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(Journal of Leukocyte Biology. 2001;69:233-240.)
© 2001 by Society for Leukocyte Biology

Differential effects of anti-Fc{gamma}RIIIb autoantibodies on polymorphonuclear neutrophil apoptosis and function

Véronique Durand*, Jacques-Olivier Pers*, Yves Renaudineau*, Alain Saraux{dagger}, Pierre Youinou* and Christophe Jamin*

* Laboratory of Immunology
{dagger} Department of Rheumatology, Institut de Synergie des Sciences et de la Santé, Brest University Medical School, Brest, France

Correspondence: P. Youinou, M.D., D.Sc., Laboratory of Immunology, Brest University Medical School Hospital, 5 av Foch, F 29 609 Brest Cedex, France. E-mail: youinou{at}univ-brest.fr


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ABSTRACT
 
Anti-Fc{gamma} receptor IIIb (Fc{gamma}RIIIb) human autoantibodies (Ab) have been classified previously into three groups, based on the results of an indirect immunofluorescence (IIF) test and an enzyme-linked immunosorbent assay (ELISA): IIF+/ELISA+ (group A), IIF+/ELISA- (group B), and IIF-/ELISA+ (group C) sera. In this study, differential effects between IIF+ autoAb, recognizing cell-bound Fc{gamma}R, and those ELISA+, recognizing only cell-free Fc{gamma}R, were studied on polymorphonuclear neutrophils (PMN). Neither group A nor B autoAb was cytotoxic, although both prolonged the survival of PMN by delaying spontaneous apoptosis. By the same extent, the PMN-binding antisera stimulated the appearance of a CD11bdim population, following a 12-h incubation. This event was associated with a lowered expression of ß2 integrin molecules, resulting in altered PMN function. Treatment with groups A and B autoAb reduced adhesiveness and respiratory burst. This impairment of the responses was more pronounced when the cells originated from donors NA1+NA1+ rather than donors NA2+NA2+. From our observations, the influences of anti-Fc{gamma}RIIIb autoAb on PMN survival, as well as function and subsequent dysregulation of the inflammatory response, have proven somewhat dependent on their target antigens, as determined by IIF coupled with ELISA and Fc{gamma}RIIIb polymorphism.

Key Words: IIF test • autoimmunity • ELISA • endothelial cells


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INTRODUCTION
 
Polymorphonuclear neutrophils (PMN), which play the major role in inflammation, can be activated by many agents, such as chemotactic molecules, cytokines, or immune complexes (IC). For example, IC elicit PMN responses via Fc{gamma} receptor (Fc{gamma}R)-transduced signals [1 ]. These cells express two classes of low-affinity Fc{gamma}R [2 ]: Fc{gamma}RII (CD32) and Fc{gamma}RIIIb (CD16) with different implications for disease [3 ]. Among multiple isoforms for Fc{gamma}R [1 ], there exist at least two major allotypes for Fc{gamma}RIIIb, NA1 and NA2, which are referred to as glycoforms [4 ]. Although Fc{gamma}RII is a transmembrane protein [5 ], thus able to convey a signal across the membrane, Fc{gamma}RIIIb is a glycophosphatidylinositol (GPI)-anchored glycoprotein [6 ], the ability of which to transduce a signal directly remains a matter of controversy. Several groups have shown that Fc{gamma}RIIIb alone is not competent for signal transduction [7 8 9 ] but rather captures IC and then links to Fc{gamma}RII, which induces the signal resulting in activation. Their observations are at variance with other studies indicating that Fc{gamma}RIIIb can transduce signals by itself [10 , 11 ].

Activation of PMN leads to the contact between PMN and endothelial cells (EC), which is initiated by L-selectin (CD62L), followed by the firm adhesion of PMN to EC [12 ]. This final attachment is mediated by the complement receptor (CR) 3, which is the prevailing adhesion molecule of PMN and binds to intercellular adhesion molecule-1 (CD54) on activated EC [13 ]. CR3 comprises two polypeptides with an {alpha}-chain (CD11b) noncovalently linked to a ß-chain (CD18) shared [14 ] by other integrins, including leukocyte function-associated antigen 1 (CD11a/CD18) and the CR4 (CD11c/CD18). CR3 participates in phagocytosis and cytolytic activities [15 ] as well as the regulation of the IC-triggered respiratory burst of PMN [16 ]. All these effects occur within a few minutes following stimulation [17 ], suggesting that relatively little is known about the role of Fc{gamma}RIIIb after a longer-term engagement.

The presence of anti-Fc{gamma}RIIIb antibodies (Ab) in autoimmune settings, such as primary Sjögren’s syndrome (pSS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), has been described recently [18 , 19 ]. We have since described [20 ] that these autoAb may be categorized on the basis of results obtained in the indirect immunofluorescence (IIF) test and the enzyme-linked immunosorbent assay (ELISA): group A autoAb are IIF+/ELISA+; group B autoAb, IIF+/ELISA-; and group C autoAb, IIF-/ELISA+. That is, group B autoAb, as well as a proportion of group A autoAb, recognizes the Fc{gamma}RIIIb anchored to the PMN membrane (IIF+ autoAb), and group C, as well as another proportion of group A autoAb, binds to a soluble form of Fc{gamma}RIIIb (ELISA+ autoAb). The production of ELISA+ autoAb is consistent with the presence of a soluble form of Fc{gamma}RIII in human serum and other body fluids because of its GPI linkage. Most of this cell-free receptor originates from release by PMN [21 ], and its level is elevated at sites of inflammation [22 ] as well as the serum of patients with various nonorgan-specific autoimmune conditions, e.g., pSS [23 ], SLE [24 ], and RA [25 ].

The shedding of surface Fc{gamma}RIIIb during apoptosis [26 ], along with our previous finding that the levels of cell-free Fc{gamma}RIIIb correlate with the titers of anti-Fc{gamma}RIIIb autoAb [25 ], raises the possibility that such autoAb could initiate apoptosis of PMN. The corollaries are that IIF+ autoAb and ELISA+ autoAb should have differential effects and that the nature of their specific target epitopes may be of the utmost importance. As an attempt to delineate the consequences of the binding of anti-Fc{gamma}RIIIb autoAb to the cells, normal NA1+NA2+, NA1+NA1+, or NA2+NA2+ PMN were incubated with serum samples from each of the three groups of autoAb, and their effect on PMN was evaluated. Unexpectedly, IIF+ autoAb (groups A and B) extended the survival of PMN. Furthermore, this delayed apoptosis was associated with reduced adhesion and respiratory burst. In addition, the surface density of CD11b, CD18, and CD62L was diminished in proportion to decreased PMN function, suggesting that the expression and, possibly, the competence of CR3 and CD62L may be modulated through Fc{gamma}RIIIb-mediated signals. All these anti-Fc{gamma}RIIIb autoAb-induced impaired responses, including delayed apoptosis, were more pronounced when the cells originated from donors NA1+NA1+ rather than donors NA1+NA2+. Taken together, these paradoxical findings establish that the functioning of membrane-anchored and soluble Fc{gamma}RIIIb may be modulated by anti-Fc{gamma}RIIIb autoAb in inflammatory autoimmune diseases and that this might be related to Fc{gamma}RIIIb polymorphism.


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MATERIALS AND METHODS
 
Patients and controls
Sera were collected from 95 pSS, 66 SLE, and 61 RA patients fulfilling the respective disease criteria [27 28 29 ], irrespective of their leukocyte count. Sera from 14 medical students and 6 members of the laboratory staff served as negative controls. Anti-PMN+/anti-Fc{gamma}RIIIb- sera from three patients with autoimmune neutropenia were kindly donated by Dr. Monique Vicariot (University Medical School, Brest, France) and used as positive controls in some experiments. For stimulation with the anti-Fc{gamma}RIIIb autoAb, immunoglobulin G (IgG) were purified on a protein-G column (Pharmacia, Uppsala, Sweden), assayed using a sandwich ELISA, and used at a final concentration of 100 µg/mL.

Cells
PMN from healthy volunteers were phenotyped by Dr. Jeanine Cartron (Le Kremlin-Bicêtre Hospital, Paris, France) to select NA1+NA2+, NA1+NA1-, and NA1-NA2+ donors. Their PMN were purified by Dextran T500 (Pharmacia) sedimentation, followed by Ficoll-Hypaque density-gradient centrifugation. Contaminating erythrocytes were lysed with hypotonic saline for 20 s. In one experiment, PMN were purified using an isotonic Percoll gradient (Sigma Chemical Co., St. Louis, MO), and PMN were harversted from the 70%/81% interface. Cell suspensions contained over 90% PMN, and 5.105 PMN/well were cultured in RPMI-1640 medium supplemented with 2.5% fetal calf serum, 2 mM L-glutamine (Biomérieux, Lyon, France), 200 U/mL penicillin (Rhône-Poulenc, Lyon, France), and 500 µg/mL streptomycin (Diamant, Paris). Peripheral blood lymphocytes (PBL) were isolated by Ficoll-Hypaque density centrifugation.

Monoclonal Ab (MoAb)
Fluorescein isothiocyanate (FITC)-conjugated anti-CD11b MoAb, termed Bear 1, anti-Fc{gamma}RIIIb MoAb (clone 3G8), FITC- and phycoerythrin (PE)-conjugated IgG1, and IgG2a isotypic controls were all purchased from Immunotech (Marseille, France). PE-conjugated anti-CD62L (clone TQ1) was from Coultronics (Miami, FL).

IIF detection of autoAb
As previously described in detail [20 ], sera diluted 1/10 in phosphate-buffered saline (PBS) were incubated with 5.106 NA1+NA2+ paraformaldehyde-fixed PMN/mL, for 30 min at 4°C. The cells were washed three times and stained with FITC-conjugated F(ab')2 anti-human IgG for 30 min at room temperature (RT). They were washed another three times, and 104 cells/test were analyzed with an Epics Elite flow cytometer (Coultronics). Control sera showed similar profiles to those obtained with cells incubated with FITC-conjugated F(ab')2 anti-human IgG alone. The IIF positivity of patients’ sera was determined using Immuno-4 software, which substracts the control profile from the test profile in each fluorescence channel.

ELISA detection of autoAb
This test has also been described in detail elsewhere [20 ]. Briefly, recombinant human Fc{gamma}RIIIb (rFc{gamma}RIIIb) produced in Escherichia coli (kindly donated by Professor David Khayat, La Pitié-Salpétrière Hospital, Paris) was heated for 5 min at 95°C, treated with 15% ß2-mercaptoethanol to prevent nonspecific binding of IgG via the Fc domain, and dispensed into half of the 96 wells of microtiter plates (Dynatech, Oslo, Norway) at 1 µg/mL in citrate buffer, pH 3.5. The other half of the wells were flooded with citrate buffer alone. After overnight incubation at 4°C, the plates were washed four times with PBS supplemented with 0.05% Tween (PBS-T) and blocked with PBS containing 5% bovine serum albumine (BSA) for 1 h at 37°C. After another four washes, serum samples (1/100 in PBS-T) were dispensed into coated and noncoated wells and left for 90 min at RT. Following three careful washes, bound autoAb were detected with horseradish peroxidase-conjugated F(ab')2, anti-human IgG (Dakopatts, Copenhagen, Denmark). MoAb 3G8, an IgG1 specific for Fc{gamma}RIIIb and revealed with horseradish peroxidase-conjugated goat F(ab')2, anti-mouse IgG (Dakopatts), was used as a positive control. After development, the optical density (OD) was read at 492 nm using an automatic microplate reader (Multiskan Labsystem, Helsinki, Finland), and specific binding was calculated as OD492-coated well–OD492-noncoated well. Cut-off for positivity was set at the mean + 2 SD of 34 normal sera. Each assay was performed in triplicate, and the means were presented.

Absorption of autoAb
To determine whether there were two distinct populations of autoAb in group A sera, one IIF+ and the other ELISA+, three sera (all from patients with pSS) were selected on the basis of their high positivity in both tests. IgG was prepared by passing the sera over a protein G column and first absorbed with soluble Fc{gamma}RIIIb. Purified IgG (100 µl), adjusted to 100 µg/mL, were dispensed into three sets of triplicate wells and coated with 0.01, 0.1, and 1 µg rFc{gamma}RIIIb, respectively. After a 90-min incubation at RT, supernatants were collected, absorbed a second time under the same conditions, and tested by IIF. The reverse experiment was also carried out. Aliquots of IgG from the same sera, adjusted at 1 mg/mL, were incubated with 104, 105, 106, or 107 PMN for 1 h at 37°C, followed by an overnight incubation at 4°C. Following a 20-min centrifugation at 2000 g, the supernatants were collected, absorbed again in the same way, and tested in the ELISA. Three group B IgG (IIF+/ELISA-) served as negative controls for the absorption with rFc{gamma}IIIb and as positive controls for that with PMN, whereas group C IgG (IIF-/ELISA+) served as positive controls for the absorption with rFc{gamma}RIIIb and as negative controls for that with PMN.

Complement-mediated cytotoxicity
The cytotoxicity of purified anti-Fc{gamma}RIIIb IgG was evaluated using a colorimetric assay for alkaline phosphatase (AP) activity. The effect of purified anti-Fc{gamma}RIIIb and control IgG on PMN was tested in microtiter plates at different dilutions in triplicate, in the presence and absence of fresh human AB serum as a source of complement (diluted 1/50 in gelatin veronal-buffered saline). After an overnight incubation at 37°C, PMN were centrifuged at 1250 g for 5 min at 4°C. AP substrate p-nitrophenyl phosphate (PNPP; 100 µl) was then added to 100 µl supernatants at a concentration of 1 mg/mL. After an overnight incubation at 37°C, plates were read at 405 nm. The release of intragranular AP by PMN was expressed as a percentage according to the formula (OD405experimental-OD405 spontaneous/OD405 maximum-OD405 spontaneous) x 100, where OD405 experimental indicates the AP activity of supernatants from PMN-containing wells in the presence of IgG plus complement, and OD405 maximum indicates that of supernatants from wells containing PMN incubated in 0.02% sodium dodecyl sulfate (SDS) in gelatin veronal-buffered saline. IgG from three patients with autoimmune neutropenia were used as controls to validate the test.

Anti-Fc{gamma}RIIIb Ab-dependent cell cytotoxicity (ADCC)
PMN were incubated with a saturating amount of purified anti-Fc{gamma}RIIIb or normal IgG for 30 min at 37°C. After three washes in Hanks’ balanced salt solution (HBSS) PMN were suspended in RPMI-1640 containing 25 mM HEPES and added to PBL at different effector/target ratios. The cells were centrifuged at 55 g for 2 min at RT and incubated at 37°C, 5% CO2for 18 h in microtiter plates. The cells were then spun down at 1250 g for 5 min at 4°C, and 100 µL supernatants were collected. Then, 100 µL PNPP was added to each sample and incubated overnight at 37°C. Release of intragranular AP of PMN cells was evaluated by the OD at 405 nm. The percentage of specific AP release was expressed as described above. AutoAb from the aforementioned three patients with autoimmune neutropenia were used to validate the test.

Measurement of apoptosis
Following a 12-h incubation in medium alone or with 100 µg/mL IgG purified from autoAb-containing sera, 5.105 PMN were washed in PBS and stained with FITC-labeled annexin V and propidium iodide (PI) according to the manufacturer’s instructions (Immunotech). After a 10-min incubation, cells were analyzed by flow cytometry. Apoptotic cells were percentages of annexin V-positive/PI-negative cells. In pilot experiments, apoptosis was confirmed by analysis of morphological features of Giemsa-stained PMN and enumeration of hypoploid cells. PMN were washed in 0.1 M sodium citrate and 0.1% Triton X-100, and incubated in 250 µL of the same buffer containing 10 µg/mL PI overnight at 4°C in the dark. Reduction in PI staining intensity compared with control cells by flow cytometry was taken as a measure of hypoploidy, characteristic of apoptotic cells.

Adhesion assay
The adhesion capacity of PMN to EC treated with anti-Fc{gamma}RIIIb or control IgG was evaluated using cells of the EC line EA.hy 926 (kindly donated by Dr. Cora-Jean S. Edgell, Chapel Hill, NC). Briefly, cells were coated onto 96-well microtiter plates and fixed with 0.1% glutaraldehyde solution for 10 min at 4°C. After three washes with PBS supplemented with 1% BSA, wells were saturated with PBS containing 3% BSA for 2 h at 37°C. Plates were washed three times with HEPES, pH 7.4, containing 0.5 mM MnCl2. Then, 5.105-stimulated PMN were dispensed into the wells, allowed to adhere for 30 min at 37°C, and washed another three times. Adhesion of PMN was quantitated by colorimetric assay for AP activity. PNPP (100 µl) was added/well at a concentration of 1 mg/mL in 0.1 M carbonate buffer, pH 9.6, containing 0.02% SDS. After an overnight incubation at 37°C, plates were read at 405 nm. Assays were performed in triplicate, and the results were averaged.

Respiratory burst assay
Following a 12-h incubation with anti-Fc{gamma}RIIIb or control IgG, 5.105 PMN were washed in HBSS and suspended in 100 µL of the same buffer. Complement-opsonized zymosan (100 µl) from Saccharomyces cerevisiae (Sigma) at 20 mg/mL, together with 100 µL of 4 µg/mL luminol, was left for 10 min at 37°C. Light emission was measured at 425 nm as millivolts using a LKB-Wallac 1250 luminometer (LKB, Turku, Finland) at peak chemiluminescence (30 s). A background substraction control zeroed the instrument. Results were compared with cells incubated in medium alone. Again, assays were performed in triplicate, and the results were averaged.

Flow cytometry analysis
For staining, 5–105 PMN were incubated on ice for 30 min with 10 µL appropriate MoAb at an optimal concentration. After extensive washing, PMN were examined by flow cytometry. Data from at least 104 events were analyzed on a logarithmic scale and expressed as the percentage of positive cells compared with isotype controls or the mean fluorescence intensity (MFI).

Statistical analysis
All the figures quoted below are arithmetic mean ± SD. Comparisons were made using the {chi}2 test with Yates’ correction when required and the Mann-Whitney U-test for unpaired data.


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RESULTS
 
Detection of anti-Fc{gamma}RIIIb autoAb
As can be seen in Table 1 , 22%, 47%, and 31% of pSS, SLE, and RA sera, respectively, were positive in the IIF test but not any of the control sera. pSS (43%), 33% of SLE, and 26% of RA were positive in ELISA, whereas, again, none of the normal sera were positive. We have demonstrated previously that these autoAb did not bind through their Fc portion [20 ]. Overall, three populations of anti-Fc{gamma}RIIIb autoAb were thus identified in each disease (Table 2 ): group A (IIF+/ELISA+), group B (IIF+/ELISA-), and group C (IIF-/ELISA+) autoAb. Reactivity in both assays was detected in 9%, 23%, and 7% of pSS, SLE, and RA sera, respectively; 13%, 24%, and 25% of pSS, SLE, and RA sera were positive only in the IIF test; and 34% of pSS sera, 11% of SLE sera, and 20% of RA sera showed reactivity only in the ELISA. As described [20 ], the titer of the PMN-reactive autoAb in groups A and B did not correlate at all with the PMN count. It is noteworthy, however, that more patients with pSS (41/95) had autoAb-to-cell-free Fc{gamma}RIIIb (p<0.01), and less patients with SLE (22/66) were positive in this test (nonsignificant, NS). As shown previously [30 ] and confirmed herein, extraglandular manifestations, such as nonerosive arthritis, Raynaud’s phenomenon, and lung involvement, were more frequent in the IIF+/ELISA+ and IIF-/ELISA+ pSS patients than in the remainder (p<0.02). In contrast, there were no differences in the SLE manifestations, severity, and presence of renal involvement between the patients with autoAb against the cell-bound Fc{gamma}RIIIb and those with autoAb against the soluble Fc{gamma}RIIIb.


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  		Table 1. Anti-Fc{gamma} receptor IIIb reactivity of patient and control sera


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  		Table 2. Relationship between IIF and ELISA detection of anti-Fc{gamma} receptor IIIb autoantibodies in autoimmune and control sera

Three IIF+/ELISA+ autoAb were selected on the basis of their strong reactivity and were further analyzed. PMN-binding autoAb were almost completely abrogated by absorption of purified IgG with soluble Fc{gamma}RIIIb (Fig. 1A ). Conversely, the OD values measured by the ELISA were reduced significantly following incubation with pelleted PMN (Fig. 1B) . These findings support the notion that group A autoAb recognize different epitopes from autoAb in groups B and C, because group B autoAb remained unchanged following incubation with soluble Fc{gamma}RIIIb, and group C autoAb were unaffected by incubation with increasing numbers of pelleted PMN.



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  		Figure 1. Absorption experiments. IgG from three IIF+/ELISA+ (group A) sera were absorbed twice with increasing amounts of rFc{gamma}RIIIb and tested again in the IIF test (A). Conversely, they were absorbed twice with increasing numbers of PMN and tested again in the ELISA (B). Three IIF+/ELISA- (group B) IgG served as negative controls for the absorption with rFc{gamma}RIIIb and as positive controls for that with PMN, and three IIF-/ELISA+ (group C) IgG served as positive controls for the former absorption and negative controls for the latter. A representative sample of each group of autoAb is shown (mean±SD of triplicate experiments).

Study of cytotoxicity
Given that there was no correlation between the presence of autoAb and the number of circulating PMN [20; present study], patients with PMN-binding autoAb (groups A and B) did not exhibit reduced numbers of circulating PMN. It could thus be argued that any deleterious effect of autoAb would be compensated for by increased production of PMN. Therefore, it was still important to determine the cytotoxicity of the different classes of autoAb. To address this question, we compared the anti-Fc{gamma}RIIIb-induced cytotoxicity of IIF+ sera (four group A and two group B) and IIF- sera (three group C). Complement-mediated cytotoxicity was first examined using IgG from three patients with autoimmune neutropenia and two normal individuals as positive and negative controls, respectively. Anti-Fc{gamma}RIIIb autoAb from none of the groups showed complement-mediated lysis of the cells (unpublished results). ADCC was then assessed. Aliquots of PMN were sensitized with autoAb from each group, incubated with PBL, and the AP activity was released from lysed PMN measured. None of the anti-Fc{gamma}RIIIb autoAb populations generated ADCC (unpublished results), irrespective of the effector/target ratio tested (6, 12.5, 25, 50, and 100 PBL/1 PMN).

Effects on apoptosis
The effects of autoAb from 12 patients (four with with pSS, five with SLE, and three with RA) were studied further using PMN from heterozygous NA1+NA1+ donors as the substrate. Four samples of each group of anti-Fc{gamma}RIIIb and six control IgG were examined for pro- or anti-apoptotic effects. Unexpectedly, autoAb did not induce apoptosis of PMN. Rather, a 12-h incubation with IIF+ IgG (group A or group B) decreased the number of cells undergoing spontaneous apoptosis, compared with those incubated in medium. Group A IgG reduced the number of spontaneously apoptotic PMN by 25.5 ± 3.6% and group B IgG, by 32.9 ± 6.3% (Fig. 2A ). This reduction was moderate but highly reproducible given that four independent, triplicate experiments with each of the four group A IgG and the four group B IgG produced comparable results. In contrast, IIF- IgG (group C: 5.5±1.0% and control IgG: -0.4±0.6%) did not influence the spontaneous apoptosis of PMN (groups A and B, p<0.05, compared with group C and controls). Representative examples are presented in Figure 2B . It is interesting that the reduction was higher when the PMN were from donors NA1+NA1+ than from donors NA2+NA2+ (Table 3 ). Importantly, patients with cell-binding autoAb did not have increased numbers of circulating PMN that could have been attributed to their prolonged survival.



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  		Figure 2. Effects of anti-Fc{gamma}RIIIb autoAb on spontaneous apoptosis of PMN. Following a 12-h incubation in medium alone or with autoAb, apoptotic PMN were enumerated as the percentage of annexin V-positive/PI-negative cells. The percentage of reduction of apoptosis by groups A, B, and C autoAb (four samples of each) and six control IgG was calculated (A). Representative examples, one for each autoAb group, are shown in B, where PMN were incubated in medium alone or with IIF+/ELISA+ IgG, IIF+/ELISA- IgG, IIF-/ELISA+ IgG, or IIF-/ELISA- IgG.


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  		Table 3. Relationship between the effect of anti-Fc{gamma}RIIIb autoAb on apoptosis and phenotype of PMN

AutoAb-mediated phenotypic changes of PMN
On the basis that lectin-polysaccharide interactions associate Fc{gamma}RIIIb with CR3 on the surface of PMN [31 ], one potential mechanism for Fc{gamma}RIIIb-mediated reduction of apoptosis is via CR3. Thus, the expression of CD11b on PMN was evaluated following a 12-h incubation with the same IgG from each group of anti-Fc{gamma}RIIIb autoAb and the same control IgG as above. Representative examples of four independent experiments for each category of autoAb are given in Figure 3 . Cell-binding autoAb (groups A and B) reduced the expression of CD11b in a fraction of PMN. This subpopulation, referred to as CD11bdim, appeared spontaneously after a 12-h incubation in medium or with control IgG (22.5±1.5 and 26.0±1.8 of PMN, respectively). Following treatment with cell-binding autoAb, i.e., groups A and B, there appeared to be a reproducible increase of CD11bdim PMN (41.8±2.1 and 40.0±1.3% of PMN, respectively, p<0.01, compared with control IgG). In contrast, CD11bdim PMN accounted for 23.7 ± 0.7% of the cells following stimulation with group C autoAb (NS, compared with control IgG). These autoAb effects were independent of the isolation procedure, because similar results were obtained with Percoll-isolated PMN (unpublished results). It is intriguing that we have described previously [32 ] and confirm in this work that the patients with PMN-binding autoAb did not exhibit reduced expression of CD11b on freshly collected PMN. Rather, it was markedly augmented.



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  		Figure 3. Anti-Fc{gamma}RIIIb IgG-induced modification of the PMN phenotype. The expression of CD11b was evaluated before and after a 12-h incubation in medium alone or with IIF+/ELISA+ (group A), IIF+/ELISA- (group B), IIF-/ELISA+ (group C), and IIF-/ELISA- (control) IgG. In each group, the results presented are representative of four independent experiments.

Functional changes in PMN
Apoptosis is associated with a loss of PMN activity [33 , 34 ]. Therefore, we determined whether some functions of PMN were modified by treatment with groups A and B autoAb. Adherence of PMN to EC following their incubation with three autoAb of each of these groups was examined. It was found that IIF+ anti-Fc{gamma}RIIIb autoAb reduced the level of adherent PMN, and control IgG did not (Fig. 4A ). Whereas no reduction was obtained with control IgG, groups A and B autoAb reduced the number of adherent PMN by 62.3 ± 4.4% and 49.4 ± 2.6%, respectively (p<0.05, compared with control IgG). A similar analysis of the respiratory burst was then conducted (Fig. 4B) . Its initiation was reduced by 83.1 ± 8.6% (group A autoAb) and 46.4 ± 6.6% (group B autoAb). In comparison, treatment with control IgG led to a reduction of <7% (p<0.05, compared with groups A and B autoAb).



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  		Figure 4. Effect of anti-Fc{gamma}RIIIb autoAb on the functions of PMN. The adhesiveness to endothelial cells (A) and respiratory burst (B) of PMN were evaluated following incubation of PMN with IIF+/ELISA+ (group A) and IIF+/ELISA- (group B) autoAb and IIF-/ELISA- control IgG. The percentages of reduction of these functions were calculated (mean±SD of three autoAb in each group).

Owing to the role of CD62L in adhesiveness, the expression of this molecule was measured, following a 14-h incubation with three group A and three group B autoAb. Freshly isolated PMN expressed CD62L with an MFI of 26.9 ± 3.2. This expression declined spontaneously to 5.7 ± 1.0 after a 14-h incubation in medium alone. When stimulated with IIF+ anti-Fc{gamma}RIIIb autoAb, the MFI of CD62L diminished dramatically to 1.5 ± 0.3 and 1.4 ± 0.5 for group A and group B autoAb (p<0.05, compared with control IgG).


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DISCUSSION
 
Following the study of Fc{gamma}RIIIb reactivity in the sera of patients with nonorgan-specific autoimmune diseases [18 19 ], we have classified these autoAb into three groups [20 ], as confirmed and extended at the beginning of the present study: IIF+/ELISA+ (group A), IIF+/ELISA- (group B), and IIF-/ELISA+ (group C) autoAb. In brief, IIF+ autoAb and ELISA+ autoAb recognized the cell-bound and the soluble forms of Fc{gamma}RIIIb, respectively. A proportion of group A sera did not contain two populations of autoAb (one IIF+ and another ELISA+), rather the IIF and ELISA reactivities overlapped. In support of this conclusion, IIF reactivity declined following absorption with rFc{gamma}RIIIb, as well as that of the ELISA reactivity following incubation with pelleted PMN. At least in part IIF+/ELISA+ (group A) autoAb recognize specific epitopes. The implication of this finding is that targeted epitope(s) are accessible on the membrane-anchored as well as the soluble form of Fc{gamma}RIIIb, and these epitopes differ from those recognized in IIF+/ELISA- (group B) and IIF-/ELISA+ (group C) sera. Our interpretation does not necessarily hold true for all group A sera or all anti-Fc{gamma}RIIIb autoAb in a given group A serum. Presumably, some of these so-called group A sera consist of a combination of group B and group C autoAb. In this respect, it is interesting that we demonstrated previously [20 ] that IIF+/ELISA+ sera (group A) recognized NA1+NA1+ as well as NA2+NA2+ cells, and IIF-/ELISA+ sera were specific for NA1+NA1+ and NA1+NA2+ cells.

On the basis that the presence of autoAb in the patients was not associated with neutropenia [20 ], we have focused on their effects on PMN apoptosis and functions. Each group of sera containing anti-Fc{gamma}RIIIb autoAb was incubated with PMN, and the survival and functions of the cells were evaluated. IIF+ sera appeared not to be cytotoxic for PMN, which is reminiscent of the fact that the amount of antineutrophil Ab detected in patients with autoimmune neutropenia does not correlate with the number of circulating PMN. These cells have the shortest longevity amongst the leukocytes and undergo spontaneous apoptosis in vitro although cultured in complete medium. In this respect, our central finding was that IIF+ sera have the capacity to delay the apoptotic process arising spontaneously after a few hours of incubation in medium. Although the biochemical pathways underlying this phenomenon remain unclear, our data suggest that in the presence of IIF+ autoAb, the survival of circulating PMN is augmented by engagement of membrane-anchored Fc{gamma}RIIIb. It is interesting that a similarly intriguing observation had been made previously with treatment of PMN with IgG used as an isotype control for MoAb (unpublished results). The antisera that inhibited spontaneous apoptosis stimulated the appearance of a CD11bdim population by approximately the same extent. Incidentally, an even greater increase was obtained using anti-CD16 MoAb F(ab')2 fragments, which exclusively target the Fc{gamma}RIIIb molecule (unpublished results). There was, however, no reduced expression of CD11b on freshly collected PMN. Rather, this expression was augmented [35; present study], indicating that the cells are activated in vivo.

Moreover, we found that functional properties, e.g., adherence and respiratory burst, declined following incubation of PMN with autoAb. Surprisingly, patients with PMN-reactive autoAb did not have raised numbers of circulating PMN that could be attributed to their prolonged survival. Should this increase occur, it might be counterbalanced by a reduced production of the cells. Inasmuch as the rate of apoptosis is influenced by PMN density and extracellular protein concentration [36 ], it is tempting to evaluate the effects of anti-Fc{gamma}RIIIb autoAb on PMN from the synovial fluid of patients with RA. Given that anti-Fc{gamma}RIIIb autoAb are associated with soluble receptors [25 ], our present negative observation is perfectly in line with the results of other investigators [21 ] who did not find a correlation between the amount of plasma Fc{gamma}RIIIb and the PMN count. Multiple functional defects of PMN have, however, been described in connective tissue diseases, particularly pSS [32 ], SLE [37 ], and RA [38 ], possibly because of PMN-binding autoAb. Furthermore, if they cover epitopes in the vicinity of the IgG-binding site, they may inhibit the binding of IC to PMN, prevent their clearance [39 ], and thereby influence the outcome of systemic autoimmune diseases.

The alteration of these functions was associated with a diminished expression of ß2 integrin CD11b and CD62L. The reduced expression of the ß2 integrins was not exclusively a result of autoAb-induced conformational changes of these molecules, because experiments with different anti-CD11b MoAb (Bear 1, Mo1, 2PLM19C, and 44) directed toward specific epitopes (unpublished results) produced shifts of the profiles to the left, although the MFI were related to the MoAb used. These findings lend weight to the concept that the PMN, GPI-anchored molecule Fc{gamma}RIIIb may be able to transduce signals. IgG-induced stimulation of PMN is mediated via Fc{gamma}RII and not via Fc{gamma}RIII. This is also in line with recent studies disclosing that anti-Fc{gamma}RII-carrying hybridoma cells are lysed readily by PMN, and hybridoma cells expressing anti-Fc{gamma}RIII are not [40 ]. With regard to autoAb-to-cell-free Fc{gamma}RIIIb, we must, however, admit that their pathogenicity remains largely unknown. Studies are now underway to delineate these mechanisms.

The respective role of each class of Fc{gamma}R in eliciting PMN responses is not well-characterized. Previous studies have, however, indicated that Fc{gamma}RIIIb and Fc{gamma}RII have unique roles in the IC-initiated stimulation of PMN and that full activation can only been achieved when both Fc{gamma}R classes are available [9 ]. Thus, it is possible that a proportion of the IIF+ anti-Fc{gamma}RIIIb autoAb might cross-react with Fc{gamma}RII, as previously described by us [35 ]. It is also of substantial interest that CD11b/CD18 influences degranulation as well as adhesiveness not simply because of its quantity on the PMN surface but rather because of an as yet unidentified molecular mechanism [41 ]. In fact, ligation of CD11b/CD18 is not required for the initiation of Fc{gamma}R-mediated PMN recruitment, and CD11b/CD18- Fc{gamma}R interactions are necessary for filamentous actin reorganization leading to sustained PMN adherence [42 ].

It is recognized that allelic polymorphisms in Fc{gamma}RIIIb have significant consequences for physiological functions [43 ]. The NA1 isoform facilitates a more robust, Fc{gamma}R-mediated phagocytosis, respiratory burst, and degranulation responses, compared with the NA2 isoform. Importantly, these donors do not show differential quantitative PMN expression of Fc{gamma}RIIIb. It is also noteworthy that the overall effects of anti-Fc{gamma}RIIIb autoAb in a NA2+NA2+ homozygote were less than those obtained in a NA1+NA1+ homozygote. Thus, it appears that Fc{gamma}RIIIb polymorphism, together with the type and quantity of autoAb, modulates the final inflammatory response.

Hence, anti-Fc{gamma}RIIIb autoAb produce different effects apparently. In the light of these results, one may conclude that autoimmune patients with anti-Fc{gamma}RIIIb autoAb have circulating PMN with prolonged longevity but, paradoxically, reduced function. Nonetheless, persistent inflammation is a feature of autoimmune conditions. The net biologic effect of these competing influences would thus be to sustain the inflammatory response. Inflammation might be modulated by autoAb, dependent on their targeted autoantigens.


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ACKNOWLEDGEMENTS
 
We gratefully acknowledge Dr. Jeanine Cartron (Le Kremlin-Bicêtre Hospital, Paris) for phenotyping the cells. Thanks are also owed to Professor David Khayat (La Pitié-Salpétrière Hospital, Paris), Dr. Monique Vicariot (Brest University Medical School Hospital, Brest, France), and Dr. Cora-Jean S. Edgell (Chapel Hill, NC) for providing valuable reagents. We are also indebted to Professor Peter M. Lydyard (UCL, London) for critical reading of the manuscript. The secretarial assistance of Mrs. Simone Forest and Sylvie Hamon is greatly appreciated.

Received December 31, 1999; revised October 2, 2000; accepted October 4, 2000.


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V. Durand, Y. Renaudineau, J.-O. Pers, P. Youinou, and C. Jamin
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