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(Journal of Leukocyte Biology. 2002;72:685-691.)
© 2002 by Society for Leukocyte Biology

Stimulation of neutrophil apoptosis by immobilized IgA

Jorge Schettini, Gabriela Salamone, Analía Trevani, Silvina Raiden, Romina Gamberale, Mónica Vermeulen, Mirta Giordano and Jorge R. Geffner

Laboratory of Immunology, Institute of Hematologic Research, National Academy of Medicine, and Laboratory of Immunogenetics, Department of Microbiology, Buenos Aires University School of Medicine, Argentina

Correspondence: Jorge Geffner, Laboratorio de Inmunología, IIHEMA, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. E-mail: geffnerj{at}fibertel.com.ar


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the current study, we analyzed whether immunoglobulin A (IgA) is able to modulate neutrophil apoptosis. We found that culture of neutrophils on immobilized plasma IgA (iIgAp) or secretory IgA (iIgAs) induced a marked increase in apoptotic rates. By contrast, soluble IgAp, IgAs, or aggregated IgAp exerted no effect. Promotion of apoptosis by iIgA was almost completely prevented by blocking antibodies directed to CD18 or CD11b and was shown to be dependent on the activation of the respiratory burst as suggested by the ability of catalase to prevent apoptosis stimulation; the effect of azide, an heme enzyme inhibitor that significantly increased promotion of apoptosis by iIgA; and the inability of iIgA to stimulate apoptosis of neutrophils isolated from chronic granulomatous disease patients. Stimulation of neutrophil apoptosis by IgA might contribute to the control of inflammatory processes in certain autoimmune diseases such as IgA nephropathy in which tissue deposits of IgA or IgA containing immune complexes are found.

Key Words: human • Fc{alpha}R • immune complexes • secretory • aggregated


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis of neutrophils plays a critical role in the resolution of acute inflammation. Apoptosis, which represents an alternative fate to necrosis, not only determines neutrophil uptake by macrophages but also is associated with a loss of neutrophil functions such as chemotaxis, phagocytosis, stimulated shape change, degranulation, and respiratory burst [1 2 3 4 ]. Signals from the microenvironment can delay or accelerate apoptosis of neutrophils. In vitro studies have shown that granuloctye macrophage-colony stimulating factor (GM-CSF), interleukin-2, leukotriene B4, corticosteroids, and lipopolysaccharides delay apoptosis [5 6 7 8 9 10 11 ], and proteolytic enzymes, tumor necrosis factor {alpha}, bacteria, and virus accelerate neutrophil apoptosis [12 13 14 15 16 ].

Previous work has stated that activation of the receptor for the Fc portion of immunoglobulin G (IgG; Fc{gamma}R) modulates the apoptotic rate of different leukocyte populations. Activated natural killer cells undergo apoptosis as a consequence of treatment with aggregated IgG (aIgG) or anti-CD16 (anti-Fc{gamma}RIIIb) antibodies (Ab) [17 18 19 ]. Conversely, observations made in human eosinophils show that cross-linking Fc{gamma}RII by soluble ligands such as anti-Fc{gamma}RII antibodies or aIgG delays apoptosis. On the contrary, when these ligands were immobilized onto tissue-culture plates, a strong stimulation of apoptosis was observed [20 ]. We have recently described that the apoptotic rate of neutrophils can also be modulated through Fc{gamma}R. Different effects were observed, however, depending on the type of immune complex (IC) used. Thus, precipitating IC, antibody-coated erythrocytes, and immobilized IgG (iIgG) dramatically stimulate apoptosis, and aIgG and soluble IC delay apoptosis [21 ]. By contrast, observations made in human monocytes indicated that exposure to iIgG increases cell survival through a mechanism that involves autocrine production of M-CSF [22 ]. Taken together, these studies identify a new role for leukocyte Fc{gamma}R: the regulation of cell survival.

In contrast with the large body of evidence supporting that activation of Fc{gamma}R modulates survival of different leukocyte populations, there are no previous reports directed to analyze whether the receptors for the Fc portion of IgA (Fc{alpha}R) could play a similar function. It is well-established that human neutrophils but not murine neutrophils constitutively express Fc{alpha}R, which have a medium affinity (Ka=5x107 mol/L-1) for IgA1, IgA2, and secretory IgA (IgAs) [23 , 24 ]. In vitro studies have shown that aIgA or IC prepared with IgA antibodies stimulates different inflammatory responses in neutrophils such as activation of the respiratory burst, phagocytosis, and degranulation [25 26 27 ]. Moreover, recent studies have demonstrated that IgA antibodies enable neutrophils to efficiently destroy a variety of tumor cells [28 , 29 ]. In the present report, we examined whether IgA regulates neutrophil survival. We found that iIgA stimulates neutrophil apoptosis through a mechanism, depending on the participation of Mac-1 and the activation of the respiratory burst.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The following agents were used: human IgG, acridine orange, ethidium bromide, propidium iodide (PI), catalase (from bovine liver; 50,000 U/mg protein), superoxide dismutase (SOD; from bovine erythrocytes; 5000 U/mg protein), and IgG1 from murine myeloma (Sigma Chemical Co., St. Louis, MO). Human plasma IgA (IgAp) and IgAs were obtained from Sigma Chemical Co. or ICN Pharmaceuticals (Aurora, OH). Electrophoresis of the IgA preparations followed by diffusion versus anti-human whole serum resulted in a single arc of precipitation. By polyacrylamide gel electrophoresis procedures, IgA was found to be at least 98% pure. The preparations of IgA were assessed for endotoxin contamination by using the Limulus amoebocyte lysate chromogenic assay (Coatest, Chromogenix, Sweden). Endotoxin content was below 25 pg/ml in all cases. The following immobilized proteins were used: iIgAp, iIgAs, iIgG, and immobilized human serum albumin (iHSA). They were prepared by incubating microplates (96-well flat-bottom, Corning Inc., Corning, NY) with IgA (100 µg/ml in saline), IgG (100 µg/ml), or HSA (1 mg/ml) for 18 h at 37°C. Before use, plates were washed six times with saline. Heat-aIgAp was prepared by heating IgAp for 20 min at 63°C, and it was characterized by size-exclusion high-pressure liquid chromatography, as previously described [30 ]. The fraction containing molecular aggregates of weight >=1,000,000 was used as aIgAp. The following blocking Ab were used: F(ab')2 fragment of IgG1 anti-CD18 (from supernatants of hybridoma TS 1/18, American Type Culture Collection, Manassas, VA), IgM anti-CD11b (Mo1, Immunotech, Marseille, France), anti-Fc{gamma}RII and anti-Fc{gamma}RIII [IV3 Fab and 3G8 F(ab')2, respectively Medarex, West Lebanon, NH), IgG1 anti-Fas ligand (FasL; NOK-1, Pharmingen, San Diego, CA), and IgG1 anti-Fas (ZB4, Immunotech). For blocking studies, neutrophils were preincubated with the corresponding monoclonal Ab (mAb) during 30 min at 4°C. Concentrations of mAb three- to fivefold higher than those needed to saturate all binding sites (1–10 µg/ml), as determined by fluorescein-activated cell sorter (FACS) analysis, were used in these studies. IgM anti-Fas mAb (CH-11) and IgG1 anti-FasL (G247-4) were from Pharmingen.

Blood samples
Blood samples were obtained from healthy donors who had taken no medication for at least 10 days before the day of sampling. Blood was obtained by venipuncture of the forearm vein, and it was drawn directly into heparinized plastic tubes.

Neutrophil isolation
Neutrophils were isolated by Ficoll-Hypaque gradient centrifugation (Ficoll Pharmacia, Upsala, Sweden; Hypake, Winthrop Products Inc., Buenos Aires, Argentina) and dextran sedimentation as described [31 ]. Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (more than 96% of neutrophils on May Grunwald/Giemsa-stained cytopreps) were resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 1% fetal calf serum (FCS; Gibco Laboratories, Grand Island, NY).

Quantitation of cellular apoptosis and viability by fluorescence microscopy
Unless otherwise stated, isolated neutrophils (2.5x106/ml) were cultured under different conditions in 96-well flat-bottom microplates, and apoptosis was evaluated after 18 h of incubation. In a separate set of experiments, the effect of IgA on neutrophil apoptosis was evaluated in whole blood cultures. To this aim, whole blood was diluted (20% v/v) in RPMI medium, and aliquots of 0.10 ml of this suspension were placed in 96-well flat-bottom microplates, which have been treated or not with IgAp, IgAs, or HSA, as described below. Quantitation of apoptosis was performed as previously described [32 ], using the fluorescent DNA-binding dyes acridine orange (100 µg/ml) to determine the percentage of cells that had undergone apoptosis and ethidium bromide (100 µg/ml) to differentiate between viable and nonviable cells. With this method, nonapoptotic cell nuclei show variations in fluorescent intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridine orange. To assess the percentage of cells showing morphologic features of apoptosis, at least 200 cells were scored in each experiment.

Quantitation of neutrophil apoptosis by annexin-V binding and flow cytometry
Annexin-V binding to neutrophils was performed using an apoptosis detection kit (Immunotech). Cells were labeled with annexin-V fluorescein isothiocyanate (FITC) and PI for 20 min at 4°C and were analyzed by two-color flow cytometry (FACScan flow cytometer, Becton-Dickinson Immunocytometry System, San Jose, CA), using CellQuest analysis software (Becton-Dickinson) as previously described [33 ]. Results are reported as percentage of annexin-V-positive cells.

Quantitation of neutrophil apoptosis by PI staining and flow cytometry
The proportion of neutrophils that display a hypodiploid DNA peak, i.e., apoptotic cells, was determined using a modification of the Nicoletti et al. protocol [34 ]. Briefly, cell pellets containing 2.5 x 106 neutrophils were suspended in 400 µl hypotonic fluorochrome solution (PI, 50 µg/ml in 0.1% sodium citrate plus 0.1% Triton X-100) and were incubated for 2 h at 4°C. The red fluorescence of PI of individual nuclei was measured using a FACScan flow cytometer (Becton-Dickinson). The forward-scatter and side-scatter of particles were simultaneously measured. Cell debris was excluded from analysis by appropriately raising the forward-scatter threshold. The red fluorescence peak of neutrophils with normal (diplod) DNA content was set at channel 1000 in the logarithmic mode.

Superoxide anion generation
Neutrophil O2- generation was assessed by the method of SOD-inhibitable reduction of cytochrome c. Briefly, neutrophils (2.5x106/ml), suspended in RPMI-1640 medium (without phenol red) and supplemented with 1% FCS and 75 µM ferricytochrome c, were placed in 96-well flat-bottom plates that have been coated or not with pIgA, as described above. After incubation for 45 min at 37°C in the presence or absence of SOD (10 µg/ml), reduction of cytochrome C was quantified using an extinction coefficient of 21 mM-1 cm-1 at 550 nm in a Hewlett Packard spectrophotometer.

Statistical analyses
Statistical analyses were performed by using one-way ANOVA, followed by a Dunnett’s test (see data from Figs. 4 and 6 ), or Student’s paired t-test, and P < 0.05 was taken as indicating statistical significance.



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  		Figure 4. Promotion of neutrophil apoptosis by iIgA involves Mac-1. Neutrophils (2.5x106/ml) were cultured for 30 min at 4°C alone (open bars) or in the presence of saturating concentrations of blocking mAb directed to CD18 [TS 1/18 F(ab)2 (hatched bars)] or CD11b (Mo1, cross-hatched bars). Then, they were placed on iIgAp or iIgAs or were cultured with PMA (5 ng/ml). Apoptosis was revealed by fluorescence microscopy after 18 h of incubation at 37°C. Results are expressed as the mean ± SEM of six experiments. *, Statistical significance (P<0.01) compared with cells cultured on iIgAp or iIgAs in the absence of anti-Mac-1 Ab.

 


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  		Figure 6. Effect of catalase, SOD, and sodium azide on the stimulation of neutrophil apoptosis by iIgA. Neutrophils (2.5x106/ml) were cultured on iIgAp in the absence (open bars) or presence (hatched bars) of catalase (200 U/ml), SOD (200 U/ml; cross hatched bars), or sodium azide (0.1 mM; solid bars). Apoptosis was revealed by fluorescence microscopy after 18 h of incubation at 37°C. Results are expressed as the mean ± SEM of six experiments. *, Statistical significance (P<0.05) compared with cells cultured on iIgAp in the absence of catalase or sodium azide.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
iIgAp and iIgAs stimulate neutrophil apoptosis
Neutrophils were cultured on untreated plates in the presence of soluble IgAp, IgAs, or aIgAp (100 µg/ml), as well as on plates that had been coated with iIgAp, iIgAs, or iHSA, as described in Materials and Methods. Apoptosis was revealed after 18 h of culture at 37°C by fluorescence microscopy using the fluorescent DNA-binding dye acridine orange. As shown in Figure 1 , treatment with iIgAp or iIgAs markedly increased the apoptotic rate of neutrophils, and no effects were observed using IgAp, IgAs, aIgAp, or iHSA. To confirm the inability of IgAp, IgAs, and aIgAp to modify the rate of neutrophil apoptosis, additional experiments were performed in which apoptosis was revealed by fluorescence microscopy after 36 h of culture. In agreement with the results described above, we found similar levels of apoptosis for controls and IgA-treated cells: % Apoptosis = 55 ± 7, 49 ± 8, 52 ± 7, and 53 ± 6 for controls and IgAp-, IgAs-, and aIgAp (100 µg/ml)-treated cells, respectively (mean±SEM, n=4–5).



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  		Figure 1. Promotion of neutrophil apoptosis by iIgA. Neutrophils (2.5x106/ml) were cultured for 18 h at 37°C in the presence of soluble IgAp, IgAs, or aIgAp (100 µg/ml), as well as on iIgAp, iIgAs, or iHSA. Then, the percentage of apoptotic cell was determined by fluorescence microscopy. Results are expressed as the mean ± SEM of seven experiments. *, Statistical significance (P<0.01) compared with the control.

 
To analyze whether our results could be related to the induction of cell injury or "priming" during neutrophil purification, we performed additional experiments in whole blood cultures, as described in Materials and Methods. Diluted blood samples (20% v/v in RPMI medium) were cultured on plates coated with IgAp, IgAs, or HSA, and apoptosis was revealed after 18 h by fluorescence microscopy. Cultures of whole blood on untreated plates served as controls. In agreement with our findings in purified neutrophils, we observed that iIgAp and iIgAs induced a marked increase in the apoptotic rate of neutrophils: % Apoptosis = 9 ± 7, 46 ± 8, 52 ± 7, and 11 ± 4 for controls and iIgAp-, iIgAs-, and iHSA-treated cells, respectively (mean±SEM, n=4, P < 0.01 controls vs. iIgAp or iIgAs).

During the course of apoptosis, phosphatidylserine (PS), a negatively charged phospholipid, becomes exposed at the cell surface [33 ]. The ability of iIgA to stimulate apoptosis was then confirmed by flow cytometry using FITC-labeled annexin-V, which specifically binds to PS in combination with PI staining. Figure 2A shows that when neutrophils were cultured with iIgAp, a marked increase in the number of apoptotic cells was observed. Apoptosis was also examined by PI staining and flow cytometry. In agreement with the observations described above, we found that treatment with iIgAp triggers a strong apoptotic response, as measured by the increased proportion of neutrophils that display a hypodiploid DNA peak (Fig. 2B) .



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  		Figure 2. Promotion of neutrophil apoptosis by iIgA analyzed by annexin-V-binding assay and PI staining. Neutrophils (2.5x106/ml) were cultured for 18 h at 37°C in the absence (left) or presence (right) of iIgAp. Then, the percentage of apoptotic cells was analyzed by the annexin-V-binding assay (A) or PI staining (B). A representative experiment is shown (n=4–5).

 
To rule out the possibility that the presence of contaminanting IgG in IgA preparations would account, at least in part, for the stimulation of neutrophil apoptosis by iIgAp or iIgAs, experiments were carried out using cells pretreated with saturating concentrations of blocking Ab directed to Fc{gamma}RIIa (IV.3 Fab) and Fc{gamma}RIIIb (3G8 Fab2), the two classes of Fc{gamma}R constitutively expressed by neutrophils [35 ]. As shown in Figure 3 , these mAb almost completely prevented stimulation of apoptosis by iIgG without affecting that induced by iIgAp or iIgAs.



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  		Figure 3. Blocking Fc{gamma}R does not prevent promotion of neutrophil apoptosis by iIgA. Neutrophils (2.5x106/ml) were cultured for 30 min at 4°C alone (open bars) or in the presence of saturating concentrations of IV.3 Fab (anti-Fc{alpha}RII) and 3G8 F(ab)2 (anti-Fc{gamma}RIII) mAb (hatched bars). Then, they were cultured on iIgG, iIgAp, or iIgAs, and apoptosis was revealed by fluorescence microscopy after 18 h of incubation at 37°C. Results are expressed as the mean ± SEM of five experiments. *, Statistical significance (P<0.01) compared with cells cultured on iIgG in the absence of Ab directed to Fc{gamma}R.

 
Promotion of neutrophil apoptosis by iIgAp and iIgAs involves Mac-1
Mac-1 (CR3, CD11b/CD18) is the predominant ß2 integrin expressed by neutrophils [36 ]. Previous work has demonstrated that Mac-1 cooperates with Fc{gamma}R and Fc{alpha}R in the induction of neutrophil responses triggered by IC [37 , 38 ]. Taking this into account and considering the ability of Mac-1 to promote apoptosis of activated neutrophils [39 ], we analyzed whether stimulation of apoptosis by IgA involved a Mac-1-dependent pathway. Experiments were performed using neutrophils treated with saturating concentrations of blocking mAb directed to CD18 [TS 1/18, F(ab')2 fragment] or CD11b (Mo1). Figure 4 shows that both mAb almost completely prevent acceleration of apoptosis triggered by iIgAp and iIgAs, without affecting the increase in the apoptotic rate of neutrophils induced by phorbol 12-myristate 13-acetate (PMA). Mouse IgG1 F(ab')2 from a murine myeloma used as irrelevant control did not exert any effect on the acceleration of apoptosis triggered by iIgAp and iIgAs (not shown).

Promotion of neutrophil apoptosis by iIgAp does not involve the Fas/FasL system
Neutrophils appear constitutively to express not only Fas but also FasL. Coexpression of these molecules appears to be responsible, at least in part, for the rapid rate of spontaneous apoptosis of neutrophils [40 ]. To explore whether the Fas/FasL system was involved in the stimulation of apoptosis induced by iIgA, we first analyzed the expression of these molecules in cells cultured for 6 h with iIgAp by using the mAb CH-11 directed to Fas and the mAb G247-4 directed to FasL. Figure 5 shows that there were no differences in the expression of Fas or FasL between untreated and iIgAp-treated cells. Moreover, we also found that when neutrophils were cultured for 18 h with iIgAp in the presence of blocking mAb directed to FasL (NOK-1) or Fas (ZB4), there was no prevention of apoptosis, as the percentage of apoptotic cells = 61 ± 11, 64 ± 10, and 58 ± 8% for iIgAp-treated neutrophils cultured in the absence or presence of the mAb NOK-1 and ZB4, respectively (mean±SEM, n=3).



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  		Figure 5. Expression of Fas and FasL in neutrophils cultured on iIgA. Neutrophils (2.5x106/ml) were cultured in the absence or presence of iIgAp for 6 h at 37°C. Then, the expression of Fas (left) and FasL (right) was evaluated by flow cytometry. Histograms of a representative experiment (n=3–4) are depicted.

 
Promotion of neutrophil apoptosis by ilgAp is dependent on the activation of the respiratory burst that involves Mac-1
We have previously shown that stimulation of neutrophil apoptosis by IgG-immune complexes is dependent on the generation of oxygen-reactive intermediates (ROI) [21 ]. To analyze whether stimulation of apoptosis by IgA also involved an oxidative-dependent pathway, we used the scavenger enzymes catalase and SOD. Moreover, we assessed the effect of the heme enzyme inhibitor sodium azide, as it has been previously found that inhibition of neutrophil catalase and myeloperoxidase activities by these compounds increases the production of hydrogen peroxide during neutrophil activation [41 , 42 ]. Figure 6 shows that catalase but not SOD almost completely prevented stimulation of neutrophil apoptosis by iIgAp, suggesting a critical role for hydrogen peroxide. Supporting this possibility, we also observed that sodium azide did not modify the spontaneous rate of apoptosis and induced a significant increase in the apoptotic rate of iIgAp-treated cells.

To further analyze whether the production of ROI was involved in the promotion of neutrophil apoptosis induced by iIgAp, we performed a new set of experiments using neutrophils isolated from two patients with chronic granulomatous disease (CGD), a rare hereditary disorder characterized by a diminished or absent production of ROI as a result of a defect in any one of the components of reduced nicotinamide adenine dinucleotide phosphate oxidase [43 ]. As expected, neutrophils isolated from these two patients produced O2- levels <1% of those produced by normal cells (not shown) in response to formyl-Met-Leu-Phe or zymosan. In contrast with the observations made in control neutrophils, we found that iIgAp was unable to promote apoptosis of CGD neutrophils, as apoptotic rates were similar in untreated or iIgAp-treated cells: apoptosis after 18 h of culture, 15 ± 8 versus 19 ± 6, respectively (mean±SEM, n=2).

We hypothesized that the role that Mac-1 plays in the stimulation of neutrophil apoptosis by iIgA could be related, at least in part, to its participation in the induction of the respiratory burst. To analyze this point, we performed another set of experiments to establish whether blockade of Mac-1 inhibited neutrophil production of O2- triggered by iIgAp. Untreated neutrophils or neutrophils treated with a blocking mAb directed to CD18 [TS 1/18, F(ab')2] were placed on plates coated with iIgAp, and the production of O2- was measured as described in Materials and Methods. Our results showed a marked inhibition in the generation of O2- for CD18-blocked neutrophils compared with unblocked cells: % Suppression = 78 ± 12 (mean±SEM, n=4, P<0.01). No inhibition was observed in cells treated with mouse IgG1 F(ab')2 used as irrelevant control (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present report, we demonstrate that iIgA promotes neutrophil apoptosis through a mechanism dependent on Mac-1 cooperation, which involves the activation of the respiratory burst. These results suggest that Fc{alpha}R can mediate not only the delivery of activating signals involved in inflammatory and cytotoxic functions but also in the control of cell survival. Stimulation of apoptosis was induced in a similar manner by IgAp or IgAs, even when suboptimal amounts of iIgA were used (unpublished results), suggesting that the secretory component does not modify the ability of IgA to promote neutrophil apoptosis. This observation is consistent with previous reports, which showed that in contrast to what was found in eosinophils, the secretory component is unable to stimulate effector functions of neutrophils [44 ].

We have previously shown that soluble IC and aIgG significantly delayed spontaneous apoptosis [21 ]. In the present work, we observed that neither soluble IgAp, IgAs, nor aIgA was able to delay apoptosis. Recent observations demonstrated that cross-linking Fc{gamma}RIIIB by specific mAb induces the production of G-CSF and GM-CSF by human neutrophils [45 ]. Consistent with these findings, we found that the expression of mRNA for GM-CSF is induced in neutrophils cultured with aIgG. By contrast, no induction in the expression of mRNA for GM-CSF was observed using aIgA as stimulus (unpublished results). Thus, contrasting results concerning the ability of aIgG and aIgA to prevent apoptosis could be related to their different abilities to trigger the production of antiapoptotic cytokines.

The fact that blocking mAb directed to CD18 or CD11b almost completely prevent stimulation of apoptosis by iIgA indicates that this response requires Mac-1 cooperation. Mac-1 (CR3, CD11b/CD18) represents the predominant ß2 integrin on polymorphonuclear neutrophils. This family of adhesion molecules, which also includes lymphocyte function-associated antigen-1 (CD11a/CD18) and gp150/95 (CD11c/CD18 or CR4), shares a common ß subunit that is noncovalently associated with unique but closely related {alpha} chains [36 ]. Mac-1 is not only able to initiate signaling, which results in neutrophil responses such as adhesion, phagocytosis, respiratory burst, and degranulation, but it has also been shown that Mac-1 acts as a signaling partner for other receptors including Fc receptors [36 37 38 , 46 ]. Findings from van de Winkel and co-workers [38 , 47 ] in transgenic mice expressing human Fc{alpha}R and in human neutrophils treated with blocking mAb directed to CD11b and CD18 demonstrated that Mac-1 is essential for Fc{alpha}R-mediated neutrophil cytotoxicity against tumor targets but not for Fc{alpha}R-mediated phagocytosis of Ab-coated particles. The mechanisms underlying the participation of Mac-1 in cytotoxicity have not been defined; however, the authors demonstrated that in the absence of Mac-1, neutrophils exhibit defective spreading on Ab-coated tumor cells [38 ]. Consistent with this result, we found that treatment with blocking mAb directed to Mac-1 inhibits not only the spreading of neutrophils on IgA-coated plates (not shown) but also the activation of the respiratory burst. It is interesting that this finding suggests that the participation of Mac-1 in the stimulation of neutrophil apoptosis by iIgA could be explained, at least in part, by the important role that Mac-1 plays in the activation of the respiratory burst.

Different studies have demonstrated the ability of Fc{gamma}R to modulate survival of neutrophils, eosinophils, or monocytes [20 21 22 , 43 , 48 ]. The ability of the high-affinity IgE receptor to modulate the apoptosis of human monocytes has also been demonstrated recently [49 ]. Our present work provides evidence indicating that Fc{alpha}R effectively promotes neutrophil apoptosis. Together, these observations suggest that FcR might modulate the course of inflammation, not only by virtue of their ability to trigger inflammatory responses mediated by phagocytic cells but also by their capacity to modulate the apoptotic rate of these cells.

IgA nephropathy (IgAN) is thought to be the most common, primary glomerulonephritis in the world [50 , 51 ]. This disease is characterized by predominant IgA deposits in the glomerular mesangium, which are able to trigger the activation of different inflammatory responses [50 51 52 53 ]. Our present results suggest that IgA deposits may also exert a regulatory activity in the course of IgAN by virtue of their ability to promote apoptosis of neutrophils, cells that have been involved in the development of the inflammatory injury in IgAN, especially during the active phase of the disease [54 , 55 ].


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires University School of Medicine, Agencia Nacional de Promoción Científica y Tecnológica (FONCyT), and Alberto J. Roemmers Foundation (Buenos Aires, Argentina). The authors thank Selma Tolosa and Nelly Villagra for their technical assistance and Maria Rita Furnkorn for her secretarial assistance.

Received April 4, 2002; revised May 22, 2002; accepted June 13, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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