TNF ligand superfamily member 13B (B lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and Ig production. While the soluble form of BLyS/BAFF is thought to be the primary biologically active form, little is known about the regulation of its cleavage and processing. We provide evidence that Fcγ receptor cross-linking triggers a rapid release of soluble, biologically active BLyS/BAFF from myeloid cells. Surprisingly, this function is primarily mediated by FcγRI, but not FcγRIIa as defined by specific mAb, and can be initiated by both IgG and C reactive protein as ligands. The generation of a B cell proliferation and survival factor by both innate and adaptive immune opsonins through engagement of an Fcγ receptor, which can also enhance Ag uptake and presentation, provides a unique opportunity to facilitate Ab production. These results provide a mechanism by which Fcγ receptors can elevate circulating BLyS levels and promote autoantibody production in immune complex-mediated autoimmune diseases.

The B lymphocyte stimulator (BLyS),3 also known as BAFF (B cell activating factor of the TNF family)/TNFSF13B, TALL-1/zTNF4/THANK, is an important member of the TNF ligand superfamily (reviewed in Refs. 1, 2, 3, 4). BLyS/BAFF regulates B cell proliferation and differentiation and regulates Ig production (5). In facilitating B cell maturation, BLyS/BAFF preferentially supports T2 cell survival compared with T1-T2 differentiation (6, 7).

Studies in both model systems and humans have suggested that BLyS/BAFF is involved in humoral immune responses and in the pathogenesis of autoantibody mediated autoimmune diseases. BLyS−/− mice show deficiencies in peripheral B cell development and maturation (8, 9, 10). Additionally, overproduction of BLyS/BAFF in transgenic mice leads to increased germinal center formation, high levels of autoantibody production (rheumatoid factor, anti-DNA) and Ig deposition in the kidney (11). Similar to observations of increased BLyS in autoimmune mice, elevations in circulating BLyS/BAFF are found in the serum of patients with a variety of autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, Sjögrens Syndrome, and Wegener’s granulomatosis (12, 13, 14).

BLyS/BAFF is expressed in cells of both hematopoietic and non-hematopoietic origins. Hematopoietic cells expressing BLyS/BAFF include monocytes/macrophages, some lymphocytes, and dendritic cells. In these cells, BLyS/BAFF is initially expressed as a cell-bound transmembrane protein that is cleaved upon cell activation and is typically found in soluble form as a BLyS/BAFF trimer. In the presence of soluble APRIL (a proliferation-inducing ligand), a trimer of BLyS/APRIL has also been demonstrated (15). Neutrophils express BLyS/BAFF, but unlike other myeloid and lymphoid cells, BLyS/BAFF is cleaved intracellularly into its truncated, soluble form (16). Non-hematopoietic cells such as astrocytes and fibroblast-like synoviocytes have been shown to express BLyS/BAFF (17, 18, 19). While the regulation of expression of BLyS/BAFF is not completely understood, both IFN-γ and G-CSF up-regulate total BLyS/BAFF protein in monocytes and neutrophils (20), and treatment of monocytes with LPS also induces an increase in soluble BLyS/BAFF levels in culture (15, 20).

Although the membrane-associated form of BLyS/BAFF has potential for biological effects, the soluble form of BLyS/BAFF appears to be the primary biologically active form (21). We demonstrate rapid receptor-specific induced release of soluble biologically active BLyS/BAFF from myeloid cells. Surprisingly, this release is triggered primarily by cross-linking FcγRI with C reactive protein (CRP) or IgG, opsonins of the innate and adaptive immune systems. FcγR ligation, especially that of FcγRI, is known to enhance Ag uptake and presentation (22, 23). Thus, the concomitant elaboration of a factor promoting B cell survival and Ig production in response to either IgG or CRP presumably provides a microenvironment that facilitates the humoral immune response. Furthermore, these observations suggest a potentially important link between innate and adaptive immune responses and between elevations in circulating BLyS/BAFF and immune complex-mediated autoimmune disease.

Blood from healthy donors was collected into heparinized tubes (BD Biosciences). PBMCs were isolated by Ficoll-Hypaque gradient centrifugation as previously described (24). CD19+ cells were purified using magnetic microbeads (Miltenyi Biotec) on positive-selection columns. All studies were approved by the Institutional Review Board for Human Use.

Human U937, THP-1, and HL-60 cell lines were maintained as suspension culture in RPMI 1640 (Invitrogen Life Technologies) medium supplemented with 10% FCS, 1 mM sodium pyruvate, penicillin (100 U/ml), and streptomycin (100 μg/ml). In brief, 293T cells were stably transfected with a cDNA-encoding BLyS and were maintained as adherent cultures in DMEM (Invitrogen Life Technologies) with 10% FBS and 0.5 mg/ml G418.

Hybridoma clones producing monoclonal anti-BLyS Abs were generated by immunizing BALB/c mice with 200 μg recombinant human BLyS (extracellular domain, aa 71–285) before fusion of lymph node cells with NS1 myeloma cells. The primary hybridoma clones were screened by ELISA for binding to the recombinant BLyS. Positive clones were further screened with the transfected 293T cells expressing the membrane form of human BLyS. Cell surface expression of human BLyS was confirmed by flow cytometry using TACI-Fc fusion protein (10 μg/ml) followed by incubation with PE-conjugated goat-anti-human IgG (Southern Biotechnology Associates) (25). The monoclonal anti-BLyS Ab (mAb 7D4) was chosen for flow cytometry and immunoprecipitation.

A blotting polyclonal anti-BLyS Ab was obtained from Alexis Biochemicals. Monoclonal anti-CRP Ab HD2–4 (26) and anti-FcγRIIb mAb 4F5 (27) were used as described. Anti-FcγRI mAb 32.2 and anti-FcγRII mAb IV.3 hybridoma cell lines were purchased from American Type Culture Collection. Anti-CD14 and anti-CD19 Abs were purchased from Caltag Laboratories. mAb Fab/F(ab′)2 fragments were prepared by Rockland Immunochemicals and were free of detectable Fc region as assessed by blotting. F(ab′)2 goat anti-mouse IgG (GαM) was obtained from Jackson ImmunoResearch Laboratories. Rabbit IgG, human IgG isotype controls, IFN-γ, and human CRP were from Sigma-Aldrich. Aggregated human IgG was prepared by heating 20 mg/ml human IgG for 30 min at 55°C followed by removal of insoluble aggregates by spinning at 2000 × g for 10 min. In addition, highly purified CRP (sodium azide free, low endotoxin) was prepared from donor sera using a standard affinity chromatography protocol (28). Recombinant BAFF-R was purchased from BioVision, and was labeled by using Alexa Fluor 488 Microscale Protein Labeling Kit from Molecular Probes.

Soluble BLyS levels were assayed in supernatants of U937 or THP-1 cells by immunoprecipitation and Western blot. Cell associated BLyS was assessed by blotting whole cell lysates or by immunoprecipitation. Cells were lysed at 5 × 107 cells/ml in PBS/1% Nonidet P-40 lysis buffer containing a protease inhibitor mixture (Roche Diagnostics). Lysates or culture supernatants were then preincubated with protein G-Agarose beads at 4°C for 1 h followed by incubation with 7D4-conjugated protein G-agarose beads (Roche Diagnostics) at 4°C overnight. After three washes with PBS containing 0.1% Tween 20, the beads were boiled in SDS loading buffer. The samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% nonfat dry milk, blots were probed with anti-BLyS polyclonal Ab followed by HRP-conjugated mouse anti-rabbit IgG and detected with ECL Western Blotting Detection Reagents (GE Healthcare).

Calcium influx was measured as previous described (29). In brief, U937 cells were incubated at 37°C for 40 min with 5 μM Indo-1/AM (Molecular Probes). After loading, the cells were washed once with modified HBSS (1.1 mM CaCl2, 1.6 mM MgCl2, and 1 mg/ml BSA (pH 7.3)) and incubated with different anti-FcγR mAbs for 30 min at 4°C followed by one cold wash. The cells were then resuspended to 5 × 106/ml and analyzed on an SLM 8000C Spectrofluorometer (SLM-Aminco). Cells were stimulated with goat anti-mouse F(ab′)2 at 60 s.

U937 cells or human PBMC were first incubated with anti-FcγRI IgG or F(ab′)2 (mAb 32.2), with anti-FcγRIIa IgG or Fab (mAb IV.3), and/or with anti-FcγRIIb IgG or F(ab′)2 (mAb 4F5) at saturating concentrations (10 μg/ml) for 30 min at 4°C, washed twice, and resuspended in modified HBSS followed by cross-linking with GαM F(ab′)2 (35 μg/ml) at 37°C for 15 min. Cells were then washed and the remaining GαM binding sites were blocked with mouse IgG1/IgG2b mixture (final concentration 10 μg/ml) before FACS analysis.

Cells were prepared for flow cytometric analysis by incubating cells with saturating concentrations of specific mAb or an isotype control for 30 min on ice, followed by incubation with goat anti-mouse F(ab′)2 conjugated with FITC for 30 min on ice. Alternatively, cells were stained with biotinylated 7D4 followed by FITC-conjugated streptavidin. In isolated PBMC, monocytes were identified with anti-CD14 and B cells were identified with anti-CD19. Cells were analyzed on a FACSCan flow cytometer and data were analyzed using Cell Quest software (BD Immunocytometry).

Survival of CD19+ B cells was assessed as previously described (20). In brief, B cells at a density of 1 × 106/ml were seeded in the upper chamber of transwell plates (Corning) in RPMI 1640 medium supplemented with 10% FBS. Medium, 5 × 106/ml CD19-depleted mononuclear cells (MNC), or 1 × 106/ml U937 cells were placed in the lower chambers with or without IgG pre-coating. After seeding, the cells were cultured for 3 days. Surviving B cells were quantified by light microscope in the presence of trypan blue.

Results are presented as mean ± SEM, and differences between means were analyzed using the Student’s t test (GraphPad Prism). A probability of 0.05 was used to reject the null hypothesis that there is no difference between the experimental and control groups.

To examine the regulation of cell surface BLyS expression, we prepared and characterized a monoclonal anti-BlyS Ab (clone: 7D4, IgG2b, κ) that was selected based on its ability to recognize both the soluble and the membrane forms of BLyS. Activity of mAb 7D4 was determined using 293T cells transfected with full length human BLyS (Fig. 1,A, upper panel). TACI-Fc fusion protein was used as a positive control for detection of membrane BLyS expression (Fig. 1,A, lower panel). No binding of mAb 7D4 to the empty vector-transfected control 293T cells was observed. Detection of soluble BLyS was performed using a previously established BLyS ELISA with mAb 7D4 as the capture Ab (12). Soluble BLyS was detected in the supernatant of cells transfected with BLyS, but no soluble BLyS was detected in culture supernatants of cells transfected with empty vector (Fig. 1 B). Immunoprecipitation of supernatant derived BLyS with mAb 7D4 followed by Western blot confirmed that the released BLyS from the transfected cells was the predicted 17 kD cleaved form of BLyS, and BLyS in transfected 293T cell lysates was the full length 34 kD form (data not shown). These results indicate that mAb 7D4 is able to bind both cell surface and released soluble BLyS.

FIGURE 1.

Characterization of the anti-BLyS Ab 7D4. A, Flow cytometry analysis of 293T cells transfected with human BLyS or empty vector. Cells were stained with 10 μg/ml TACI-Fc fusion protein followed by PE-conjugated anti-human IgG (lower panel) or 5 μg/ml 7D4 followed by PE-conjugated anti-mouse IgG2b (upper panel). B, Expression levels of BLyS in culture supernatants of 293T cells (5 × 105/ml) cultured in fresh medium for 24 h. Soluble BLyS in culture supernatants was measured by sandwich ELISA using mAb 7D4 as capture Ab and rabbit anti-human BLyS polyclonal Ab as the detection Ab.

FIGURE 1.

Characterization of the anti-BLyS Ab 7D4. A, Flow cytometry analysis of 293T cells transfected with human BLyS or empty vector. Cells were stained with 10 μg/ml TACI-Fc fusion protein followed by PE-conjugated anti-human IgG (lower panel) or 5 μg/ml 7D4 followed by PE-conjugated anti-mouse IgG2b (upper panel). B, Expression levels of BLyS in culture supernatants of 293T cells (5 × 105/ml) cultured in fresh medium for 24 h. Soluble BLyS in culture supernatants was measured by sandwich ELISA using mAb 7D4 as capture Ab and rabbit anti-human BLyS polyclonal Ab as the detection Ab.

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Circulating monocytes and monocytic cell lines constitutively express BLyS/BAFF on the cell surface and, as previously reported, we found that stimulation of these cells with IFN-γ induced a marked increase in the level of surface BLyS expression (Fig. 2). Because CRP, an acute phase reactant and opsonin of the innate immune system, may be an important modulator of acquired immune responses, we determined whether CRP could alter cell surface BLyS/BAFF expression. Indeed, membrane BLyS/BAFF expression on both constitutive and IFN-γ stimulated human monocytic cell line U937 is decreased by cross-linking of CRP with F(ab′)2 fragments of a monoclonal anti-CRP Ab HD2–4. In cells with constitutive BLyS expression, cross-linked CRP induced a 16.2 ± 5.8% decrease (p = 0.026, n = 8) (Fig. 2,A) and in IFN-γ stimulated cells cross-linked CPR induced a 23.1 ± 5.6% decrease in surface BLyS expression (p = 0.014, n = 5) (Fig. 2 B).

FIGURE 2.

CRP and IgG induced reduction in membrane BLyS (mBLyS) expression. U937 cells (A), or U937 cells pretreated with IFN-γ overnight (B), were incubated with CRP (10 μg/ml) and then stimulated with anti-CRP F(ab′)2 mAb HD2–4 (10 μg/ml) followed by cross-linking with GαM F(ab′)2 (35 μg/ml), and surface BLyS expression was measured with mAb 7D4. C and D, U937 cells were incubated overnight alone or in the presence of rabbit IgG (rIgG) (200 μg/ml pre-coated on the plate surface), IFN-γ (200 U/ml), or aggregated human IgG (agg IgG)(200 μg/ml) as indicated, and cells were stained for surface mBLyS expression. Data presented are representative of three or more independent experiments. E, U937 cells were incubated 30 min alone or in the presence of agg IgG, followed by determination of surface mBLyS expression by Alexa 488-conjugated BAFF-R recombinant protein. Alexa 488-conjugated BSA was used as background staining control. F, Human PBMC were incubated with or without aggregated human IgG for 30 min, followed by determination of surface mBLyS expression on CD14+ cells by flow cytometry. Stimulated vs control, p = 0.012, n = 6.

FIGURE 2.

CRP and IgG induced reduction in membrane BLyS (mBLyS) expression. U937 cells (A), or U937 cells pretreated with IFN-γ overnight (B), were incubated with CRP (10 μg/ml) and then stimulated with anti-CRP F(ab′)2 mAb HD2–4 (10 μg/ml) followed by cross-linking with GαM F(ab′)2 (35 μg/ml), and surface BLyS expression was measured with mAb 7D4. C and D, U937 cells were incubated overnight alone or in the presence of rabbit IgG (rIgG) (200 μg/ml pre-coated on the plate surface), IFN-γ (200 U/ml), or aggregated human IgG (agg IgG)(200 μg/ml) as indicated, and cells were stained for surface mBLyS expression. Data presented are representative of three or more independent experiments. E, U937 cells were incubated 30 min alone or in the presence of agg IgG, followed by determination of surface mBLyS expression by Alexa 488-conjugated BAFF-R recombinant protein. Alexa 488-conjugated BSA was used as background staining control. F, Human PBMC were incubated with or without aggregated human IgG for 30 min, followed by determination of surface mBLyS expression on CD14+ cells by flow cytometry. Stimulated vs control, p = 0.012, n = 6.

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CRP can exert biological effects on myeloid cells through engagement of Fcγ receptors as cognate receptors (30). To directly determine whether the decrease in BLyS/BAFF surface expression can be modulated by Fcγ receptors, we incubated U937 cells with plate-bound IgG. In contrast to the induction of surface BLyS expression by IFN-γ, overnight culture of U937 on IgG-coated wells resulted in a marked decrease in cell surface BLyS/BAFF expression (52.2 ± 3.0%, p = 0.003, n = 3) (Fig. 2,C). In addition to surface-bound IgG, soluble aggregated human IgG also resulted in a marked decrease in cell surface BLyS/BAFF expression (Fig. 2,D), demonstrating that adherence to a surface is not necessary for the IgG-induced decrease in surface BLyS expression. To confirm that the Fc portion of the anti-BLyS Ab 7D4 was not contributing to the apparent IgG-induced decrease in cell surface BLyS, we utilized a soluble recombinant BLyS receptor (TNFSF13C or BAFF-R) corresponding to the extracellular domain for detection of surface BLyS. Indeed, using the soluble BAFF-R, we observed the same IgG-induced decrease in surface BLyS expression after IgG stimulation (54.7 ± 5.8%, p = 0.016, n = 4) (Fig. 2 E). Comparable results with surface-bound IgG were observed in two other human monocytic cell lines, THP-1 and HL-60 (data not shown). Overnight culture of U937 cells on mouse IgG F(ab′)2 coated wells did not change BLyS/BAFF expression (data not shown).

To examine whether the effect of FcγR cross-linking on monocytic cell lines could be extended to primary human cells, PBMC were isolated and stimulated with aggregated human IgG, and surface BLyS/BAFF expression on CD14+ monocytes was measured by flow cytometry. In concert with the cell line data, monocyte cell surface BLyS was consistently decreased by FcγR engagement (Fig. 2 F). These results indicate that FcγR stimulation of both monocytes and monocytic cell lines induces a marked decrease in cell surface BLyS/BAFF.

The kinetics of loss of cell surface BLyS were determined (Fig. 3). In contrast to the transcriptional regulation of BLyS expression by IFN-γ which peaks at 24 h (Fig. 3,A) (15, 31), FcγR cross-linking resulted in a rapid loss of cell surface BLyS/BAFF from the surface of U937 cells. Indeed, stimulation with surface IgG resulted in the rapid down-regulation of membrane BLyS/BAFF expression within 5 min, reaching a maximum by 15 min (Fig. 3,B). The decreased levels of cell surface BLyS/BAFF did not rebound during continued exposure to surface IgG over a period of 48 h (Fig. 3 A). We did not detect any change in BLyS mRNA levels by quantitative PCR during the 8–24 h incubation with IgG (data not shown). These results, together with the kinetic pattern of BLyS/BAFF loss from the cell surface, rule out the possibility that the FcγR-induced change on BLyS/BAFF expression is transcriptional.

FIGURE 3.

Kinetic analysis of mBLyS expression. U937 cells were incubated in the presence or absence of plate-bound rIgG (200 μg/ml precoated on the plate surface) or IFN-γ (200 U/ml) for varying times (A and B), followed by determination of surface mBLyS expression by flow cytometry. Data are presented relative to control mBLyS expression vs stimulated cells. Three to five independent time course studies were performed.

FIGURE 3.

Kinetic analysis of mBLyS expression. U937 cells were incubated in the presence or absence of plate-bound rIgG (200 μg/ml precoated on the plate surface) or IFN-γ (200 U/ml) for varying times (A and B), followed by determination of surface mBLyS expression by flow cytometry. Data are presented relative to control mBLyS expression vs stimulated cells. Three to five independent time course studies were performed.

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To determine the mechanism of loss of cell surface BLyS/BAFF, we considered the possibility that membrane BLyS/BAFF was released from the cell surface upon cleavage. Accordingly, we immunoprecipitated soluble BLyS from cell-free supernatants collected after FcγR ligation. Both THP-1 (Fig. 4,A) and U937 cells (Fig. 4,B) constitutively express the 32 kD membrane BLyS as seen by immunoprecipitation from cell lysates. In contrast, only the 17 kD soluble form could be immunoprecipitated from the cell-free culture supernatants after overnight IgG stimulation, demonstrating the appearance of a cleaved form of soluble BLyS/BAFF. The kinetics of the appearance of detectable cleaved soluble BLyS/BAFF demonstrated increasing protein at 8 and 20 h after stimulation (Fig. 4 B). A low level of cleaved BLyS/BAFF protein was observed in the non-stimulated cells after overnight culture consistent with a low level of constitutive BLyS/BAFF shedding. While we cannot formally exclude the possibility that some cell surface BLyS/BAFF is also internalized after FcγR cross-linking, these results clearly show the FcγR activation results in the generation of cleaved soluble BLyS/BAFF.

FIGURE 4.

Soluble BLyS released into supernatants upon FcγR cross-linking. Culture supernatants of THP-1 (A) or U937 cells (B) at varying time points were analyzed by immunoprecipitation of soluble BLyS. Cells were incubated with or without plate-bound rIgG for 24 h (A) or for three different time points (B). BLyS was immunoprecipitated from culture supernatants (A and B) and whole cell lysates (A) with anti-BLyS mAb 7D4 bound to protein G-agarose. The membrane 32 kD and cleaved soluble 17 kD forms of BLyS are indicated. Recombinant BLyS (extracellular domain) is also shown. As a loading control, the membrane in A was stripped and reprobed with an anti-actin Ab. Data are one representative experiment of three independent experiments.

FIGURE 4.

Soluble BLyS released into supernatants upon FcγR cross-linking. Culture supernatants of THP-1 (A) or U937 cells (B) at varying time points were analyzed by immunoprecipitation of soluble BLyS. Cells were incubated with or without plate-bound rIgG for 24 h (A) or for three different time points (B). BLyS was immunoprecipitated from culture supernatants (A and B) and whole cell lysates (A) with anti-BLyS mAb 7D4 bound to protein G-agarose. The membrane 32 kD and cleaved soluble 17 kD forms of BLyS are indicated. Recombinant BLyS (extracellular domain) is also shown. As a loading control, the membrane in A was stripped and reprobed with an anti-actin Ab. Data are one representative experiment of three independent experiments.

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Among different families of FcγRs, two activating receptors FcγRI and FcγRIIa and the inhibitory FcγRIIb, are expressed on the surface of U937 cells, THP-1 cells, and circulating monocytes. To determine which of those FcγRs is responsible for BLyS/BAFF release, receptor-specific activation of cells with specific anti-receptor mAbs was performed. To our surprise, using U937 cells, cross-linking of FcγRI but not FcγRIIa induced loss of BLyS/BAFF expression. FcγRI (CD64) cross-linking with mAb 32.2 IgG or F(ab′)2 resulted in a rapid and significant loss of surface BLyS/BAFF expression while cross-linking of FcγRIIa (CD32A) with mAb IV.3 IgG or Fab induced little to no change in surface BLyS/BAFF expression (Fig. 5,A). Heterotypic cross-linking of both FcγRI and FcγRIIa with anti-receptor mAbs resulted in similar loss of surface BLyS/BAFF expression relative to cross-linking of FcγRI alone. As a positive control for mAb IV.3 IgG and Fab activity, we tested the ability of cross-linked IV.3 and of 32.2 to elicit a rise in intracellular calcium levels. Homotypic ligation of either FcγRI or FcγRIIa was capable of inducing a calcium influx with FcγRIIa generating a stronger response than FcγRI (Fig. 5,C). Heterotypic cross-linking of both FcγRI and FcγRIIa enhanced the calcium influx to levels higher than FcγRI alone (Fig. 5 D). Together, these results document a unique role for FcγRI over FcγRIIa in the induction of BLyS/BAFF release, despite the fact that both receptors utilize an ITAM and that FcγRIIa resulted in a stronger Ca2+ influx than FcγRI.

FIGURE 5.

FcγRI is the major inducer of BLyS release from U937 cells. A, U937 cells (5 × 106/ml) were stimulated by receptor specific cross-linking of FcγRs. FcγRs were cross-linked using anti-FcγRI mAb 32.2 (10 μg/ml), anti-FcγRIIa mAb IV3 (10 μg/ml), and/or anti-FcγRIIb mAb 4F5 (10 μg/ml) followed by GαM F(ab′)2. B, PBMC were stimulated with anti-FcγRI mAb32.2 F(ab′)2 or anti-FcγRIIa mAb IV3 Fab followed by GαM F(ab′)2. CD14-positive cells were analyzed for mBLyS expression by flow cytometry. C, Homotypic cross-linking of 32.2 IgG or IV3 IgG induced a transient rise in intracellular Ca2+ in U937 cells. D, Calcium influx induced by heterotypic ligation of 32.2 F(ab′)2 (CD64) and either IV3 Fab (CD32A) or 4F5 Fab (CD32B) compared with 32.2 F(ab′)2 alone in U937 cells. Data presented are representative of three independent experiments.

FIGURE 5.

FcγRI is the major inducer of BLyS release from U937 cells. A, U937 cells (5 × 106/ml) were stimulated by receptor specific cross-linking of FcγRs. FcγRs were cross-linked using anti-FcγRI mAb 32.2 (10 μg/ml), anti-FcγRIIa mAb IV3 (10 μg/ml), and/or anti-FcγRIIb mAb 4F5 (10 μg/ml) followed by GαM F(ab′)2. B, PBMC were stimulated with anti-FcγRI mAb32.2 F(ab′)2 or anti-FcγRIIa mAb IV3 Fab followed by GαM F(ab′)2. CD14-positive cells were analyzed for mBLyS expression by flow cytometry. C, Homotypic cross-linking of 32.2 IgG or IV3 IgG induced a transient rise in intracellular Ca2+ in U937 cells. D, Calcium influx induced by heterotypic ligation of 32.2 F(ab′)2 (CD64) and either IV3 Fab (CD32A) or 4F5 Fab (CD32B) compared with 32.2 F(ab′)2 alone in U937 cells. Data presented are representative of three independent experiments.

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U937 cells and THP-1 cells also express the inhibitory Fc receptor, FcγRIIb. Using the recently described anti-FcγRIIb mAb 4F5 IgG or F(ab′)2 (27), we cross-linked FcγRIIb alone or in combination with the anti-FcγRI mAb 32.2 F(ab′)2. Homotypic cross-linking of FcγRIIb did not result in any change in cell surface BLyS expression (Fig. 5,A). Surprisingly, though heterotypic cross-linking of FcγRI and FcγRIIb resulted in diminished rise of intracellular Ca2+ (Fig. 5 D), there was no change in the magnitude of BLyS loss from the cell surface. These results suggest that BLyS cleavage is resistant to FcγRIIb-mediated inhibition.

To confirm the unique role of FcγRI in inducing BLyS/BAFF shedding on primary myeloid cells, we performed the anti-receptor mAb cross-linking studies using peripheral blood monocytes. Rapid loss of BLyS/BAFF expression was observed after stimulation of the cells through FcγRI. There was little reduction in BLyS/BAFF expression after stimulation of cells through FcγRIIa (Fig. 5 B). Consistent with the data in the U937 cell line, heterotypic ligation of FcγRI with FcγRIIa or FcγRIIb showed little alteration of the level of FcγRI induced BLyS/BAFF reduction (data not shown).

The functional significance of Fcγ receptor BLyS/BAFF shedding was determined by testing B cell viability as previously described (20) in cocultures with either U937 cells or primary MNC. In these experiments, the U937 cells or MNC were cultured in wells in the presence or absence of pre-coated IgG and the isolated B cells were cocultured in the same well separated by a 0.4-μm transwell membrane. As previously shown (18), B cells cultured alone had poor survival with nearly 60% of cells non-viable after 3 days in culture while addition of soluble rBLyS maintained 100% cell viability (Fig. 6). In the absence of FcγR ligation, U937 cells and primary MNC increased B cell survival (Fig. 6, A and B), presumably due in part to constitutive BLyS shedding (see Fig. 4). However, with IgG stimulation, both U937 cells and MNC significantly enhanced B cell viability above levels observed in the absence of IgG stimulation (p < 0.002). Not only was viability maintained, the higher levels of BLyS/BAFF shedding from U937 cells resulted in a slight increase in B cell numbers suggesting proliferation. Enhanced B cell survival after U937 cell/MNC FcγR stimulation was reversed with the inclusion of the neutralizing anti-BLyS Ab 7D4 (FcγR stimulation vs FcγR stimulation plus 7D4 neutralization; p < 0.018) (Fig. 6, A and B). These results confirm that biologically active soluble BLyS/BAFF is released from monocytes by FcγR cross-linking.

FIGURE 6.

Released soluble BLyS by FcγR stimulation supports B cell survival in vitro. Freshly isolated CD19+ B cells from healthy adults were cultured in the upper chamber of a transwell chamber. The bottom chamber contained either medium alone, U937 cells (± rIgG) (A) or primary CD19 depleted MNC (± rIgG) (B) for 72 h. Trypan blue-negative B cells were determined. As a positive control, recombinant soluble BLyS (200 ng/ml) was used (A). BLyS activity was neutralized with a blocking Ab (mAb 7D4, 50 μg/ml) (A and B). A, n = 3–10 independent determinations; B, n = 4–7 independent determinations. ∗, p < 0.018; ∗∗, p < 0.002.

FIGURE 6.

Released soluble BLyS by FcγR stimulation supports B cell survival in vitro. Freshly isolated CD19+ B cells from healthy adults were cultured in the upper chamber of a transwell chamber. The bottom chamber contained either medium alone, U937 cells (± rIgG) (A) or primary CD19 depleted MNC (± rIgG) (B) for 72 h. Trypan blue-negative B cells were determined. As a positive control, recombinant soluble BLyS (200 ng/ml) was used (A). BLyS activity was neutralized with a blocking Ab (mAb 7D4, 50 μg/ml) (A and B). A, n = 3–10 independent determinations; B, n = 4–7 independent determinations. ∗, p < 0.018; ∗∗, p < 0.002.

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Our study reports for the first time that the shedding of soluble BLyS/BAFF from myeloid cells is regulated in an Fcγ receptor specific manner by opsonins of both innate and acquired immune origins. Both CRP and IgG triggered rapid and significant shedding of soluble BLyS/BAFF from the surface of myeloid cells. A unique role for FcγRI (CD64) over FcγRII is shown by direct anti-receptor mAb engagement. The shed BLyS/BAFF is biologically active and directly promotes B cell survival.

Of interest, our results show that FcγRI (CD64) is much more effective in IgG-induced shedding process compared with FcγRIIa (CD32A). Both FcγRI and FcγRIIa are ITAM-bearing activating FcγRs, although the ITAM in the cytoplasmic domain of FcγRIIa differs in primary sequence from the ITAM-containing common FcRγ-chains that associate with FcγRI. There are now several lines of evidence, suggesting that the ligand binding α-chain of FcγRI can modulate FcRγ-chain-associated receptor functions such as phagocytosis, regulation of Th2 cytokines production (IL-6 and IL-10), and Ag presentation (32, 33, 34). Receptor-induced shedding of BLyS/BAFF is not a property of all FcRγ-chain-associated receptors because we observed no change in BLyS/BAFF expression after cross-linking of another γ-chain-associated receptor CD89 (data not shown).

It is known that ITAM-dependent activation can be inhibited by concomitant ligation of ITIM-bearing receptors such as FcγRIIb (35, 36). However, when we cross-linked FcγRI with FcγRIIb, the level of membrane BLyS/BAFF expression was indistinguishable from ligation of FcγRI alone despite significant reduction in the FcγRI induced Ca2+ flux. This result suggests that FcγR-mediated BLyS/BAFF shedding may be an ITAM-independent process (as shown in Fig. 4 A). While we cannot exclude the possibility that myeloid cells do not express sufficient FcγRIIb to inhibit the cleavage signaling pathway, these levels of FcγRIIb were sufficient to mediate >70% inhibition of the FcγRI triggered Ca2+ influx. Thus, either BLyS shedding is independent of ITAM signaling or it requires minimal levels of ITAM activation. Indeed, the tyrosine kinase inhibitor genistein achieved <50% reduction of the IgG-induced membrane BLyS/BAFF release (data not shown), suggesting that γ-chain tyrosine phosphorylation is not the only event contributing to BLyS/BAFF shedding.

BLyS/BAFF is critical for maintaining homeostasis of primary B cells. Deficiency of BLyS in the mouse causes a block in B cell development, and blocking of the BLyS/APRIL receptor TACI can reverse the autoantibody mediated autoimmune phenotype in the NZB/WF1 mouse (9, 37). Conversely, recent reports suggested that excess amounts of BLyS can rescue self-reactive B cells from peripheral deletion and allow them to enter forbidden follicular and marginal zone niches (38) and overexpression of BLyS/BAFF in the mouse causes autoimmune symptoms characteristic of systemic lupus erythematosus (39). It is likely that a furin-like protease plays a role in the cleavage of both cell surface BLyS/BAFF from myeloid cells and of intracellular BLyS/BAFF in neutrophils (16), but the exact enzyme or other factors that regulate proteolytic activity toward BLyS/BAFF remain unclear. Indeed, we observed that the furin inhibitor CMK and the metalloprotease inhibitor 1,10-phenanthroline demonstrated only marginal inhibition of FcγR-induced BLyS/BAFF shedding (data not shown). Interestingly, a similar protease-resistant FcγRI induced cleavage of CR1 (CD35) has been observed (40, 41, 42).

CRP circulates at a very low level in most healthy individuals, but in response to inflammation or infection, CRP levels rise rapidly and to a high level (43, 44, 45). CRP is traditionally regarded as an innate immune opsonin that acts in part through activation of the classical complement cascade and also interacts with myeloid receptors including FcγR. Previous work indicates that CRP can induce secretion of IL-6 and matrix metalloproteinase 1 (46), triggers shedding of L-selectin and up-regulation of the β2 integrin CD11b expression in myeloid cells (47), and triggers IL-6 receptor shedding from granulocytes (42). It is now recognized that many of these events are due to CRP engagement of Fc receptors on myeloid cells. Our observation raises the possibility that CRP could link innate responses to acquired immune responses by engaging FcγRs and modulating BLyS/BAFF cleavage resulting in promotion of B cell maturation.

Together, our studies demonstrate that the level of soluble functional BLyS/BAFF can be up-regulated by myeloid FcγRs, with a primary role for FcγRI. A central role of FcγRI allows for the regulation of soluble-cleaved BLyS/BAFF by both IgG and by the innate acute phase-reactive CRP. Furthermore, a unique role for FcγRI in the regulation of Ab production is suggested through enhancement of Ag presentation (23) and through cleavage of membrane BLyS making soluble BLyS available for B cell activation. This role for myeloid FcγRI contrasts with the ability of B cell FcγRIIb to inhibit BCR signaling and Ab production. The production of an Ab/autoantibody promoting B cell survival factor by Fcγ receptors provides a possible mechanism for known elevations in BLyS/BAFF in patients with autoantibody-associated autoimmune disease and supports current therapeutic BLyS/BAFF blockade in these diseases.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by grants from the National Institutes of Health (AR42476, AR33062, P01-AR49084, and M01-RR00032).

3

Abbreviations used in this paper: BLyS, B lymphocyte stimulator; BAFF, B cell activating factor; CRP, C reactive protein; GαM, goat anti-mouse IgG; MNC, mononuclear cell.

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