RP105-lacking B cells from lupus patients are responsible for the production of immunoglobulins and autoantibodies.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 46, No. 12, December 2002, pp 3259–3265 DOI 10.1002/art.10672 © 2002, American College of Rheumatology RP105-Lacking B Cells From Lupus Patients Are Responsible for the Production of Immunoglobulins and Autoantibodies Yuji Kikuchi,1 Syuichi Koarada,2 Yoshifumi Tada,2 Osamu Ushiyama,2 Fumitaka Morito,2 Noriaki Suzuki,2 Akihide Ohta,2 Kensuke Miyake,2 Masao Kimoto,2 Takahiko Horiuchi,3 and Kohei Nagasawa2 Objective. We previously reported that B cells lacking the RP105 molecule, which proved to be highly activated B cells, are increased in the peripheral blood of patients with systemic lupus erythematosus (SLE). In the present study, we attempted to determine whether RP105-negative B cells obtained from SLE patients would be capable of producing autoantibodies as well as immunoglobulins. Methods. RP105-positive and RP105-negative B cells, sorted by cell sorter, were cultured for 5 days without stimulation, or were stimulated with Staphylococcus aureus Cowan 1 strain (SAC) or recombinant interleukin-6 (IL-6). For the assay of autoantibodies, RP105-positive and RP105-negative B cells were cultured separately for 10 days with anti-CD3 antibody– stimulated T cells. The production of immunoglobulins and autoantibodies was determined by enzyme-linked immunosorbent assay. Results. We demonstrated that RP105-negative B cells, but not RP105-positive B cells, obtained from SLE patients could spontaneously produce IgG and IgM in vitro until day 5. SAC and IL-6 enhanced production of IgG and IgM by RP105-negative B cells but failed to induce such production by RP105-positive B cells. The latter cells, however, when cocultured with activated T cells in the presence of IL-10, produced IgG, although the amount was very small compared with that produced by RP105-negative B cells. Most important, under these conditions, anti–double-stranded DNA antibodies were produced only by the RP105-negative B cells obtained from SLE patients. Conclusion. These data indicate that RP105negative B cells, constituting a subset of B cells in SLE patients, are highly activated and may be responsible for the production of autoantibodies as well as polyclonal immunoglobulins. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysfunction of T cells and polyclonal B cell activation, resulting in production of pathogenic IgG autoantibodies such as anti–doublestranded DNA (anti-dsDNA) antibody. High-affinity anti-dsDNA IgG in SLE bears characteristics of an antigen-driven, T cell–dependent immune response. Such autoantibodies may play a critical role in the progression of lupus nephritis. In fact, exacerbations of the disease are preceded by increasing levels of antidsDNA (1–4). It is known that peripheral B cells obtained from patients with SLE contain populations that spontaneously produce immunoglobulins, including autoantibodies, and also can mature into antibodysecreting cells when cultured in vitro in the absence of obvious activators of B cell differentiation (5–7). In vivo, antigen-specific activation and differentiation of B cells occur in germinal centers in the lymphoid system, where naive B cells undergo activation, proliferation, somatic hypermutation of rearranged V region genes, immunoglobulin isotype switching, and Supported in part by grant-in-aid 13670459 from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 1 Yuji Kikuchi, MD: Saga Medical School, Saga, Japan, and Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; 2Syuichi Koarada, MD, Yoshifumi Tada, MD, Osamu Ushiyama, MD, Fumitaka Morito, MD, Noriaki Suzuki, MD, Akihide Ohta, Kensuke Miyake, MD, Masao Kimoto, MD, Kohei Nagasawa, MD: Saga Medical School, Saga, Japan; 3Takahiko Horiuchi, MD: Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan Address correspondence and reprint requests to Yuji Kikuchi, MD, Department of Medicine and Biosystemic Science, Kyushu University, Graduate School of Medical Sciences, Fukuoka 812-8582, Japan. E-mail: Kikuchin@intmed1.med.kyushu-u.ac.jp. Submitted for publication March 5, 2002; accepted in revised form August 29, 2002. 3259 3260 KIKUCHI ET AL subsequent positive and negative selection by antigen. Moreover, activated B cells mature into antibodyproducing plasma cells, or, alternatively, become memory B cells within germinal centers (8–11). RP105, a novel molecule on B cells identified in 1994 (12), transmits an activation signal that leads to B cell proliferation and protection from apoptosis in mice. The extracellular domain of RP105 is structurally similar to that of Toll-like receptors, suggesting that RP105 senses pathogen invasion and activates B cells (13–15). Mice devoid of RP105 were recently generated, and it has been shown that RP105 regulates lipopolysaccharide signaling in B cells (16). In contrast to these findings in mice, little is known about RP105 in humans: the human homologue of RP105 has been identified and its monoclonal antibody established (17,18). Histologic studies showed that RP105 was expressed mainly on mature B cells in mantle zones, and that germinal center cells were negative for RP105 (19). It is known that virtually all mature B cells have RP105 molecules on their surface, in humans as well as in mice (19). We previously demonstrated that the number of RP105-negative B cells is significantly increased in the peripheral blood of patients with SLE, Sjögren’s syndrome, and dermatomyositis, the pathophysiology of which is characterized by highly activated B cells (20–22). Particularly in SLE, the increase in RP105-negative B cells was well correlated with disease activity (20). We defined the phenotype of RP105negative B cells as CD95-positive, CD86-positive, and CD38-bright, which is consistent with that of activated B cells or germinal center B cells. Moreover, RP105negative B cells had intracellular IgG, suggesting that they could produce IgG (20). It was of great interest, therefore, to determine whether RP105-negative B cells obtained from patients with SLE would be responsible for the production of autoantibodies. In this study, we demonstrated that RP105-negative B cells, but not RP105-positive B cells, produced immunoglobulins without stimulation and even produced anti-dsDNA antibody, with activated T cell help. PATIENTS AND METHODS Patients. Seven patients who had active SLE and were attending our hospital were enrolled in this study. All patients were female and met the American College of Rheumatology criteria for SLE (23). The activity of SLE was assessed by the SLE Disease Activity Index (SLEDAI) (24), and we defined the active disease state as SLEDAI scores of ⱖ14. All patients in this study had anti-dsDNA antibody in their sera. In patients 1, 2, 4, 5, and 7, SLE had not been diagnosed previously, and the diagnosis was first made at the time of this analysis. Although patient 3 had been on a maintenance dosage of prednisolone (15 mg/day), progressive proteinuria occurred. Patient 6 had been given a maintenance dosage of predonisolone (5 mg/day), but slight fever and decreased serum level of complement emerged. Patients ranged in age from 17 to 65 years (mean 36 years). Cell preparation. Samples of peripheral venous blood obtained from patients with SLE were collected into tubes containing heparin. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation over FicollHypaque (Pharmacia Biotech, Uppsala, Sweden) gradient. Immunofluorescence analysis of B cells. PBMCs were stained with fluorescein isothiocyanate–conjugated antihuman RP105 monoclonal antibody (mAb) (20) and phycoerythrin-conjugated anti-human CD19 mAb (BD PharMingen, San Diego, CA), and analyzed using FACScan. Statistical analyses were performed using CellQuest software (Becton Dickinson, Mountain View, CA). Cells were then sorted into 2 fractions, RP105-negative CD19-positive B cells, and RP105-positive CD19-positive B cells, using the Epics Elite cell sorter (Coulter, Hialeah, FL). Preparation of T cells. PBMCs obtained from a healthy donor were isolated by centrifugation over FicollHypaque (Pharmacia Biotech) gradient. CD19-negative cells were isolated by negative selection using immunomagnetic beads (Dynal, New Hyde Park, NY). CD19-negative cells were allowed to adhere to plastic dishes. Nonadherent cells were used as T cells. T cells were stimulated with 10 g/ml of phytohemagglutinin (Difco, Detroit, MI) and 100 units/ml of recombinant interleukin-2 (IL-2) (Takeda Chemical Industries, Osaka, Japan) every 4 days. These activated T cells were irradiated at 3,000 rad, and 2 ⫻ 105 cells were cultured in anti-human CD3 antibody–coated 96-well plates. Production of total IgG and IgM. RP105-positive B cells and RP105-negative B cells were separately suspended in RPMI 1640 containing 10% fetal calf serum and incubated in a 5% CO2 incubator at 37°C in 96-well plates (Nalgene Nunc, Milwaukee, WI). Cells (2 ⫻ 104) were cultured for 5 days without stimulation, or were stimulated with 0.001% Staphylococcus aureus Cowan 1 strain (SAC), or 1 ng/ml recombinant IL-6 (rIL-6) (Genzyme, Cambridge, MA) with or without 50 g/ml anti–IL-6 antibody (Genzyme). The cultured supernatants were harvested on day 3 and day 5 and added to goat anti-human immunoglobulin–coated (Biosource, Fleurus, Belgium) 96-well Nunc Maxisorb immunoplates (Nalgene Nunc) with control human IgG and IgM (Chemicon, Hofheim, Germany) overnight at 4°C. After discarding the supernatants and washing with 0.05% Tween in phosphate buffered saline (PBS), the bound human immunoglobulin was detected with peroxidase-labeled goat anti-human IgG ␥ chain and IgM chain (BioSource) at a dilution of 1:2,000. The plates were washed again and developed with o-phenylendiamine. The reaction was stopped with 3N HCl solution, and the optical density at 490 nm was read with an ImmunoMini NJ-2300 microplate reader (System Instruments, Tokyo, Japan). Production of IL-6. The supernatants cultured with no stimulation were measured using an ultrasensitive IL-6 enzyme-linked immunosorbent assay (ELISA) kit (BioSource). SIGNIFICANT ROLE OF RP105-NEGATIVE B CELLS IN SLE Table 1. Demographic data and frequency of RP105-negative B cells in the peripheral blood of female patients with systemic lupus erythematosus Patient 1 2 3 4 5 6 7 Age, years Treatment at study entry % RP105-negative B cells 17 32 26 36 39 40 65 – – Prednisolone, 15 mg/day – – Prednisolone, 5 mg/day – 20.8 19.9 20.0 19.0 31.0 8.8 16.7 Production of anti-DNA IgG. Monoclonal anti-human CD3 antibody (UCHL1; Genzyme/Techne, Minneapolis, MN) was diluted to a concentration of 1 g/ml in PBS. A total of 100 l was added to each culture well, and the plates were incubated for at least 2 hours at room temperature, then washed with PBS to remove any anti-CD3 antibody that was not bound to the plates. RP105-positive and RP105-negative B cells were separately cultured for 10 days at a density of 5–10 ⫻ 104 cells/well with 2 ⫻ 105 T cells stimulated with immobilized anti-CD3 antibody and 10 ng/ml of recombinant IL-10 (Genzyme/Techne), and the supernatants were harvested. Single-stranded DNA (ssDNA) was obtained by boiling calf thymus DNA (Worthington, Freehold, NJ) for 5 minutes, followed by rapid cooling on ice. Double-stranded DNA was obtained by treating calf thymus DNA with S1 nuclease. The plates were coated with 10 g/ml of calf thymus DNA. The plates were washed and blocked with PBS containing 1% bovine serum albumin. The samples of culture supernatants were incubated in the plates overnight at 4°C. The bound human immunoglobulin was detected as described above. In the serum obtained from 1 patient with SLE, the titer of anti-dsDNA antibody was 500 IU/ml, and the titer of antissDNA antibody was 500 AU/ml; this serum was used as a control. 3261 produced neither IgG nor IgM (detection threshold for IgG 150 ng/well, for IgM 7.5 ng/well). IgG production by RP105-negative B cells obtained from patients 1–3 was not increased on day 5 (13.5 ⫾ 6.7 g/ml) compared with day 3 (13.4 ⫾ 6.2 g/ml). In contrast, IgG production by RP105-negative B cells obtained from patient 4 increased on day 5 (13.0 g/ml) compared with day 3 (7.50 g/ml) (Figure 1). IgM production by RP105-negative B cells from patients 1, 3, and 4 was not significantly increased on day 5 (245.0 ⫾ 74.6 ng/ml) compared with day 3 (213.0 ⫾ 61.8 ng/ml) (Figure 1). The effect of SAC and rIL-6 on in vitro immunoglobulin synthesis by RP105-positive and RP105negative B cells. Addition of 0.001% SAC enhanced total IgG production by RP105-negative B cells from patients 1–3 by an average of 2.7-fold above the mean value of controls. It did not, however, influence total IgG production by RP105-negative B cells from patient 4. Similarly, SAC increased total IgM production by RP105-negative B cells from patients 1 and 3 by an average of 1.9-fold above the mean value of controls. Conversely, SAC could not induce IgG from RP105positive B cells, although it induced a small amount of IgM production by RP105-positive B cells from patient 3 (Figure 2). Like SAC, rIL-6 (1 ng/ml) increased total IgG production by RP105-negative B cells from patients 1–3 by an average of 3.1-fold above the mean value of controls, but exerted no effect on total IgG production by RP105-negative B cells from patient 4. Similarly, RESULTS RP105-negative B cells in SLE patients. The frequency of peripheral RP105-negative B cells ranged from 8.8% to 31.0% (mean 19.5%) (Table 1). RP105negative B cells, which are rare in normal subjects, appeared in significant numbers in SLE patients, as previously described (20). IgG and IgM production by RP105-positive and RP105-negative B cells obtained from SLE patients. RP105-positive and RP105-negative B cells obtained from SLE patients with active disease (patients 1–4) were cultured separately without stimulation, and IgG and IgM produced in the supernatant were measured on days 3 and 5. RP105-negative B cells spontaneously produced IgG and IgM (13.4 ⫾ 5.8 g/ml and 245 ⫾ 74.6 ng/ml, respectively, on day 5), but RP105-positive B cells Figure 1. Spontaneous production of polyclonal immunoglobulins by RP105-negative B cells obtained from 4 patients with systemic lupus erythematosus. Highly purified RP105-positive and RP105-negative B cells were cultured separately for 5 days. Supernatants were harvested after 3 and 5 days of culture, and IgG and IgM secretion was measured by enzyme-linked immunosorbent assay. 3262 Figure 2. The effect of Staphylococcus aureus Cowan 1 (SAC) on immunoglobulin production in RP105-positive and RP105-negative B cells obtained from 4 patients with systemic lupus erythematosus. RP105-positive and RR105-negative B cells were cultured separately for 5 days in the presence of 0.001% SAC. The culture supernatants were assessed for total IgG and IgM by enzyme-linked immunosorbent assay. rIL-6 enhanced total IgM production by RP105-negative B cells from patients 1 and 3 by an average 2.0-fold above the mean value of controls. In RP105-positive B cells, however, the addition of rIL-6 did not induce production of either IgG or IgM (Figure 3). In an attempt to confirm the effect of IL-6, anti–IL-6 antibody was added in this system. Anti–IL-6 Figure 3. The effect of interleukin-6 (IL-6) or anti–IL-6 antibody on immunoglobulin production in RP105-positive and RP105-negative B cells obtained from 4 patients with systemic lupus erythematosus. RP105-positive and RP105-negative B cells were cultured separately for 5 days in the presence of IL-6 (1 ng/ml) or anti–IL-6 antibody (50 ng/ml). The culture supernatants were assessed for total IgG and IgM by enzyme-linked immunosorbent assay. KIKUCHI ET AL Figure 4. Spontaneous interleukin-6 (IL-6) production by RP105positive and RP105-negative B cells obtained from patients with systemic lupus erythematosus. The culture supernatants that were harvested as described in Figure 1 were measured for the quantity of IL-6. antibody inhibited both IgG and IgM production (an average of 18% and 30%, respectively) by RP105negative B cells from patients 1–4 (Figure 3). IL-6 production by RP105-positive and RP105negative B cells. Although SAC and IL-6 exerted no effect on IgG and IgM production by RP105-negative B cells from patient 4, anti–IL-6 antibody reduced production of both IgG and IgM. This suggested that endogenous production of IL-6 would contribute to spontaneous production of immunoglobulins. Therefore, we measured IL-6 production in the supernatants of unstimulated cultures, using an IL-6–specific ELISA. It was demonstrated that both RP105-positive and RP105negative B cells (patients 2–4) produced IL-6. In particular, the concentration of IL-6 produced by RP105negative B cells obtained from patient 4 was extremely high (Figure 4). These results suggest that a large amount of endogenous IL-6 production increased production of immunoglobulins, rendering the extra SAC and IL-6 stimulation ineffective in patient 4. In this respect, the results suggest that RP105-negative B cells produce immunoglobulins, with help from autocrine and paracrine IL-6. Anti-DNA antibody production by RP105negative B cells obtained from SLE patients. Among several autoantibodies, anti-DNA antibodies are particularly important in the pathophysiology of SLE (1–4). It was of great interest, therefore, to determine whether SIGNIFICANT ROLE OF RP105-NEGATIVE B CELLS IN SLE these activated and immunoglobulin-producing RP105negative B cells would also be capable of producing anti-DNA antibodies. We measured the amount of anti-ssDNA and anti-dsDNA antibodies in the culture supernatant of RP105-positive and RP105-negative B cells obtained from patients 1, 3, 5, 6, and 7. In this system, however, the amount of these specific antibodies was too small to be compared, although the antibodies were detected. It has been shown that anti-CD3– stimulated T cells induce terminal differentiation of B cells into nondividing high-rate immunoglobulinsecreting plasma cells (25), and that IL-10, present at an elevated level in the sera of SLE patients, may contribute to the abnormal production of immunoglobulins and autoantibodies in SLE (26). This prompted us to culture RP105-positive and RP105-negative B cells obtained from SLE patients together with anti-CD3–activated T cells obtained from normal subjects, in the presence of IL-10 for 10 days. On day 10, both subsets of B cells were still viable (⬎50%), using trypan blue dye exclusion. With the help of activated T cells and IL-10, even RP105-positive B cells became capable of producing total IgG, although the amount was apparently small compared with that produced by RP105-negative B cells (mean ⫾ SD 16.0 ⫾ 6.5 g/ml versus 56.2 ⫾ 20.6 g/ml) (Figure 5). It was also observed that a very small amount of anti-ssDNA 3263 Figure 6. Anti–double-stranded DNA (anti-dsDNA) antibody production by RP105-negative B cells. Culture supernatants collected as described in Figure 5 were assayed for anti–single-stranded DNA (anti-ssDNA) antibody and anti-dsDNA antibody. Bars show the mean ⫾ SD. antibodies was detected in the supernatant of RP105positive B cells obtained from 2 (patients 3 and 5) of 5 SLE patients. In contrast, however, RP105-negative B cells from all 5 patients produced a significant amount of anti-ssDNA antibodies (29.4 ⫾ 18.1 AU/ml) (Figure 6). Most importantly, RP105-negative B cells obtained from all SLE patients were found to be capable of producing anti-dsDNA antibodies (0.133 ⫾ 0.107 IU/ml), whereas RP105-positive B cells from any of the 5 patients did not produce the antibodies (Figure 6). B cells from normal subjects did not produce either anti-ssDNA or antidsDNA antibodies (data not shown). DISCUSSION Figure 5. IgG production by RP105-positive and RP105-negative B cells with T cell help. RP105-positive and RP105-negative B cells (obtained from patients 1, 3, 5–7) were cultured for 10 days with activated T cells obtained from the normal subject, together with interleukin-10. The culture supernatants were assayed for total IgG. Bars show the mean ⫾ SD. RP105, detected on B cells, has been shown to be associated with B cell proliferation and resistance to apoptosis in mice (12). We previously demonstrated that most human B cells also have RP105 molecules (20). Interestingly, however, we found that a significant percentage of B cells obtained from patients with SLE, Sjögren’s syndrome, and dermatomyositis are devoid of RP105; all of these diseases are known to be characterized by B cell activation (21,22). In particular, in SLE, the number of RP105-negative B cells was well correlated with disease activity and the serum level of IgG. Characterization of RP105-negative B cells revealed that they were IgD-negative and CD38-bright with the expression of CD95 and CD86, being consistent with activated B cells or germinal center B cells (20). Moreover, RP105-negative B cells had intracellular IgG, suggesting that RP105-negative B cells can be induced to produce class-switched immunoglobulin (IgG) (20). 3264 Harada et al reported that early plasma cells, in which the intensity of expression of CD38 antigen was between that of germinal center B cells and that of bone marrow plasma cells, were significantly increased in the peripheral blood of patients with active SLE, bacterial septicemia, or liver cirrhosis (27). The phenotype of RP105-negative B cells appears to be similar to that of the early plasma cells. In this study, we demonstrated that only a subset of RP105-negative B cells, which represented ⬃20% of B cells (Table 1), produced a significant amount of IgG and IgM by day 3 without any stimulation. It is known that peripheral B cells from patients with SLE contain populations that spontaneously produce immunoglobulins and also mature into antibody-secreting cells when cultured in vitro in the absence of obvious activators of B cell differentiation (5–7). The ability of these cell populations to spontaneously produce immunoglobulins may be equal to or very close to that of RP105-negative B cells. Conversely, RP105-positive B cells are quite different in that they did not produce any immunoglobulins, either spontaneously or even with stimulation by IL-6 or SAC. IL-6 is a main cytokine that induces terminal differentiation of B cells into plasma cells and also protects early plasma cells from apoptosis (28). It is known that the serum concentration of IL-6 is elevated in patients with active SLE (29,30). In this study, we demonstrated that IL-6 enhanced production of both IgG and IgM by the RP105-negative B cells obtained from 3 of 4 SLE patients tested, and that this effect of IL-6 was abrogated by anti–IL-6 antibody. Interestingly, RP105-negative B cells from patient 4, which did not respond to exogenous IL-6, produced, by themselves, a significant amount of IL-6 (Figure 4), suggesting the existence of IL-6–mediating autocrine B cell activation. This possibility seems to be supported by Nagafuchi et al, who demonstrated that constitutive expression of IL-6 receptors on B cells in conjunction with spontaneous IL-6 production by B cells induces autocrine B cell activation, which may lead to B cell hyperactivity and autoantibody secretion in patients with SLE (31,32). In this respect, immunoglobulin production by RP105negative B cells could be enhanced by the autocrine/ paracrine IL-6 loop. Our result, that RP105-positive B cells did not respond to IL-6 to produce immunoglobulins despite the ability to produce a certain amount of IL-6, suggests that those cells may not be differentiated enough to mediate an IL-6 signal into the cells. It is now delineated that in patients with SLE, a B cell subset lacking RP105 molecules can produce auto- KIKUCHI ET AL antibodies such as anti-ssDNA and anti-dsDNA antibodies as well as nonspecific total IgG. Our results suggest that anti-DNA antibodies are disease-specific, because RP105-negative B cells obtained from patients with Sjögren’s syndrome or dermatomyositis failed to produce these antibodies (data not shown). With respect to autoantibody formation, however, T cell help seems to be of great importance, because RP105-negative B cells alone produced a very small amount of antibodies. In this system, the T cells should only be activated and are not necessarily required to be autoreactive. In contrast, RP105-positive B cells were shown to be activated to produce IgG with T cell help, probably by CD40–CD40 ligand interaction, but failed to produce anti-dsDNA antibodies. These findings indicate that RP105-negative B cells obtained from SLE patients are already activated, and that some of them, if not all, are committed to differentiate to produce autoantibodies. In lupus mice, a B cell subset that expresses CD5 is known to produce autoantibodies and to be associated with the pathogenesis of the disease (33–35), whereas in other mouse models the autoantibody production is independent of CD5-positive B cells (36). In humans, however, CD5-positive B cells are clearly distinct from RP105-negative B cells and do not seem to be associated with the pathogenesis of SLE (20,37–40). So far, despite extensive efforts, any B cell subset that can produce autoantibodies in SLE has not been identified. 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