA) to increase PMN numbers (29, 30). Blood was collected from the retro-orbital plexus 3 days later. Erythrocytes were removed by hypotonic lysis, followed by washing remaining leukocytes with RPMI 1640 medium (Life Technologies, Grand Island, NY) with 10% FCS. FACS analyses, performed on a FACScan (BD Biosciences, San Jose, CA), revealed leukocytes to consist of ~60% PMN, ~35% lymphocytes, ~3% monocytes, and ~1% eosinophils. Cell viability determined by trypan blue exclusion was always >95%.

PMN binding and spreading on IgA

To exclude interference with complement, all Ab preparations were heat-inactivated before use (30 min, 56°C). Glass slides (Menzel, Braunschweig, Germany) were coated with 0.5% (w/v) BSA (Boehringer Mannheim, Mannheim, Germany), 100 µg/ml human serum IgA (ICN Pharmaceuticals, Costa Mesa, CA), dIgA (kindly provided by Dr. C. van Kooten, Leiden University Medical Center, Leiden, The Netherlands), SIgA (Sigma-Aldrich), or IgG (CLB, Amsterdam, The Netherlands) for 3 h at 37°C, and rinsed with PBS. Isolated human or mouse PMN (2 x 105 cells) were incubated in RPMI 1640 medium (with 10% FCS) on coated slides for 10 or 30 min at 37°C. Cells were fixed in 3.7% paraformaldehyde, and stained for actin with phalloidin-FITC (1:200; Sigma-Aldrich), or stained for Fc{alpha}RI and Mac-1 (see Colocalization studies) for 30 min at 20°C. Samples were mounted in Mowiol (with 2.5% 1,4-diazobicyclo-[2.2.2]-octane), and PMN binding and spreading was analyzed by confocal laser scanning microscopy using a Leitz DMIRB fluorescence microscope (Leica, Voorburg, The Netherlands) interfaced with a Leica TCS4D confocal laser microscope (Heidelberg, Germany). Cell morphology was imaged just above (0.2 µm) the coated surfaces. PMN diameters were measured orthogonally, and then averaged. At least 30 PMN from three different experiments were analyzed. In Mac-1 blocking experiments, PMN were preincubated with 10 µg/ml anti-CD11b mAb for 30 min at 4°C before SIgA binding. In additional experiments, PMN were preincubated with 0.1 M N-acetyl-D-glucosamine (NADG; Sigma-Aldrich) for 10 min at 20°C, and plated on SIgA-coated slides in the presence of NADG. Cytochalasin D (Sigma-Aldrich) was used at 10 µg/ml to study the role of actin microfilament polymerization in Fc{alpha}RI-IgA binding.

Respiratory burst measurements

The luminol-ECL method was used for analysis of real-time respiratory burst activity. Polystyrene tubes were coated with PBS, 100 µg/ml serum IgA, or SIgA for 3 h at 37°C, and blocked with HEPES+ buffer (containing 20 mM HEPES, pH 7.4, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM NaH2PO4, 1 mM CaCl2, 5 mM glucose, and 0.5% (w/v) BSA) for 1 h at 37°C. Isolated mouse PMN (4 x 105 cells) in HEPES+ buffer were incubated in these tubes, and placed in a 953 LB Biolumat (Berthold, Wildbad, Germany). Luminol (150 µM) was injected in all tubes and light emission was recorded continuously for 30 min at 37°C. As a positive control, PMN were stimulated with 100 ng/ml PMA (Sigma-Aldrich), and PMN incubated with luminol only served as negative control. In blocking experiments, PMN were incubated with Fc{alpha}RI mAb My43 supernatant during the assay (25).

SC binding to cells

Purified recombinant human SC (kindly provided by Dr. B. Corthesy, State University Hospital, Lausanne, Switzerland) was incubated at 50 µg/ml with isolated human or mouse PMN (2 x 105 cells) for 60 min at 4°C. PMN were washed with FACS buffer (0.5% (w/v) BSA, 0.05% azide in PBS), incubated with mouse IgG1 anti-human SC (Sigma-Aldrich) (1:50) for 45 min at 4°C, washed, and incubated with FITC-labeled goat anti-mouse IgG F(ab')2 (Jackson ImmunoResearch Laboratories, West Grove, PA) (1:100) for 30 min at 4°C. Controls included PMN incubated with both mouse anti-human SC, and FITC-labeled goat anti-mouse IgG F(ab')2. FITC fluorescence of PMN was determined by flow cytometry.

Colocalization studies

Human or mouse PMN were incubated on BSA or IgA-coated glass slides for 15 min at 37°C, washed with PBS, and incubated with 20 µg/ml of mAb 14.1 and mAb M1/70 in PBS containing 0.2% (w/v) BSA and 5% rabbit serum (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. PMN were then fixed in 3.7% paraformaldehyde (30 min, 20°C), quenched with 20 mM NH4Cl in PBS (5 min, 20°C), washed, and incubated with F(ab')2 of FITC-labeled rabbit anti-human IgG (DAKO, Glostrup, Denmark) (1:50) and F(ab')2 of TRITC-labeled donkey anti-rat IgG (Jackson ImmunoResearch Laboratories) (1:125) in PBS with 0.2% BSA for 45 min at 20°C. Slides were washed, mounted, and analyzed by confocal microscopy (see above). As a control, colocalization between Fc{alpha}RI and the {beta}2 integrin LFA-1 (CD11a/CD18) was analyzed using mAb M17/4 (rat IgG2a anti-mouse CD11a), which was a kind gift from Dr. G. Kraal (Vrije Universiteit, Amsterdam, The Netherlands).

Statistical analyses

Data are expressed as means ± SD. Statistical significance was determined by two-tailed unpaired Student’s t tests. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Role of Mac-1 in Fc{alpha}RI-IgA binding

To assess Mac-1 involvement in Fc{alpha}RI-ligand binding, we studied PMN from human Fc{alpha}RI-Tg mice crossed with CD11b knock-out mice (Mac-1-/-). Because Fc{alpha}RI represents a low affinity IgAR, we analyzed PMN binding to IgA-coated surfaces. Tg Mac-1-expressing PMN efficiently bound and spread onto serum IgA (IgA), dIgA, and SIgA-coated slides within 10 min (Fig. 1Go, upper panels). PMN spreading capacity on IgA-coated surfaces varied among different IgA forms, and was quantified by measuring PMN diameters (18 ± 4.2 µm on serum IgA, 15 ± 3.3 µm on dIgA, and 14.6 ± 3.7 µm on SIgA). Tg Mac-1-/- PMN bound properly to serum IgA and dIgA, but did not interact with SIgA (Fig. 1Go, lower panels). Furthermore, Mac-1-/- PMN, exhibited impaired spreading on slides coated with serum IgA (12.1 ± 2.5 µm) or dIgA (11.9 ± 4.2 µm). PMN of Ntg Mac-1+/- littermates, expressing Mac-1, but not Fc{alpha}RI, served as controls and did not bind human IgA. These data were supported by studies with human PMN. Binding and spreading capacity of human PMN on serum IgA, dIgA, and SIgA was similar to that of Tg Mac-1+/- PMN. However, F(ab')2 of {alpha}-Mac-1 mAb inhibited spreading of human PMN on SIgA, but not on serum IgA slides (data not shown). In addition, all phagocytes bound IgG- or BSA-coated surfaces (Fig. 1Go), irrespective of Fc{alpha}RI/Mac-1 expression. Binding of both human and Tg mouse PMN to SIgA was abolished in the presence of cytochalasin D. These results indicate Mac-1 to be required for phagocyte-Fc{alpha}RI binding of SIgA.



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  		FIGURE 1. Mac-1 is crucial for Fc{alpha}RI-SIgA interaction. Isolated Tg Mac-1+/- (upper panels) and Tg Mac-1-/- (lower panels) PMN were incubated on BSA (control), serum IgA- (IgA), dIgA-, or SIgA-coated slides for 10 min at 37°C, and stained for actin. Cell morphology was imaged in x/y direction in the FITC (upper panel) and transmission (lower panel) channel by confocal microscopy. Experiments were repeated four times yielding comparable results. Cell diameters (mean micrometers ± SD) of Tg Mac-1+/- (upper panels) were 7.6 ± 1.0 (control), 18.0 ± 4.2 (serum IgA), 15 ± 3.3 (dIgA), 14.6 ± 3.7 (SIgA), and of Tg Mac-1-/- (lower panels) were 7.7 ± 1.6 (control), 12.1 ± 2.5 (serum IgA), and 11.9 ± 2.1 (dIgA). Bar represents 15 µm.

 
Respiratory burst activity

To study the significance of PMN interaction with immobilized IgA, we investigated respiratory burst activity of mouse (Ntg Mac-1+/-, Tg Mac-1+/-, Tg Mac-1-/-) and human PMN. Both serum IgA and SIgA induced comparable oxygen radical production in Tg Mac-1+/- PMN (Fig. 2Go, left graph). However, Tg Mac-1-/- PMN were stimulated by serum IgA, but not by SIgA (Fig. 2Go, middle graph). Importantly, Mac-1-expressing and Mac-1-deficient PMN exhibited similar respiratory burst activity in response to PMA. Ntg Mac-1-expressing PMN did not initiate respiratory activity in response to serum IgA or SIgA (Fig. 2Go, right graph). Human PMN exhibited comparable oxygen radical production upon interaction with serum IgA or SIgA, albeit that serum IgA-induced respiratory burst activity was significantly faster (data not shown). Blocking studies with My43, an IgM recognizing the Fc{alpha}RI ligand-binding domain (25), decreased oxygen radical production close to background levels (data not shown), showing IgA-induced respiratory burst to depend on interaction with Fc{alpha}RI. These data demonstrate requirement of both Fc{alpha}RI and Mac-1 for SIgA-induced respiratory burst activity.



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  		FIGURE 2. IgA-induced respiratory burst activity. Isolated PMN of Tg Mac-1+/-, Tg Mac-1-/- (left and middle graph) and Ntg Mac-1+/- mice (right graph) were incubated in HEPES (control, {blacktriangleup})-, serum IgA (IgA, •)-, or SIgA ({circ})-coated tubes. As positive control, PMN were stimulated with PMA (x). Oxygen radical production of PMN was measured in time by chemiluminescence detection. Experiments were performed four times yielding essentially identical results. Statistical analyses of areas below the curve verified Tg Mac-1+/- PMN to bind SIgA significantly better (p = 0.001) than Tg Mac-1-/- PMN.

 
Colocalization of Mac-1 and Fc{alpha}RI

Because our data pointed to an essential role for Mac-1 in Fc{alpha}RI-IgA interactions, we next studied the distribution of Mac-1 and Fc{alpha}RI in PMN membranes. Isolated Tg Mac-1+/- PMN were incubated on surfaces coated with BSA, serum IgA, or SIgA, whereupon Fc{alpha}RI and Mac-1 were stained with FITC (green), and TRITC (red), respectively. Tg Mac-1+/- PMN mediated spreading on both serum IgA (Fig. 3GoA, upper panel) and SIgA (Fig. 3GoA, middle panel). Fc{alpha}RI was expressed on the plasma membrane of PMN, but also in filipodia-like outgrowth of cells. A similar staining was found for Mac-1, and colocalization was indicated in yellow (Fig. 3Go; merged pictures). Tg Mac-1+/- PMN did not spread on BSA-coated slides, and exhibited overall membrane staining of Fc{alpha}RI and Mac-1 (Fig. 3GoA, lower panel). As a control, Fc{alpha}RI and {beta}2 integrin LFA-1 membrane staining was examined, which revealed no colocalization (Fig. 3GoB). Importantly, experiments performed with human PMN revealed Fc{alpha}RI and Mac-1 expression to colocalize as well (see below).



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  		FIGURE 3. Colocalization of Fc{alpha}RI and Mac-1 in PMN membranes. A, Tg Mac-1+/- PMN were incubated on immobilized serum IgA (upper panel), SIgA (middle panel), or BSA (lower panel) for 15 min at 37°C, stained for Fc{alpha}RI (green) and Mac-1 (red), and analyzed by confocal microscopy. Right panels show merges of the two signals (indicating receptor colocalization). B, Fc{alpha}RI (green) and {beta}2 integrin LFA-1 (red) localization in Tg Mac-1+/- PMN bound to IgA. Experiments were repeated four times yielding similar results. Bars represent 15 µm.

 
Mac-1 binds SC

Our results demonstrated only Mac-1-expressing PMN capable of binding SIgA-coated surfaces. Next, we evaluated the capacity of Mac-1+/- and Mac-1-/- PMN to bind aggregated IgA by flow cytometry. Ntg Mac-1+/- PMN were ineffective in binding serum IgA-complexes, but interacted well with SIgA complexes. On the contrary, Ntg Mac-1-deficient PMN were unable to bind SIgA complexes (data not shown). These observations pointed to a role for the SC in PMN-SIgA binding. Therefore, we examined the capacity of recombinant SC to interact with isolated human and mouse PMN. As shown in Fig. 4GoA, Mac-1-expressing PMN readily bound SC, irrespective of the presence of Fc{alpha}RI. However, binding of SC to Mac-1-deficient Tg and Ntg PMN, was abrogated. SC interacted with human PMN as well (Fig. 4GoB). These data show SC, either recombinant or present in its normal configuration (i.e., complexed with dIgA), to be capable of interacting with Mac-1.



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  		FIGURE 4. PMN binding of SC is dependent on Mac-1. Binding of recombinant SC to mouse (Tg Mac-1+/-, Tg Mac-1-/-, Ntg Mac-1+/-, and Ntg Mac-1-/-) PMN (A) and to human PMN (B) was assayed by flow cytometry. PMN were incubated with SC, mouse IgG1 anti-SC, and were stained with FITC-conjugated anti-mouse IgG (thick lines). Controls were incubated with mouse IgG1 anti-SC and FITC-conjugated anti-mouse IgG (thin lines). Representative histograms of one of three experiments are shown.

 
Involvement of Mac-1 lectin-binding site in SIgA binding

We next evaluated the region of Mac-1 involved in SIgA-Fc{alpha}RI binding. Human PMN were plated on immobilized serum IgA or SIgA in the absence (control) or presence of NADG, which interacts with the Mac-1 lectin-binding domain (31), and were stained for both Fc{alpha}RI and Mac-1 (Fig. 5Go). Human PMN exhibited spreading on both serum IgA and SIgA, like Tg Mac-1+/- PMN. Incubation of PMN with NADG resulted in a blockade of PMN binding to SIgA-coated surfaces, whereas binding to serum IgA was barely affected by NADG (Fig. 5Go, lower panel). The partial colocalization of Fc{alpha}RI and Mac-1 observed in control PMN bound to serum IgA-coated slides was decreased in the presence of NADG. PMN incubated on BSA-coated surfaces in the presence of NADG exhibited no change in binding. Furthermore, NADG impaired binding of Tg Mac-1+/- to SIgA, but not to serum IgA (data not shown). In contrast, Abs directed against the I-domain of Mac-1, although partly inhibiting PMN spreading, did not inhibit binding of human PMN to serum and SIgA (data not shown). These results implicate the Mac-1 lectin-binding domain to be involved in Fc{alpha}RI-SIgA interaction.



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  		FIGURE 5. Mac-1 lectin-binding site involved in Fc{alpha}RI-IgA interaction. Purified human PMN were plated on serum IgA- (left panels) or SIgA- (right panels) coated surfaces in the absence (upper panels) or presence (lower panels) of NADG for 15 min at 37°C. PMN were stained for membrane Fc{alpha}RI (green) and Mac-1 (red) expression, and analyzed by confocal microscopy. Pictures show overlays of the staining, and bars indicate 15 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The significance of IgA in immune defense is well-recognized, but incompletely understood (32). Although a number of studies focused on the molecular characterization of Fc{alpha}RI and IgA, relatively little is known about the biology of Fc{alpha}RI-IgA interactions. The present study documents a new role for Mac-1, a {beta}2 integrin, in ligand binding of Fc{alpha}RI. Mac-1 was found to be crucial for interaction of PMN Fc{alpha}RI with SIgA, and subsequent PMN activation. Our data support Mac-1 binding to SC to underlie this phenomenon, and implicate the Mac-1 lectin-binding domain to be involved.

Modulation of receptor-ligand interactions by accessory molecules is well-documented for a number of immune receptors, including cytokine receptors, integrins, and FcR (28, 33, 34, 35, 36). IgG binding to Fc{gamma}RI (CD64) and Fc{gamma}RIII (CD16a) is dependent on association with the FcR {gamma}-chain (36, 37, 38). In this study, Mac-1 is identified as a novel accessory protein, crucial for Fc{alpha}RI-SIgA interaction. In SIgA, SC is covalently ligated to the C{alpha}2 and C{alpha}3 domains within the dIgA Fc region (39), which may interfere with the affinity of Fc{alpha}RI for SIgA (40, 41, 42). The present study demonstrates SC binding to PMN in a Mac-1-dependent manner. When one appreciates the structure of SC, Mac-1 interaction with SC seems rational. SC represents a heavily glycosylated protein belonging to the Ig superfamily (3). Moreover, we observed Mac-1 lectin-binding domain, bearing carbohydrate-binding specificity (31), involved in PMN binding of SIgA.

Physical association between leukocyte membrane proteins (such as FcR-FcR {gamma}-chain, and Fc{gamma}RIIIb-Mac-1) has been documented before (22, 37, 38). Despite clear colocalization of Fc{alpha}RI and Mac-1, a direct Fc{alpha}RI-Mac-1 interaction was not apparent from immunoprecipitation experiments (our unpublished data).

Importantly, Mac-1 modulates PMN function upon IgA binding, as indicated by stimulation of a potent respiratory burst. Our data show requirement of both Fc{alpha}RI and Mac-1 for SIgA-induced respiratory burst activity, because Mac-1-deficient Tg PMN and Ntg PMN were unable to elicit oxygen radical production in response to SIgA. A role for Mac-1 in Fc{gamma}R functions, including respiratory burst has been shown before (28, 43, 44, 45). CD18 interactions with the actin cytoskeleton and associated proteins may enable Mac-1 signaling (46, 47). Furthermore, the present work is consistent with earlier studies documenting a role for {beta}2 integrins in superoxide production of eosinophils mediated by SIgA, and not serum IgA (48). SC by itself was described to activate eosinophil functions, but not PMN functions, which was proposed to relate to an additional (15 kDa) "SC receptor" on eosinophils (49). Indeed, we observed Mac-1-deficient eosinophils capable of interacting with both SC and SIgA (our unpublished data).

In conclusion, this study provides evidence for a crucial role of Mac-1 in phagocyte SIgA binding. Enhanced IgA responses upon incubation of PMN with GM-CSF, TNF-{alpha}, or IL-8 have been reported (50, 51, 52), although the underlying mechanisms remain to be addressed. We propose Mac-1 involvement in these phenomena to be likely, because of the well-recognized ability of these cytokines and chemokines to activate Mac-1 (53, 54, 55). Requirement of two molecules interacting with one ligand may provide immune effector cells with an extra way to regulate their activity. When "first-line" defense fails, mucosal inflammation leads to PMN recruitment and priming, accompanied by up-regulation of Mac-1, which may trigger more potent Fc{alpha}RI-mediated responses. We hypothesize Mac-1 to increase affinity of Fc{alpha}RI-SIgA interaction via binding of SC.


   Acknowledgments
 
We thank Tanya Mayadas for providing CD11b-deficient mice, Jacques Duijs and Cees van Kooten for human dIgA, and Bruce Wines and Jos van Strijp for critically reading the manuscript. Furthermore, we thank Arnout Gerritsen and Marc van Dijk for assistance in generating mAb 14.1, and Toon Hesp, Herma Boersma, and Sabine Versteeg for excellent animal care.


   Footnotes
 
1 This work was supported by the Netherlands Organization for Scientific Research (Fellow Grant No. 901-07-229, to J.H.W.L.). Back

2 Address correspondence and reprint requests to Dr. Jan G. J. van de Winkel, Immunotherapy Laboratory, Department of Immunology, University Medical Center, Room KC02.085.2, Lundlaan 6, 3584 EA, Utrecht, The Netherlands. E-mail address: JanvandeWinkel{at}aol.com Back

3 Abbreviations used in this paper: SIgA, secretory IgA; dIgA, dimeric IgA; SC, secretory component; pIgR, polymeric IgR; PMN, polymorphonuclear leukocyte; Tg transgenic; Ntg, nontransgenic; NADG, N-acetyl-D-glucosamine. Back

Received for publication December 7, 2001. Accepted for publication July 26, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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