Antibody response against Betaferon® in immune tolerant mice: involvement of marginal zone B-cells and CD4+ T-cells and apparent lack of immunological memory.

PURPOSE: The immunological processes underlying immunogenicity of recombinant human therapeutics are poorly understood. Using an immune tolerant mouse model we previously demonstrated that aggregates are a major trigger of the antidrug antibody (ADA) response against recombinant human interferon beta (rhIFNß) products including Betaferon®, and that immunological memory seems to be lacking after a rechallenge with non-aggregated rhIFNß. The apparent absence of immunological memory indicates a CD4+ T-cell independent (Tind) immune response underlying ADA formation against Betaferon®. This hypothesis was tested. METHODS: Using the immune tolerant mouse model we first validated that rechallenge with highly aggregated rhIFNß (Betaferon®) does not lead to a subsequent fast increase in ADA titers, suggesting a lack of immunological memory. Next we assessed whether Betaferon® could act as Tind antigen by inactivation of marginal zone (MZ) B-cells during treatment. MZ B-cells are major effector cells involved in a Tind immune response. In a following experiment we depleted the mice from CD4+ T-cells to test their involvement in the ADA response against Betaferon®. RESULTS: Inactivation of MZ B-cells at the start of Betaferon® treatment drastically lowered ADA levels, suggesting a Tind immune response. However, persistent depletion of CD4+ T-cells before and during Betaferon® treatment abolished the ADA response in almost all mice. CONCLUSION: The immune response against rhIFNß in immune tolerant mice is neither a T-cell independent nor a classical T-cell dependent immune response. Further studies are needed to confirm absence of immunological memory (cells).

New targets in systemic lupus (part 2/2).

Glucocorticoids, aspirin, conventional antimalarials and immunosuppressants are the mainstay of treatment of Systemic Lupus Erythematosus (SLE). Until recently, the first three were the only agents approved for treatment. A better understanding of the pathophysiology of the immune system has identified new therapeutic targets. In fact, belimumab, a human monoclonal antibody to BLyS inhibitor has become, in recent months, the first drug approved for the treatment of SLE since 1957, underscoring difficulties of all kinds, including economic and organizational ones inherent to clinical trials on this disease. Many other molecules are in various stages of development and soon will have concrete results. In this review, we examined the mechanism of action and most relevant clinical data for these molecules.

TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+ NKT cells to initiate contact sensitivity in vivo.

LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Valpha14+Jalpha18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.

Human CD57+ germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination.

BACKGROUND: The function of CD57+ CD4+ T cells, constituting a major subset of germinal center T (GC-Th) cells in human lymphoid tissues, has been unclear. There have been contradictory reports regarding the B cell helping function of CD57+ GC-Th cells in production of immunoglobulin (Ig). Furthermore, the cytokine and co-stimulation requirement for their helper activity remains largely unknown. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57+ GC-Th cells in inducing B cell Ig synthesis. RESULTS: We demonstrated that CD57+ GC-Th cells are highly efficient in helping B cell production of all four subsets of Ig (IgM, IgG, IgA and IgE) compared to other T-helper cells located in germinal centers or interfollicular areas. CD57+ GC-Th cells were particularly more efficient than other T cells in helping GC-B cells but not naive B cells. CD57+ GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and class switch recombination in developing B cells. IgG1-3 and IgA1 were the major Ig isotypes induced by CD57+ GC-Th cells. CD40L, but not IL-4, IL-10 and IFN-gamma, was critical in CD57+ GC-Th cell-driven B cell production of Ig. However, IL-10, when added exogenously, significantly enhanced the helper activity of CD57+ GC-Th cells, while TGF-beta1 completely and IFN-gamma partially suppressed the CD57+ GC-Th cell-driven Ig production. CONCLUSIONS: CD57+CD4+ T cells in the germinal centers of human lymphoid tissues are the major T helper cell subset for GC-B cells in Ig synthesis. Their helper activity is consistent with their capacity to induce AID and class switch recombination, and can be regulated by CD40L, IL-4, IL-10 and TGF-beta.

Agonists of toll-like receptors 2 and 4 activate airway smooth muscle via mononuclear leukocytes.

RATIONALE: Toll-like receptors 2 and 4 (TLR2, TLR4) enable cellular responses to bacterial lipoproteins, LPS, and endogenous mediators of cell damage. They have an established role in the activation of leukocytes, endothelial cells, and some smooth muscle cell types, but their roles in airway smooth muscle are uncertain. OBJECTIVES: To determine the roles of TLRs in activation of airway smooth muscle. METHODS: Airway smooth muscle cells were cultured with TLR agonists, in the presence or absence of mononuclear leukocytes. MEASUREMENTS AND MAIN RESULTS: We observed expression of TLR2 and TLR4 mRNAs, which could be upregulated by treatment with proinflammatory cytokines in primary human airway smooth muscle, but no important functional responses to agonists of these TLRs were seen. Coincubation of airway smooth muscle with peripheral blood mononuclear cells, at concentrations as low as 250 mononuclear cells/ml, resulted in a marked cooperative response to TLR stimuli, and synergistic production of cytokines, including chemokines (interleukin [IL-]-8) and IL-6. This cooperative response was greater when monocytes were enriched and was transferable using supernatants from LPS-stimulated peripheral blood mononuclear cells. Activation of cocultures required IL-1 generation from mononuclear cells, and was blocked by IL-1 receptor antagonist, though IL-1 generation alone was not sufficient to account for the magnitude of mononuclear cell-dependent coculture activation. CONCLUSIONS: These data indicate that potent amplification of inflammation induced by TLR agonists, such as LPS, may be achieved by cooperativity between airway smooth muscle and leukocytes involved in immune surveillance or inflammation.

[Assessment of the rhythm of lymphocyte stimulation and suppression as a criterion of prognosis of the effects of immunomodulatory drugs]. / Otsenka ritma stimuliatsii limfotsitov kak kriterii prognoza deistviia immunomoduliatorov.

The authors discuss mechanisms of action of immunocorrectors the targets of which are various populations of antigen-presenting lymphoid cells and approaches to application of modulators with different mechanisms of action in disease. To assess a long-term effect of the drug which produces an oscillatory effect (activation/suppression) on T and B cell activity, competitive relations between T and B cells should be tested and taken into account. Common practice of immunocorrection on the basis of T cell activity without consideration of the functional state of other lymphoid sets, especially B cells, deteriorates efficacy of immunocorrection. It is necessary to know the time of T- and B-cell activation and suppression to schedule combined use of drugs with different mechanisms of action.

Bromelain modulates T cell and B cell immune responses in vitro and in vivo.

The ability to modulate immune responses is a major aim of many vaccine and immunotherapeutic development programs. Bromelain, a mixture of cysteine proteases, modulates immunological responses and has been proposed to be of clinical use. However, the identity of the immune cells affected by bromelain and the specific cellular functions that are altered remain poorly understood. To address these shortcomings in our knowledge, we have used both in vitro and in vivo immunological assays to study the effects of bromelain. We found that bromelain enhanced T cell receptor (TCR) and anti-CD28-mediated T cell proliferation in splenocyte cultures by increasing the costimulatory activity of accessory cell populations. However, despite increased T cell proliferation, bromelain concomitantly decreased IL-2 production in splenocyte cultures. Additionally, bromelain did not affect TCR and CD28-induced proliferation of highly purified CD4+ T cells, but did inhibit IL-2 production by these cells. In vivo, bromelain enhanced T-cell-dependent, Ag-specific, B cell antibody responses. Again, bromelain induced a concomitant decrease in splenic IL-2 mRNA accumulation in immunized mice. Together, these data show that bromelain can simultaneously enhance and inhibit T cell responses in vitro and in vivo via a stimulatory action on accessory cells and a direct inhibitory action on T cells. This work provides important insights into the immunomodulatory activity of bromelain and has important implications for the use of exogenous cysteine proteases as vaccine adjuvants or immunomodulatory agents.

Total serum IgE levels in chronic hepatitis C: influence of interferon alpha therapy.

BACKGROUND: Liver disease has been considered a prominent cause of IgE elevation. No data on serum IgE levels in chronic hepatitis C have been reported. Interferon-alpha is a standard therapy for chronic hepatitis C. Cytokine use is a promising type of immunomodulation in the treatment of IgE-mediated diseases. The effects of interferon-alpha therapy on serum IgE have not been fully evaluated. The aim of the study was to evaluate both serum IgE levels in patients with chronic hepatitis C and the course of these levels after interferon-alpha therapy. PATIENTS AND METHODS: Serum IgE was determined in 100 adult patients with chronic hepatitis C (24 atopics according to positive skin prick tests and 76 nonatopics) and in 75 healthy controls (25 atopics and 50 nonatopics). Serum IgE measurements were repeated at 1 and 3 months of therapy with recombinant interferon-alpha (3 x 106 units s.c. 3 times weekly) in 34 of these patients. RESULTS: Serum IgE levels were similar in chronic hepatitis C patients and in controls when adjusted for atopic status. Among patients with chronic hepatitis C, serum IgE levels were unrelated to liver necroinflammatory activity. A modest but statistically significant increase of IgE values was observed after interferon-alpha therapy, particularly in patients with no virological response. CONCLUSIONS: Chronic hepatitis C is not a significant cause of increased total serum IgE values. Serum IgE increase in some patients with liver disease may be related to the cause of liver injury and not to liver disease per se. Interferon-alpha therapy in patients with chronic hepatitis C is followed by no modification or even a moderate increase of serum IgE values.

Ganglioside GD1a enhances immunoglobulin production by human peripheral blood mononuclear cells.

We previously reported that ganglioside GD1a greatly enhanced spontaneous immunoglobulin (Ig) production by human peripheral blood mononuclear cells (PBMC) in vitro. We herein examined the mechanism for the stimulatory effect of GD1a.PBMC from healthy volunteers were cultured with GD1a. The amounts of IgG, IgM, and IgA and cytokine activity in the culture supernatants were measured by enzyme-linked immunosorbent assays. Proliferation was determined by [3H] thymidine uptake.GD1a at 10(-6) M increased IgG, IgM, and IgA production by PBMC 2.10-fold, 2.10-fold, and 2.23-fold above the control values, respectively. GD1a did not affect the proliferation and viability of PBMC. GD1a did not alter Ig production of B cells alone. Anti-interleukin-6 (IL-6) or anti-IL-10 antibody each partially blocked the GD1a-induced enhancement of Ig production by PBMC, and the addition of both antibodies completely blocked the enhancement. GD1a increased IL-6 and IL-10 production of monocytes without altering those of T cells or B cells. The supernatant from GD1a-treated monocytes enhanced B cell Ig production to a greater extent than that from medium-treated monocytes. The supernatant-mediated effect of GD1a was partially blocked by anti-IL-6 or anti-IL-10 antibody, and the addition of both antibodies completely blocked the GD1a effect. GD1a-induced increases of IL-6 and IL-10 production in monocytes were both blocked by Ca(2)+/calmodulin (CaM)-dependent phosphodiesterase (PDE) inhibitors, 8-methoxymethyl-3-isobutyl-1-methylxanthine and vinpocetin, but not by other signal-transducing enzyme inhibitors. The culture with GD1a enhanced Ca(2)+/CaM-dependent PDE activity in monocytes. These results suggest that GD1a may indirectly enhance B cell Ig production in whole PBMC by increasing IL-6 and IL-10 production of monocytes via promoting Ca(2)+/CaM-dependent PDE activity.

Dysregulation of B7.2 (CD86) expression on monocytes of HIV-infected individuals is associated with altered production of IL-2.

T helper (Th) responses are mediated in part by immunoregulatory cytokines and the signals delivered by the costimulatory CD28-B7 pathway. In this study, we have investigated the relationship between the regulation of B7 isoform expression on antigen-presenting cells from HIV+ individuals and the production of Th cytokines. The level of expression of both B7.1 and B7.2 isoforms as measured by mean channel fluorescence was significantly decreased on freshly isolated monocytes from HIV+ individuals compared with HIV- controls. However, the levels of expression of B7.1 and B7.2 on both B cells and monocytes increased significantly following culture in HIV+ individuals compared with HIV- controls. B7 expression is subject to regulation by immunoregulatory cytokines. Therefore, we analysed the regulation of B7 expression by cytokines, namely IL-10 and tumour necrosis factor-alpha (TNF-alpha), the production of which is enhanced in HIV infection and have similar inhibitory effects on B7 expression. Two groups of HIV+ individuals were distinguished on the basis of the inhibitory effect of IL-10 and TNF-alpha on monocyte B7.2 expression. IL-10 inhibited B7.2 expression on monocytes from some HIV+ individuals (termed responders) like the HIV- controls. However, in a subset of HIV+ individuals (non-responders) this inhibitory effect was lost. Loss of inhibition of B7.2 expression by IL-10 was associated with significantly reduced IL-2 production by phytohaemagglutinin (PHA)- stimulated peripheral blood mononuclear cells (PBMC). These observations showing an association of B7 dysregulation on monocytes and B cells with altered production of IL-2 may have implications in HIV immunopathogenesis.

Interleukin-10 abrogates the inhibition of Epstein-Barr virus-induced B-cell transformation by memory T-cell responses.

In vitro infection of human B lymphocytes by Epstein-Barr virus (EBV) results in their growth transformation and establishment of immortalized lymphoblastoid cell lines. The virus was found to encode a homologue of the pleiotropic cytokine interleukin-10 (IL-10), which has wide-ranging effects on the immune system. We investigated the effect of human IL-10 (hIL-10) and viral IL-10 (vIL-10) on EBV-specific immunological memory, as assessed by the inhibition of EBV-induced B-cell transformation by the autologous T cells. We found that IL-10 abrogates the inhibitory capacity of T cells. This IL-10 effect is mediated through suppression of T-cell activation-induced IL-2 and interferon-gamma production and through a direct enhancement of EBV-infected B-cell growth.

IL-12 up-regulates CD40 ligand (CD154) expression on human T cells.

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4+ Th1 cells and their IFN-gamma production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA level. T cells costimulated by IL-12 provided more efficient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction.

The effects of T cell subpopulations and recombinant interleukin (IL)-2 on peripheral B cell function in patients with myasthenia gravis.

Myasthenia gravis is an autoimmune disease mediated by antibodies to acetylcholine receptors (AChR) of skeletal muscle. The production of anti-AChR antibodies has been shown to be T cell dependent. To elucidate the mechanism(s) of anti-AChR antibody production in myasthenic patients, we studied the effects of regulatory T cells and/or IL-2 on the differentiation of AChR-primed B cells, with use of AChR stimulation for the induction of anti-AChR antibodies in vitro. Our data suggest that CD8+ T cells possess some complicated functions. CD8+ T cells could not only provide help for B cells to secrete anti-AChR antibody, but also possibly inhibit response of CD4+ T cells or kill B cells, then repress anti-AChR antibody production in MG patients. There might be some defect either in the number or function of CD8+ T cell in MG patients. Exogenous IL-2 could completely restore the suppression activity of CD8+ T cells in anti-AChR antibody production in vitro.

A role for phosphatidylinositol 3-kinase in generating T cell help for B cell growth and differentiation.

Evidence supporting the importance of the 3-phosphoinositide signaling pathway in lymphocyte activation is rapidly accumulating. In our study, we assessed the effects of two PI 3-kinase inhibitors, wortmannin and LY294002, on T cells as a means to analyze the role of the PI 3-kinase-signaling pathway in the generation of T cell help for B cell growth and differentiation. For these studies, B cells were cocultured with CD3-activated mitomycin C-treated T cells to induce B cell responsiveness. Of interest, wortmannin or LY294002 pretreatment of the T cell population significantly inhibited T cell-dependent induction of B cell proliferation and differentiation. The failure of wortmannin-treated CD3-activated mitomycin C-treated T cells to provide help in driving the differentiation of B cells to Ig-secreting cells could not be corrected by the addition of exogenous IL-2. Further studies designed to elucidate the mechanism by which wortmannin-treated T cells failed to provide B cell help indicated that wortmannin and LY294002 significantly inhibited the induction of CD40 ligand and, to a lesser extent, intercellular adhesion molecule-1 expression. These results suggest that the PI 3-kinase-signaling pathway, or other wortmannin- and LY294002-sensitive pathways, may be important for the induction of expression of crucial interaction molecules, such as CD40 ligand, on T cells and thus indicates that D-3 phosphoinositides play a pivotal role in regulating T cell-dependent B cell activation.

Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response.

Systemic lupus erythematosus is characterized by B cell production of pathogenic autoantibodies dependent upon cooperation from CD4+ Th cells. The interaction between CD40 on B cells and CD40 ligand (CD40L) on Th cells is necessary for normal thymus-dependent Ab production. An anti-murine CD40L mAb blocks binding of CD40L to CD40 and prevents primary and secondary immune responses to thymus-dependent Ags. In this study, New Zealand Black x New Zealand White lupus-prone mice treated with this anti-CD40L Ab from ages 4 to 10 mo had reduced anti-DNA autoantibody production and renal disease and significantly prolonged survival compared with control mice. Pathologic examination verified the absence of significant renal damage or immune deposition in responding mice. Mice that responded to treatment did not develop an Ab response to the administered Ab. Long-term survivors mounted a substantial Ab response to keyhole limpet hemocyanin after completion of anti-CD40L Ab treatment, suggesting that some of the immunosuppressive effects of the Ab may be reversible. These results suggest a human form of this Ab may have therapeutic utility in human systemic lupus erythematosus.

Nebulized IFN-gamma inhibits the development of secondary allergic responses in mice.

The effects of nebulized IFN-gamma on primary and secondary IgE production and development of airway hyper-responsiveness (AHR) were investigated. BALB/c mice received primary exposure to aerosolized OVA daily for 10 days and developed anti-OVA IgE responses, immediate cutaneous reactivity to OVA, and altered airway function when assayed on day 12. After secondary exposure to OVA challenges on days 30 and 31, these mice developed an amplified IgE response, heightened cutaneous reactivity to OVA and AHR when measured on day 37. Administration of IFN-gamma for 13 days, beginning 3 days prior to and during primary OVA sensitization, resulted in a decrease in anti-OVA IgE, increases in serum anti-OVA IgG2a levels, a decrease in cutaneous reactivity to OVA, and normal airway function when assessed on day 12 after primary sensitization. This treatment also prevented the development of secondary anti-OVA IgE responses and altered airway responsiveness but did not induce a secondary rise in anti-OVA IgG2a in the serum measured on day 37. Treatment with IFN-gamma on days 26 to 30, well after primary responses were established but just prior to secondary OVA challenge, abolished the development of secondary anti-OVA IgE responses, resulted in an increase in anti-OVA IgG2a in the serum, and prevented the development of AHR. In vitro, CD4+ T cells obtained from OVA-sensitized mice treated with either "early" or "late" IFN-gamma inhibited IgE production. Delivery of IFN-gamma to the airways can prevent secondary allergen sensitization even after primary sensitization has been achieved and this effect is mediated by CD4+ T cells.

In vitro inhibition by intravenous immunoglobulin of human T cell-dependent B cell differentiation induced by staphylococcal superantigens.

Treatment with intravenous Ig (IVIG) is efficacious not only in humoral immunodeficiency diseases but in several nonimmunodeficiency disorders as well. Since microbial superantigens (SAg) have been postulated to play a role in promoting in vivo pathogenic autoantibody production and since IVIG preparations are rich in anti-SAg antibodies, we tested whether IVIG could inhibit in vitro SAg-driven human T cell-dependent B cell differentiation. We demonstrate that IVIG inhibits such B cell differentiation by at least three different mechanisms. Early addition of IVIG inhibits B cell differentiation not only in SAg-stimulated PBMC cultures but in anti-CD3- and pokeweed mitogen (PWM)-stimulated cultures as well, pointing to a SAg-nonspecific inhibitory effect. However, anti-SAg antibodies contained in IVIG can also effect SAg-specific inhibition, since polyclonal rabbit anti-SAg antisera added early to peripheral blood mononuclear cell (PBMC) cultures inhibit neither anti-CD3- nor PWM-driven B cell differentiation and inhibit B cell differentiation triggered only by the specific SAg against which the individual antiserum was raised. Finally, late addition of IVIG at a time at which B cells have already committed to terminal differentiation inhibits SAg-driven, but not anti-CD3- or PWM-driven, generation of Ig-secreting cells (IgSC). This late inhibition is associated with enhanced SAg-dependent cytolytic activity against Raji cell targets which is dramatic in PBMC cultures but is often not detectable in T + B cell cultures. Reconstitution of T + B cell cultures with natural killer cells restores the enhancing capacity of IVIG on SAg-dependent cytolytic activity as well as the late inhibitory effects of IVIG on IgSC generation. Understanding the multiple mechanisms through which IVIG can inhibit SAg-driven B cell differentiation may offer a rational basis for determining which patients are likely to favorably respond to IVIG administration.

[Effects of beta-adrenergic compounds on IgE production]. / Effets des composés beta 2-adrénergiques sur la production d'IgE.

We studied the effects of beta 2-adrenoceptor agonist on IgE production in vitro in human and in vitro and in vivo in mouse. We observed that salbutamol and fenoterol potentiate the IL-4-induced IgE production from peripheral blood mononuclear cells. This effect is associated by an enhanced mRN expression for IgE. Fenoterol also potentiated, but in a lesser extent, the IgE production from purified B lymphocytes stimulated by both IL-4 and CD40, suggesting that the activity of beta 2-adrenoceptor agonist is mediated through T lymphocyte or monocyte modulation. Fenoterol also inhibited the PHA-induced IFN-gamma production by T lymphocytes. Analogues of cAMP or activator of PKA also elicited an increase in IgE production. Moreover, the effect of fenoterol on IgE production was suppressed in the presence of PKA inhibitor. Salbutamol also potentiated the IL-4-induced IgE production from murine splenocytes activated by LPS. Furthermore, mice sensitized to ovalbumin elicited increased IgE responses after daily injection of salbutamol. This was accompanied by an increased in cytokines of Th2 subtypes. Our results showed that beta 2-adrenoceptor agonist, which are currently used in the treatment of asthma, potentiate the IgE production in vitro and in vivo.

Endogenous cytokine expression profiles in retinoic acid-induced IgA production by LPS-stimulated murine splenocytes.

All-trans-retinoic acid (RA) enhances IgA production by LPS-stimulated murine splenocytes. After stimulation by RA and LPS, or by LPS alone, total RNA was extracted from cultured cells on Days 1 to 4, and the kinetics of expression of various cytokine mRNAs were analyzed by the RT-PCR method. RA induced the expression of IL-5 and TGF-beta 2 mRNAs in the LPS-stimulated cells. In addition, the expression of IL-6 and IL-2 mRNAs was more intensive in RA-stimulated cells than in unstimulated cells. TGF-beta 1 and TGF-beta 3 mRNAs were constitutively expressed in both culture groups. RA enhanced IgA production by LPS-stimulated spleen cells but not that by LPS-stimulated mu(+) naive splenic B-cells. For RA-induced IgA production, the B-cells required T-cells or the culture supernatant from RA-stimulated T-cells. Furthermore, exogenous IL-5 replaced the T-cell requirement, at least in part, in RA-induced IgA production by LPS-stimulated B-cells. This reaction was partially inhibited by anti-TGF-beta-neutralizing antibodies. These findings suggest that RA induces IgA production by (IL-5 + LPS)-stimulated B-cells in TGF-beta-independent and TGF-beta-dependent manners.