Mouse IgA inhibits cell growth by stimulating tumor necrosis factor-{alpha} production and apoptosis of macrophage cell lines

Rajko Reljic1, Carol Crawford1, Stephen Challacombe1 and Juraj Ivanyi1

1 Department of Oral Medicine and Pathology, Guy’s Campus of King’s College London, Guy’s Hospital, London SE1 9RT, UK

Correspondence to: J. Ivanyi; E-mail: juraj.ivanyi{at}kcl.ac.uk
Transmitting editor: M. Feldmann


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Potent Fc{alpha}-mediated actions of IgA have previously been shown for myeloid cells from man, but much less is known in relation to murine cells. Here, we report that mouse monoclonal IgA, irrespective of their antigenic specificity, inhibit the proliferation of mouse macrophage cell lines. The anti-proliferative activity was manifested by both monomeric and polymeric mouse IgA, but not by mouse monoclonal IgG and IgM. Growth of J774 cells was significantly inhibited during the 4–8 days of logarithmic growth, followed by a subsequent recovery of cell numbers prior to the stationary phase. We demonstrated that IgA binds to J774 cells, stimulates tumor necrosis factor (TNF)-{alpha} production and induces apoptosis which is not dependent on NO or FAS/CD95. We also demonstrated that IgA, in synergy with IFN-{gamma}, induced TNF-{alpha} production and apoptosis of thioglycollate-elicited mouse peritoneal macrophages. Thus, the in vitro actions of IgA described may also play a regulatory role for mouse macrophages in vivo.

Keywords: apoptosis, Fc receptor, macrophage cell line, mouse IgA, tumor necrosis factor-{alpha}


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of secretory IgA antibodies at mucosal surfaces, their transcytosis through epithelial cells, and hepatobiliary transport mediated by the poly-Ig and asialoglycoprotein receptors have been previously well characterized (13). The effector actions of human serum IgA on human macrophages and other myeloid cells have also been investigated extensively (46). Interaction of IgA with the poly-Ig receptor is non-inflammatory, while the Fc{alpha}RI, CD89 receptor on human myeloid cells, mediates a broad spectrum of pro-inflammatory and immunomodulatory effects, such as the respiratory burst, production of cytokines and chemokines (7), antibody-dependent cell cytotoxicity (8), antigen presentation (9), and activation of dendritic cells (10). Resistance against bacterial infections was conferred to mice by transgenic expression of CD89 on myeloid and Kupffer cells (5,11). With regard to the different isoforms of Fc{alpha}RI expressed in various cells (12), some associated with Fc{gamma}RII (13), while those without {gamma} chains mediated endocytosis, recycling of IgA and a non-inflammatory response of colostral neutrophils (14).

In contrast to these extensive studies in man, there is surprisingly little knowledge about the structure and function of Fc{alpha} receptors in mice. Transcripts of two cDNAs (PIR-A and PIR-B), isolated from a mouse splenic library using the human Fc{alpha} receptor probe, were detected in mouse lymphoid cells (15), but the expressed gene products failed to bind to IgA. Recently, six genes of a diverse family of Fc receptor homologues (16) and a common Fc{alpha}/µ receptor (17) were found expressed mainly on B cells, while previous studies reported Fc{alpha} receptor expression on activated mouse T lymphocytes (18).

In view of the capacity of IgA antibodies to confront viral and bacterial intracellular infections, we were interested in exploring their possible role in mycobacterial infections. Using IgA mAb (19), we demonstrated their passive protective capacity upon intranasal inoculation of mice (20). However, when attempting to modulate tuberculous infection of macrophage cell lines with IgA mAb, we observed an unexpected inhibition of cell growth even in the absence of any bacilli. We report in this paper the basic features and characteristics of this ‘anti-proliferative’ activity of IgA on mouse macrophage cell lines. We also demonstrate that IgA stimulated tumor necrosis factor (TNF)-{alpha} production and apoptosis of a substantial portion of cells, and propose that these events are involved in the inhibition of the logarithmic phase of cell growth. Finally, induction of TNF-{alpha} and apoptosis of mouse peritoneal macrophages by IgA in synergy with IFN-{gamma} indicated the biological relevance of these mechanisms.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and other reagents
The anti-mycobacterial mAb TBA61 (IgA) and TBG65 (IgG), both anti-acr, and TBA84 (IgA) anti-PstS1 (19), were purified from tissue culture supernatants by affinity chromatography on antigen-coupled Affigel-15 Bio-Rad (Hercules, CA) columns. The purchased reagents were: MOPC 315 (IgA anti-nitrophenylated protein; cat. no. M2046), TEPC 183 (IgM of unknown specificity; cat. no. M3795), mouse TNF-{alpha} (cat. no. T7539), lipopolysaccharide (LPS) from Escherichia coli (cat. no. L-2654) and polymyxin B from Sigma (Poole, UK); rat anti-mouse F4/80–R-phycoerythrin (PE) (cat. no. MCA1125PE), rat anti-mouse IgG2b–PE (cat. no. MCA497PE) and mouse IFN-{gamma} from Serotec (Oxford, UK); FITC-conjugated hamster anti-mouse CD95 (cat. no. 554257) and isotype control IgG2a (cat. no. 553964) from BD PharMingen (Oxford, UK); rabbit anti-mouse TNF-{alpha} neutralizing antibody from Genzyme (Cambridge, MA); Vivapure Q ion-exchange columns for removal of LPS from Vivascience (Hanover, Germany); and N-monomethyl- L-arginine (L-NMMA) from Alexis (San Diego, CA).

Cell growth inhibition assays
J774A.1 (referred to as J774 in the text) and MH-S macrophage cell lines and M15 epithelial cell line were obtained from the European Type Tissue Culture collection (CAMR, Salisbury, UK). The cells were grown in DMEM supplemented with 10% heat-inactivated FBS. In most experiments, 5 x 104–5 x 105 cells were seeded in 12.5-cm2 tissue culture flasks in triplicate in 1 ml of medium and antibodies were added at 50 µg/ml or as stated in the text. In some experiments, the NO synthesis inhibitor, L-NMMA, was added at 0.5 mM concentration. Following incubation for 4 days, or as indicated in each experiment, the cultures were harvested in cell dissociation buffer and the remaining adherent cells collected by scraping, using rubber scrapers. The viable cells were counted after negative staining with Trypan blue.

Proliferation was tested by adding 1 µCi of [methyl-3H]thymidine/well (TRK120; Amersham, Little Chalfont, UK) to cultures in 96-well plates, 24 h before harvest. Arithmetic means of incorporation counts of radioactivity from triplicate samples were determined by standard procedures. Limiting dilution analysis was performed with six dilutions of cells, each one plated out in 48 wells, and the fraction of negative wells following incubation for 2 weeks was evaluated and plotted (21).

Cell culture of peritoneal macrophages
BALB/c mice were injected i.p. with 1 ml of 4% thioglycollate medium, 5 days before harvesting the peritoneal washings with 5 ml of culture medium (RPMI 1640, supplemented with 10% FBS and 0.1 mg/ml gentamicin). The cells were seeded in 96-well dishes and allowed to adhere for 4 h, after which time the medium was replaced with reagents, as indicated.

Flow cytometry, TNF-{alpha} and apoptosis assays
Cells (1 x 106) were incubated with 50 µg IgA/ml in the presence of 5% BSA, for 1 h at 4°C, washed and then incubated with FITC-conjugated goat anti-mouse IgA antibody (Sigma) for 1 h and washed again prior to FACS analysis by a Becton Dickinson (Mountain View, CA) apparatus. For detection of CD95 expression, cells were grown with or without IgA for 4 days and stained with 1 µg of FITC-conjugated anti-CD95 antibody/106 cells, in 100 µl of PBS/BSA buffer, for 1 h and washed three times prior to analysis. TNF-{alpha} production was tested by ELISA using anti-TNF-{alpha} antibodies (Serotec, Oxford, UK): MCA1487-coated microtiter plates were incubated with cell culture media and developed with the biotinylated MCA1488B antibody followed by streptavidin–peroxidase. Apoptosis was determined by staining of cells for expression of Annexin-V using an apoptosis detection kit (Sigma) and flow cytometry. Alternatively, culture media were tested for the cytosolic lactate dehydrogenase (LDH) enzyme using the CytoTox 96 Non-radioactive Cytotoxicity Assay Kit (Promega, Madison, WI), according to the manufacturer’s instructions.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
IgA inhibits the growth of macrophage cell lines
The presence of the purified mouse IgA mAb TBA61 in the tissue culture medium was found to inhibit the growth of J774 and MH-S mouse macrophage cell lines, but not the M15 mouse epithelial cell line (Fig. 1A). This ‘anti-proliferative’ effect on the macrophage cell lines was demonstrable after 4 days of cell culture, although incubation of these macrophages with the same antibody for a few hours had no immediate cytotoxic effect, as determined by the Trypan blue exclusion assay. Growth of J774 cells was also inhibited by the TBA84 IgA mAb against the PstS-1 mycobacterial antigen and by the MOPC 315 mouse myeloma protein, but not by the TBG65 IgG1 mAb and the TEPC 183 IgM mouse myeloma protein (Fig. 1B). These results suggested the mandatory role of the IgA isotype, irrespective of its combining site specificity. The further finding that both monomeric and polymeric forms of the TBA61 mAb inhibited the growth of J774 cells (Fig. 1C) suggested the functional role of IgA binding to an Fc{alpha}, rather than poly-Ig type of receptor on J774 cells.



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Fig. 1. IgA isotype-dependent inhibition of the growth of mouse macrophage cell lines. Cell lines seeded at 1 x 105 cells/ml/flask were incubated for 4 days in the presence of 50 µg/ml mouse monoclonal or myeloma Ig. Columns represent mean values ± SD of viable cell numbers determined by exclusion of Trypan blue staining from triplicate cultures. (A) Macrophage (J774 and MH-S) and epithelial (M15) cell lines grown in the presence of PBS-control (open columns) or TBA61 IgA mAb (filled columns). (B) J774 cultures incubated with three different IgA (MOPC315 anti-nitrophenylated protein myeloma or TBA61:anti-acr and TBA84:anti-PstS1 mAb), IgM (TEPC183) myeloma or IgG (TBG65:anti-acr) mAb. (C) J744 cells cultured in the presence of unfractionated TBA61 IgA or its monomer (M) and polymer (P) fractions obtained by gel filtration on a Superdex-200 column. (D) Polymyxin B inhibition of the LPS-induced, but not the IgA TBA61-induced, anti-proliferative effects. Viable counts of J774 cells after 4 days of culture with or without TBA61 (50 µg/ml), LPS (10 or 100 ng/ml) and polymyxin B (20 µg/ml).

 
The growth of J774 cells can also be suppressed by incubation in the presence of 10 or 100 ng/ml LPS to a similar extent as by TBA61 IgA (Fig. 1D). Thus, it seemed desirable to investigate whether LPS contamination could have contributed to the J744 growth inhibition by the IgA preparations. However, this possibility has been excluded on the grounds of results that showed that the LPS inhibitor polymyxin B did not reverse the growth inhibitory effect of TBA61 IgA, while it blocked the inhibitory effect of LPS (Fig. 1D). Furthermore, TBA61 mAb retained its full growth-inhibitory activity after depletion of LPS by passage through the Vivapure Q ion-exchange column (results not shown).

Time and dose requirements for the IgA effect on the growth of J744 cells
The time course of IgA inhibition of J774 cells was examined over a 10-day culture period with cells seeded at 5 x 104 cells/flask/ml and the TBA61-containing culture medium replaced every 3 days (Fig. 2A). When comparing cell counts in cultures with and without TBA61, inhibition by IgA became apparent at 4 days, the difference was the largest at 6 days and was reduced at 8 days of culture. However, compensatory growth of IgA refractory cells resulted in cell counts equal to those in the control culture between 8 and 10 days of incubation. Thus, the presence of IgA in culture had delayed the stationary phase of J774 cells by 2–4 days.



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Fig. 2. Time course and TBA61 dose dependence of inhibition of J774 cell growth. Mean counts ± SD of viable cells from triplicate cultures of 5 x 104 seeded cells. (A) Cells grown for 2–10 days (media replaced on days 3, 6 and 9). TBA61, 50 µg/ml (solid symbols); PBS (open symbols). (B) TBA61 (50 µg/ml) was added at the onset of culture. At times given on the horizontal scale, the medium was replaced with fresh medium without antibody and then further incubated until the day 4 cell harvest. (C) The effect of 1–100 µg TBA61/ml concentrations on day 4 harvested cells.

 
We then examined how long the contact between TBA61 IgA and J774 cells (followed by washing and incubation without IgA) is required for the inhibition of J774 cell growth (Fig. 2B). When counting the cells after 4 days of culture, the results showed that an initial 1-h contact was partially inhibitory, while 24 h incubation of cells with IgA achieved maximal inhibition. In a separate experiment, the IgA dose–response analysis showed that inhibition of J774 growth was partial at 10 µg/ml and complete at 20 µg/ml TBA61 (Fig. 2C).

Cell proliferation, limiting cell dilution assay and synergy with IFN-{gamma}
Confirmation of the anti-proliferative effect of TBA61 IgA was shown using the [3H]thymidine incorporation assay. After seeding 4.5 or 9.0 x 104 J774 cells/flask, cells were harvested after 5 days, using trypsin–EDTA, either alone or combined with mechanical scraping (Table 1). Inhibition by IgA was found to be stronger in the ‘scraped’ fraction than in the less adherent fraction of cells when measured by either [3H]thymidine uptake (76–89 versus 22–39%) or Trypan blue staining (93–98 versus 63–84%). This was observed in cultures at both cell seed densities, the higher seed cultures containing a greater proportion of strongly adherent cells. The stronger IgA inhibition of the viable counts than [3H]thymidine uptake could be attributed to the 24-h delay between the addition of [3H]thymidine and cell harvest or to the reduced [3H]thymidine uptake of the control culture at confluence.


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Table 1. TBA61 inhibition of thymidine incorporation and viable cell counts: association with cell adherence to plastic plates
 
Limiting cell dilution was performed after 24 h incubation of J774 cells with or without 100 µg TBA61 and cells were plated out in the absence or presence of 500 peritoneal washing cells from normal BALB/c mice. The results plotted in Fig. 3(A) showed that in the cultures with IgA, the number of cells yielding 37% negative wells was reduced 4.4- or 4.5-fold in the presence or absence of feeder cells. This result confirmed that the IgA inhibitory effect was imparted during the 24-h incubation period and that the progenitors of proliferating cells were particularly susceptible to inhibition.



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Fig. 3. Limiting dilution assay of the IgA effect on macrophages and synergy with IFN-{gamma}. (A) Limiting dilution assay. J774 cells were incubated in the absence (squares) or presence of 100 µg TBA61 (circles) for 24 h. Subsequently, equal numbers of viable cells were plated in the absence (full lines) or presence (broken lines) of peritoneal washings at limiting dilution and the numbers of negative cultures were evaluated. Figures in brackets represent the numbers of plated cells yielding 37% negative wells. (B) Synergy with IFN-{gamma}. Cells were grown in the presence of 0.33 ng/ml (triangles), 1 ng/ml (squares) or without IFN-{gamma} (open circles) and indicated concentrations of TBA61 IgA for 4 days. Viable cells were counted and percent inhibition of cell growth was calculated compared to untreated cells.

 
The IgA-mediated growth inhibitory effect could be further enhanced by co-incubation of cells with IgA and IFN-{gamma}. At the concentration of 1 ng/ml, IFN-{gamma} exhibited potent growth inhibition of J774 cells on its own (Fig. 3B). However, at a lower concentration (0.33 ng/ml) IFN-{gamma} imparted only a low-level inhibition, but synergistically enhanced the inhibitory effect of IgA, thus suggesting a possible regulatory role of IFN-{gamma} for IgA receptor on macrophages or the convergence of signaling pathways of IFN-{gamma} and IgA.

IgA binds to J774 cells, stimulates TNF-{alpha} production, and induces NO- and FAS-independent apoptosis
Using flow cytometry, we observed no significant staining by TBA61 IgA of the control M15 epithelial cells, while 48% of J774 cells were prominently stained (Fig. 4A). In the several experiments using different J744 and IgA preparations, the extent of binding varied between 20 and 70%. Notably, the binding of TBA61 to J774 cells could be inhibited neither by the TBA61 corresponding acr antigen nor by the TBG65 IgG antibody, which binds to the same epitope of acr as TBA61 (Fig. 4B) (19). These results strongly suggest that the TBA61 binding to J744 cells was not due to cross-reactivity of the antibody with some surface components of J744 cells.



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Fig. 4. Detection of TBA61 IgA binding to J774 cells by flow cytometry. (A) M15 epithelial cells and J774 macrophages (1 x 106) were incubated in 100 µl of PBS buffer containing 1% FBS and sodium azide on ice for 1 h with TBA61 (open histograms) or with FITC-conjugated secondary antibody alone (filled histograms). The numbers represent the proportion of stained cells. (B) Lack of inhibition of TBA61 binding in the presence of TBG65 or acr antigen. J774 cells were incubated with 50 µg/ml TBA61 alone (filled histogram, 32%) or in the presence of 500 µg/ml (10-fold molar excess) TBG65 IgG1 mAb (thick line, 30%) or recombinant acr antigen at 200 µg/ml (40-fold molar excess) (thin line, 45%).

 
As TNF-{alpha} can kill transformed cell lines (22), we examined the effect of IgA and IgG mAb on TNF-{alpha} production by J774 cells. Indeed, the results showed that TBA61 IgA, but not TBG65 IgG, stimulated TNF-{alpha} production by J774 cells to almost the same extent as LPS (Fig. 5A). Reciprocally, only TBA61 and LPS suppressed J774 cell growth, thus indicating a possibly causal relationship between the elevated TNF-{alpha} and reduced cell growth. We also found that IgA does not significantly induce CD95/FAS expression in macrophages (Fig. 5B). Thus, after 4 days of incubation in the presence of IgA, J774 cells showed only marginal (11%, compared to 6% in control cultures) increase of CD95 expression. Furthermore, addition of NO synthesis inhibitor, L-NMMA, did not reverse growth inhibitory effect of IgA (Fig. 5C).



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Fig. 5. IgA stimulates TNF-{alpha}, but not FAS/CD95, expression. Failure of NO synthesis inhibitor, L-NMMA, to reverse growth inhibition of J744 mouse macrophages. (A) Inverse relationship between cell growth and TNF-{alpha} production. J774 cells (1 x 105) were seeded in 48-well plates in 300 µl of medium and the following day 50 µg/ml mAb (TBA61 IgA or IgG TBG65) or 100 ng LPS /ml was added. Open columns: viable cell counts after 4-days of culture. Shaded columns: TNF-{alpha} concentrations in supernatants after 24 h incubation. Mean ± SD values from triplicate cultures. (B) FAS expression in J774 cells. Paraformaldehyde-fixed cells were analysed for FAS expression by anti-FAS antibody staining and flow cytometry; negative isotype antibody did not stain (not shown). Percentages of stained cells for control (filled histogram) and TBA61 IgA-treated cultures (open histogram) are indicated. (C) NO synthesis inhibition. L-NMMA (0.5 mM) was added to cultures of cells with or without TBA61 and viable cells were counted after 4 days; mean values ± SD of triplicates cultures grown with (filled columns) or without L-NMMA (open columns) are shown.

 
We also analysed if TBA61 induced the apoptosis of J774 cells by staining for Annexin-V expression (Fig. 6). The results showed that incubation of J774 cells in the presence of the TBA61 mAb significantly increased the percentage of apoptotic cells. The proportion of cells stained for Annexin-V alone (early apoptotic) increased from 6 to 22% and the proportion of double-positive cells stained for Annexin-V and PI (late apoptotic and necrotic) increased from 16 to 31%. These results suggest that apoptosis probably plays a role in IgA-mediated inhibition of growth of J774 cells.



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Fig. 6. IgA-mediated induction of apoptosis of J774 cells. Annexin-V and propidium iodide staining of control (A) and TBA61 IgA-treated cultures (B); the numbers represent percentage of single, Annexin-V-stained (early apoptotic) and double, Annexin-V/propidium iodide (late apoptotic/necrotic)-stained cells.

 
The effects of IgA and IFN-{gamma} on peritoneal macrophages: IgA binding, TNF-{alpha} production and apoptosis
We further ascertained the biological relevance of the effects of IgA using freshly explanted mouse peritoneal macrophages. Since these cells do not proliferate ex vivo and TBA61 IgA did not diminish their stationary numbers up to 7 days of culture (results not shown), we investigated the induction of TNF-{alpha} production and apoptosis. The initial experiments failed to show a significant stimulatory activity of IgA on unstimulated macrophages from peritoneal washings of BALB/c mice. When using thioglycollate-induced peritoneal washings, IgA induced only low levels of TNF-{alpha} production in macrophages (results not shown), despite the fact that 41% of F4/80+ cells (Fig. 7A) bound IgA as assessed by flow cytometry (Fig. 7B).



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Fig. 7. IgA binding to mouse thioglycollate-elicited peritoneal macrophages. Thioglycollate-elicited macrophages were purified from peritoneal washings by adherence to plastic culture dishes for 2 h and tested by flow cytometry for F4/80 (macrophage cell marker) staining (A) or TBA61 IgA binding (B). The percentage values above the horizontal bars indicate staining with F4/80+ (A) or with the secondary antibody alone (filled histogram) and IgA binding for cells incubated without (thin line) or with 10 ng/ml IFN-{gamma} (thick line) (B).

 
In the light of the finding that IgA antiproliferative action on J774 cells was enhanced by IFN-{gamma} (see Fig. 3B), we examined if the effects of IgA on peritoneal macrophages could also be amplified by their incubation in the presence of recombinant IFN-{gamma}. IgA binding to thioglycolate-induced macrophages was tested after 36 h incubation without or with 10 ng/ml IFN-{gamma}. We found that IFN-{gamma} increased the level of IgA binding from 41 to 54% (Fig. 7B). However, incubation of macrophages in the presence of both TBA61 IgA and IFN-{gamma} substantially amplified TNF-{alpha} production, when compared with levels in the presence of IgA or IFN-{gamma} alone (Fig. 8A). Furthermore, cell death reflected by cytosolic LDH levels was also the highest in cultures containing both IgA and IFN-{gamma} (Fig. 8B). These results demonstrating a synergistic action of IgA and IFN-{gamma} on freshly explanted peritoneal macrophages suggest that a regulatory function of IgA may play a role on some macrophage and other monocyte-derived cell populations in vivo.



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Fig. 8. Synergistic effect of IgA and IFN-{gamma} on TNF-{alpha} production and apoptosis of BALB/c peritoneal macrophages. Thioglycollate-elicited peritoneal macrophages were cultured in 96-well plates (at 105/well) with TBA61 or TBG65 (50 µg/ml each) alone or together with IFN-{gamma} (1 µg/ml) for 48 h. Culture supernatants were analysed for TNF-{alpha} concentration (A) and LDH activity, reflecting cell death (B). Mean values ± SD from triplicate cultures.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The experimental evidence reported herein emanated from the initial observation that mouse IgA can inhibit the logarithmic growth phase of transformed mouse macrophage cell lines, as demonstrated by the Trypan blue dye exclusion, proliferation and limited cell dilution assays. The profound extent of this effect is, however, alleviated by the recovery of cell counts after a delay of ~4 days in bulk cell cultures. This recovery may emanate either from a proportion of cells that down-regulated their IgA receptor or from a fraction of cells which did not display a sufficient density of the relevant IgA-binding receptors. These effects may depend on cytokine-mediated expression and metabolism of the responsible Fc{alpha} receptor on some populations of macrophages in vivo in certain anatomical locations (23).

Our search for such a receptor revealed that the constituent in a detergent lysate from the J774 cells which bound to IgA is galectin-3 (Mac-2), a ß-galactoside-binding S-type lectin, expressed among several other cell types on activated macrophages (24). In these experiments, IgA bound more to intracellular than to the cell-surface galectin-3 of J774 macrophages, indicating that the intracellular interaction of IgA with galectin-3 may interfere with its known anti-apoptotic function (25). We speculate that IgA binding with any surface receptor could be followed by galectin-3-mediated sequestration of IgA-containing complexes to phagosomes. However, full understanding of the mechanisms leading to the IgA-induced apoptosis of macrophages has yet to be examined.

The growth suppression by IgA was found to be associated with the stimulation of TNF-{alpha} production and apoptosis of J774 cells. However, we attribute such action exclusively or predominantly to intracellular TNF-{alpha}, because addition of anti-TNF antibody to J744 cell cultures failed to reverse the anti-proliferative action of IgA and addition of recombinant TNF-{alpha} was growth inhibitory only at non-physiologically high concentrations (results not shown). Such an interpretation is corroborated by the reported apoptotic effect of TNF-{alpha} following its microinjection into J774 cells (22). An alternative explanation could be the antibody-dependent cellular cytotoxicity of IgA (8) with macrophages acting as both effector and target cells, and it could also involve an effector role of TNF-{alpha} (26). As this mechanism would lead to relatively fast killing of cells, it is not corroborated by the requirement of the anti-proliferative action for a protracted (i.e. 24 h) exposure of cells to IgA. Therefore, we consider it to be a more likely explanation that growth inhibition is mediated by TNF-{alpha}-induced apoptosis. We found no evidence that CD95/FAS expression or NO synthesis may be responsible for IgA-induced apoptosis of macrophages. Furthermore, our finding that monomeric and polymeric IgA had a similar action on J774 cells suggests that murine IgA effector actions do not need the cross-linking of Fc receptors, unlike IgG which can activate macrophages only when immobilized with antigen or inert particles (27).

As permanent activation and apoptosis of normal macrophages by IgA would be deleterious, physiological protective mechanisms involving the rapid membrane integration of TNF-{alpha} and internalization of its receptor (28,29) may be operational in normal macrophages, but lost in the transformed macrophage cell lines. Alternatively, the regulatory role may concern the expression during cell maturation of the relevant macrophage receptors, which may become adequately expressed only upon direct or cytokine-mediated stimulation during microbial infections (9,30,31). Indeed, we found that IFN-{gamma} induced a small increase in IgA binding to macrophages and that IgA acted synergistically with IFN-{gamma} to induce TNF-{alpha} production in thioglycollate-elicited macrophages. In the light of this observation, although most of our results were obtained using a transformed macrophage cell line, they could reflect pertinent mechanisms which are finely regulated in macrophage populations during a wide range of protective or pathogenic host–parasite interactions.

In conclusion, this study demonstrates a novel, potentially important action of mouse IgA. The predominant emphasis in the study of the IgA isotype originally rested on its mucosal and hepatobiliary transport functions. Potent effector functions mediated by the Fc{alpha} receptor have also been identified, but only for human IgA and human myeloid cells, while a lack of corresponding knowledge for mouse IgA and mouse cells is a hindrance to analysis in murine experimental models. The in vitro activity of murine IgA on mouse macrophages described in this paper is probably mediated by a yet unknown IgA receptor, whose structure and function are amenable for further studies.


    Acknowledgements
 
This work was supported by grant QLK2-1999-00367 from the Fifth Framework of the European Commission, and grant Ro11208 from the Guy’s and St Thomas Charitable Foundation.


    Abbreviations
 
LDH—lactate dehydrogenase

L-NMMA—N-monomethyl-L-arginine

LPS—lipopolysaccharide

PE—phycoerythrin

TNF—tumor necrosis factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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