IL-6 is a potent cofactor of IL-1 in IgM synthesis and of IL-5 in IgA synthesis

In these studies we determined the capacity of IL-6 to act as a differentiation cofactor for murine Peyer's patch B cells producing different Ig classes and subclasses. In preliminary studies we determined that sufficient endogenous IL-6 was produced in LPS-induced cell systems to obscure responses to exogenous IL-6. We therefore studied IL-6 effects on Peyer's patch B cells (T cell-depleted cell populations) in the absence of LPS, relying on responses of in vivo-activated cells. rIL-1 alpha or purified IL-6 only slightly enhanced synthesis of IgM over minimal baseline levels in Peyer's patch T cell-depleted cell cultures; however, when IL-6 was added to cultures also containing rIL-1, IgM synthesis was very substantially increased. In addition, rIL-5 alone gave rise to a modest increase in IgM synthesis and its effect was not enhanced by either rIL-1 or IL-6. IgG production (mainly IgG3) followed a similar pattern. In contrast, IgA production was only modestly increased above baseline by rIL-1, rIL-5, or IL-6 alone or by rIL-1 and IL-6 in combination, but was greatly increased by rIL-5 and IL-6 in combination. The effect of IL-6 on Ig synthesis in the above studies was not due to an effect on cell proliferation. In summary, these data indicate that B cells differ in respect to the cytokines supporting maximal terminal differentiation and thus the class of Ig produced may depend on the presence of a particular combination of cytokines and lymphokines.     

IgG1 and IgG2a production by autoimmune B cells treated in vitro with IL-4 and IFN-gamma

Autoimmune MRL-lpr/lpr and NZB/W mice spontaneously secrete large quantities of pathogenic IgG1 and IgG2a autoantibodies. NZB mice also produce autoantibodies but these tend to be of the IgM H chain class. This work examines whether differences in the isotype of autoantibody produced by lupus-prone mice reflects differences in the sensitivity of autoreactive B cells to lymphokine-mediated IgG secretion. Twenty-five percent of normal BALB/c B cells produced IgG1 when stimulated in vitro with IL-4 plus LPS. This was comparable with the effect of IL-4 on small resting B cells from MRL-lpr/lpr and NZB/W mice. In contrast, less than 8% of the resting B cells from NZB mice produced IgG1 under these conditions. LPS plus IFN-gamma induced 5% of BALB/c and NZB/W but only 1% of NZB B cells to secrete IgG2a. Because lymphocytes from both young and old NZB mice showed diminished IgG1 and IgG2a secretion after lymphokine treatment, B cells from this strain appeared to be intrinsically resistant to the effects of IL-4 and IFN-gamma. In contrast, a disproportionately large proportion (22%) of B cells from adult MRL-lpr/lpr mice produced IgG2a when treated with IFN-gamma in vitro. Only B cells from MRL-lpr/lpr mice with active disease responded with such high levels of IgG2a production: cells from animals that had not yet developed clinical disease produced normal levels of IgG2a. Within each strain, B cells producing antibodies against autoantigens such as DNA, bromelain-treated mouse RBC and Sm responded to treatment with IL-4 and IFN-gamma in a manner indistinguishable from B cells producing antibodies against conventional Ag such as TNP and ARS.     

Transforming growth factor-beta 1 is a costimulator for IgA production

Transforming growth factor-beta 1 (TGF-beta 1) belongs to a family of polypeptides involved in the regulation of cell growth and differentiation. We have examined the ability of TGF-beta 1 to regulate isotype specific Ig secretion by murine spleen B cells. TGF-beta 1, in the presence of rIL-2, induced a synergistic 10-fold or greater increase in IgA secretion by LPS-stimulated spleen B cells. TGF-beta 1 alone had little to no effect on IgA secretion. In contrast, TGF-beta 1, with or without rIL-2, markedly inhibited IgG1 and IgM secretion under the same conditions. The costimulatory activity of TGF-beta 1 and rIL-2 on IgA secretion was seen in cultures of surface IgA negative B cells and was inhibited by anti-TGF-beta 1 antibody in a dose dependent manner. Vicia villosa agglutinin non-adherent Peyer's patch T cells, which secrete IL-2, also synergized with TGF-beta 1 and could substitute for the activity of LPS and rIL-2 on the IgA response. Finally, IL-5 added after 2 days of culture, but not at the beginning of culture, synergized with TGF-beta 1 on the IgA response. These studies indicate that TGF-beta 1 can interact with other lymphokines and selectively modulate the IgA response.     

Regulation of isotype production by IL-4 and IL-5. Effects of lymphokines on Ig production depend on the state of activation of the responding B cells

The effects of IL-4 and IL-5 on the production of Ig of different isotypes was investigated. We compared B cells from spleen and from Peyer's patches either stimulated with LPS or without added polyclonal stimulation. We also compared high density (small) and low density (large) B cells. The effect of lymphokines depended on the size and source of the B cells as well as on whether LPS was added. As expected, small B cells from either lymphoid compartment responded to LPS alone and IL-4 suppressed IgM and IgG3 production and enhanced IgG1. In contrast, when large B cells were examined, the suppressive effects of IL-4 were much less apparent but the enhancement of IgG1 was still marked. IL-5 alone had only minimal effects in LPS-stimulated cultures but the combination of IL-4 plus IL-5 appeared to overcome much of the IL-4-mediated suppression of IgM, and IgA production was enhanced. In the absence of LPS, a quite different profile is seen. First, small B cells make little if any response. Second, there is dramatic synergy between IL-4 and IL-5 in the response of large B cells, which is independent of isotype. Third, IL-4 does not suppress any isotype in the absence of LPS. Fourth, IL-4 plus IL-5 stimulate large Peyer's patch B cells to produce 10 times more IgA but three times less IgM than large spleen B cells. Fifth, Th2 cells directly stimulate both large and small B cells.     

Defective isotype-specific regulation of IgA anti-erythrocyte autoantibody-forming cells in NZB mice

B cell hyperactivity characterizes many autoimmune diseases. In NZB mice this is manifested by a variety of immunologic aberrations, including increased B cell proliferation and hyper IgM and IgA secretion in vitro. Recent studies have shown that IgA secretion can be suppressed or enhanced in an isotype-specific manner by a soluble factor(s), called IgA-binding factor (IgABF), produced by IgA FcR-bearing T cells. We now show that T cells from young NZB mice, cultured with high concentrations of IgA, produce an IgABF that has aberrant biologic activity when compared to IgABF produced from IgA FcR+ T cells of BALB/c mice. Although BALB/c IgABF normally suppresses proliferation and secretion by IgA-producing B cells, neither proliferation nor IgA secretion from normal murine IgA-B cells is suppressed by NZB IgABF. In fact, IgA secretion is significantly enhanced by NZB IgABF. We also present the first evidence of IgA anti-mouse erythrocyte (anti-MRBC) autoantibody-forming cells present in the spleens of NZB mice. Whereas BALB/c IgABF suppresses the in vitro generation of IgA anti-MRBC autoantibody-forming cells by NZB spleen cells, NZB IgABF enhances this response. Of particular interest is the development of IgA anti-MRBC autoantibody-forming cells in cultures of spleen cells from nonautoimmune BALB/c mice in the presence of NZB IgABF. These studies suggest that isotype-specific T cells factors might play an important role in the development of autoantibody-forming cells.     

Defective B cell clonal regulation and autoantibody production in New Zealand black mice

By using the splenic fragment assay in a KLH-primed host, we have evaluated the clonal anergy model of tolerance in DBA/2 and spontaneously autoimmune NZB mice. Unlike immature B cells from DBA/2 mice (which are tolerized by encounter with TNP-OVA), SIg- B cells from NZB mice respond to TNP-KLH with equal precursor frequency in TNP-OVA-tolerized or control fragments. In additional experiments, SIg- bone marrow or mature spleen cells of DBA/2 or NZB origin were adoptively transferred into irradiated (DBA/2 X NZB) F1 X xid hosts, and host-derived splenic fragments were stimulated in vitro with LPS and growth factors. These experiments revealed a substantial anti-ssDNA precursor frequency in NZB marrow and spleen (2.5 and 5.1, respectively, per 10(7) transferred cells). In DBA/2 SIg- marrow cells, there was an anti-ssDNA precursor frequency of 1.3 to 3.5/10(7) transferred cells; however, anti-ssDNA-producing clones were reduced in fragments derived from recipients of DBA/2 as compared with NZB spleen cells (0.2 to 1.9/10(7) transferred cells). By using a replica plate technique, we evaluated fragments from recipients of DBA/2 SIg- marrow cells or mature spleen cells for anti-TNP reactivity. In fragments derived from recipients of DBA/2 SIg- marrow cells, 92% of anti-TNP-producing fragments also bound ssDNA. In fragments derived from recipients of DBA/2 spleen cells, only 43% of anti-TNP-producing fragments also bound ssDNA. Our findings document that NZB marrow-derived immature B cells abnormally resist tolerance induction, and that clonal anergy/selection operates in directing the B cell repertoire away from autoantibody formation.     

The effects of IL-4 and IL-5 on the IgA response by murine Peyer's patch B cell subpopulations

IL-5 has been shown to specifically enhance IgA secretion in LPS-stimulated splenic B cell cultures. Maximum enhancement of IgA in such cultures, however, requires IL-4 in addition to IL-5. Because the Peyer's patches (PP), compared with spleen and lymph nodes, are enriched for precursors of IgA-secreting cells, we tested whether IL-4 and IL-5 would have a more profound effect on IgA secretion by polyclonally stimulated PP cells than spleen cells. The combination of IL-4 and IL-5 causes a comparable enhancement of IgA secretion in both LPS-stimulated PP and splenic B cell cultures. The majority of IgA secreted in LPS-stimulated PP cell cultures is derived from the sIgA- population. Furthermore, the binding high level of peanut agglutinin, germinal center subpopulation of PP cells is essentially nonresponsive to LPS, even in the presence of lymphokines; the majority of secreted IgA in these cultures is derived from the binding low level of peanut agglutinin population. In contrast to LPS-stimulated cultures, PP B cells secrete considerably more IgA than splenic B cells when polyclonally stimulated by a clone of autoreactive T cells in the presence of IL-4 and IL-5. The majority of IgA made by T cell-stimulated PP cell cultures is derived from the sIgA+ population. In these cultures, sIgA- PP cells and spleen cells secrete comparable levels of IgA and other non-IgM isotypes suggesting that sIgA- PP B cells are similar to splenic B cells in their potential to switch to IgA. In T cell-stimulated cultures the majority of IgA as well as of all other isotypes is also derived from the nongerminal center, binding low level of peanut agglutinin population.     

Alternate mucosal immune system: organized Peyer's patches are not required for IgA responses in the gastrointestinal tract

The progeny of mice treated with lymphotoxin (LT)-beta receptor (LTbetaR) and Ig (LTbetaR-Ig) lack Peyer's patches but not mesenteric lymph nodes (MLN). In this study, we used this approach to determine the importance of Peyer's patches for induction of mucosal IgA Ab responses in the murine gastrointestinal tract. Immunohistochemical analysis revealed that LTbetaR-Ig-treated, Peyer's patch null (PP null) mice possessed significant numbers of IgA-positive (IgA+) plasma cells in the intestinal lamina propria. Further, oral immunization of PP null mice with OVA plus cholera toxin as mucosal adjuvant resulted in Ag-specific mucosal IgA and serum IgG Ab responses. OVA-specific CD4+ T cells of the Th2 type were induced in MLN and spleen of PP null mice. In contrast, when TNF and LT-alpha double knockout (TNF/LT-alpha-/-) mice, which lack both Peyer's patches and MLN, were orally immunized with OVA plus cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were induced. On the other hand, LTbetaR-Ig- and TNF receptor 55-Ig-treated normal adult mice elicited OVA- and cholera toxin B subunit-specific mucosal IgA responses, indicating that both LT-alphabeta and TNF/LT-alpha pathways do not contribute for class switching for IgA Ab responses. These results show that the MLN plays a more important role than had been appreciated until now for the induction of both mucosal and systemic Ab responses after oral immunization. Further, organized Peyer's patches are not a strict requirement for induction of mucosal IgA Ab responses in the gastrointestinal tract.     

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel IgA receptor expressed on a murine B cell lymphoma.

The specificity and properties of a novel IgA receptor expressed on the surface of a tissue culture-adapted B cell lymphoma, T560, that originated in murine gut-associated lymphoid tissue, have been explored. Like the IgA receptors of murine T and splenic B cells studied by others, the T560 IgA receptor is trypsin sensitive and neuraminidase resistant and is up-regulated on T560 cells by exposing them overnight to high concentrations of polymeric IgA. Unlike them, the T560 IgA receptor is inhibited by low concentrations of IgM and high concentrations of IgG2a and IgG2b, binds at pH 4.0 but not at pH 8.0, is down-regulated by activation of protein kinase C and is sensitive to phosphatidylinositol-specific phospholipase C, indicating that it is glycosyl phosphatidylinositol-linked to the cell membrane. It is not a cell-bound form of galactosyl transferase, does not appear to bind to Ig through carbohydrate residues and does not react specifically with antibody to secretory component. It may be a completely new, cross-reactive receptor, perhaps related in some way to the polymeric Ig receptor or to the receptor for IgA expressed on the apical surface of Peyer's patch M cells, which is known to cross-react with IgG. Alternatively, it may be homologous to the highly IgA-specific Fc alpha R of T cells but, perhaps because of its glycosyl phosphatidylinositol linker, may have an ability to move and interact with other Ig receptors on the cell surface such that Ig bound to them are cross-inhibitory.     

Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

Regulation of mucosal IgA production in vitro by autoreactive immunoregulatory T cells from murine Peyer's patches

In this study, we investigated whether Peyer's patch-derived T-cell subsets participate in vitro in self major histocompatibility (MHC) class II antigen (Ag)-mediated immunoregulatory circuits for gut-mucosal IgA isotype selection in the presence of Peyer's patch (PP)-derived syngeneic surface immunoglobulin M (sIgM)-bearing B cells. When fresh (in vitro unstimulated) sIgM-bearing B cells were cocultured with fresh, PP-derived L3T4+ Vicia villosa-nonadherent (VV-) T cells (T helper (Th) cells), the production of all class-specific immunoglobulins (Ig), but, in particular, IgA, was enhanced two- to sixfold. This augmented Ig production was, however, reduced by nearly 50% when fresh PP-derived Lyt2+ VV-T cells (suppressor T cells) were added. Furthermore, addition of PP-derived L3T4+ VV+ and Lyt 2+ VV+ T cells abrogated, by nearly 100%, the suppression induced by the Lyt 2+ VV-T cells (contrasuppression). When lipopolysaccharide (LPS)-stimulated, PP-derived sIgM-bearing B cells were cocultured with the Th cells, the production of each class-specific Ig was similarly enhanced, but Ig levels exceeded those obtained with cultures of the unstimulated B cells (P less than 0.001). Anti-I-A or anti-I-E monoclonal antibody (mAb) inhibited the induction of each immunoregulatory T-cell effector activity (P less than 0.001), and anti-I-A/E inhibited it synergistically. Thus, unstimulated fresh PP-derived T cells appear to be activated and then to exert T-cell effector functions in the sequential development of helper, suppressor, and contrasuppressor immunoregulatory networks in the presence of PP-derived sIgM B cells and, particularly, LPS-preactivated sIgM B cells. Based on the blocking effect of anti-I-A and/or anti-I-E mAb on the induction of each T-cell-mediated immunoregulatory function in class-specific Ig production, it appears that the autoreactive (self MHC class II Ag-reactive) activation of PP T cells evoked by Ia Ag on PP sIgM B cells largely controls mucosal IgA production by the latter cells. Furthermore, this immunoregulation by autoreactive effector T cells, especially the L3T4+ VV- helper T cell, may play a significant role in vivo in gut-mucosal IgA isotype production.     

Synthetic Fc peptide-mediated regulation of the immune response. II. Analysis of secreted immunoglobulin classes, accessory cell contribution, and lymphocyte proliferation in p23-stimulated spleen cell cultures

The synthetic peptide p23 representing residues 335 to 357 in the CH3 domain of human IgG1 was able to increase levels of secreted Ig in murine spleen cell cultures. This in vitro response was optimal in the presence of between 10(-4) and 10(-3) micron p23/ml and the levels of secreted Ig reached a maximum on day 4 or day 5 of culture. Supernatants from p23-treated cell cultures generally contained more IgM than IgG and undetectable levels of IgA. Induction of Ig secretion by p23 was macrophage-independent but T cell-dependent and, with respect to the latter case, removal of T cells from spleen cells reduced the levels of both IgM and IgG. Although maintaining the B cell differentiation-inducing quality of its progenitor molecule, the Fc gamma fragment, p23 appeared to have lost the ability to induce B cell proliferation. Evidence is presented that a sequence functionally similar to p23 is extant in mouse IgG by showing that murine Fc gamma fragments were also able to induce increases in Ig-secreting cells in murine spleen cell cultures.     

Mucosal immunoregulation: environmental lipopolysaccharide and GALT T lymphocytes regulate the IgA response

In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection. Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/ H3J B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern. Murine PP contain accessory cells (approximately 1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/ H3J PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fc alpha receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/ H3J mice are considerably higher than those in identical cultures from LPS responsive mice. In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5+ B cell subpopulation capable of both TI-2 and TD responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Secretory immunoglobulin A antibodies against the sigma1 outer capsid protein of reovirus type 1 Lang prevent infection of mouse Peyer's patches

Reovirus type 1 Lang (T1L) adheres to M cells in the follicle-associated epithelium of mouse intestine and exploits the transport activity of M cells to enter and infect the Peyer's patch mucosa. Adult mice that have previously cleared a reovirus T1L infection have virus-specific immunoglobulin G (IgG) in serum and IgA in secretions and are protected against reinfection. Our aim in this study was to determine whether secretory IgA is sufficient for protection of Peyer's patches against oral reovirus challenge and, if so, against which reovirus antigen(s) the IgA may be directed. Monoclonal antibodies (MAbs) of the IgA isotype, directed against the sigma1 protein of reovirus T1L, the viral adhesin, were produced and tested along with other, existing IgA and IgG MAbs against reovirus T1L outer capsid proteins. Anti-sigma1 IgA and IgG MAbs neutralized reovirus T1L in L cell plaque reduction assays and inhibited T1L adherence to L cells and Caco-2(BBe) intestinal epithelial cells in vitro, but MAbs against other proteins did not. Passive oral administration of anti-sigma1 IgA and IgG MAbs prevented Peyer's patch infection in adult mice, but other MAbs did not. When anti-sigma1 IgA and IgG MAbs were produced in mice from hybridoma backpack tumors, however, the IgA prevented Peyer's patch infection, but the IgG did not. The results provide evidence that neutralizing IgA antibodies specific for the sigma1 protein are protective in vitro and in vivo and that the presence of these antibodies in intestinal secretions is sufficient for protection against entry of reovirus T1L into Peyer's patches.     

Functional dissection of lupus susceptibility loci on the New Zealand black mouse chromosome 1: evidence for independent genetic loci affecting T and B cell activation

In previous work, we demonstrated linkage between a broad region on New Zealand Black (NZB) chromosome 1 and increased costimulatory molecule expression on B cells and autoantibody production. In this study, we produced C57BL/6 congenic mice with homozygous NZB chromosome 1 intervals of differing lengths. We show that both B6.NZBc1(35-106) (numbers denote chromosomal interval length) and B6.NZBc1(85-106) mice produce IgG anti-nuclear autoantibodies, but B6.NZBc1(35-106) mice develop significantly higher titers of autoantibodies and more severe renal disease than B6.NZBc1(85-106) mice. Cellular analysis of B6.NZBc1(85-106) mice revealed splenomegaly and increased numbers of memory T cells. In addition to these features, B6.NZBc1(35-106) mice had altered B and T cell activation with increased expression of CD69, and for B cells, costimulatory molecules and MHC. Introduction of an anti-hen egg white lysozyme Ig transgene, as a representative nonself-reactive Ig receptor, onto the B6.NZBc1(35-106) background corrected the B cell activation phenotype and led to dramatic normalization of splenomegaly and T cell activation, but had little impact on the increased proportion of memory T cells. These findings indicate that there are multiple lupus susceptibility genes on NZB chromosome 1, and that although B cell defects play an important role in lupus pathogenesis in these mice, they act in concert with T cell activation defects.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

In vitro production of anti-histone antibodies by spleen cells from young autoantibody negative NZB/NZW mice

Anti-histone antibodies (AHA) are spontaneously produced in NZB/NZW mice as part of their autoimmune disease. IgM AHA are usually not detected until after 4 mo of age, and older female mice switch to the production of IgG AHA. We studied the in vitro production of AHA by spleen cells from young (less than or equal to 12-wk-old) NZB/NZW mice. Despite the absence of elevated serum AHA activity, spleen cells from these mice demonstrated marked spontaneous autoantibody production in culture. In kinetic studies, little in vitro production was detectable after 1 day of culture, and maximal accumulation occurred on day 5. Elevated AHA production was apparent by cells from 2-wk-old NZB/NZW mice, and an age-dependent increase in autoantibody production was also noted. Only AHA of the IgM class were detected in cultures of young spleen cells. The in vitro production of IgM AHA in culture was T cell dependent, depletion of T cells resulting in a 70 to 90% reduction in production, which was corrected by the readdition of T cells. In cultures where both IgM AHA and total IgM secretion were measured, a much greater T cell dependence for AHA production was apparent. The requirement for T cells could also be partially replaced by factors present in concanavalin A supernatant. AHA secretion was induced by lipopolysaccharide by using cells from both NZB/NZW and non-autoimmune mice. Although production was greater with NZB/NZW cells, the difference was much less than that for spontaneous production. Thus, AHA-secreting cells that are dependent on in vitro T cell help are present in young NZB/NZW mice. These studies may help define the mechanisms responsible for selective autoantibody secretion in lupus-like disease.     

B cell response to fresh and effector T helper cells. Role of cognate T-B interaction and the cytokines IL-2, IL-4, and IL-6

The helper activity of resting T cells and in vitro generated effector T cells and the relative roles of cognate interaction, diffusible cytokines, and non-cognate T-B contact in B cell antibody responses were evaluated in a model in which normal murine CD4+ T cells (Th), activated with alloantigen-bearing APC, were used to support the growth and differentiation of unstimulated allogeneic B cells. Both "fresh" T cells, consisting of memory and naive cells, stimulated for 24 h, and "effector" T cells, derived from naive cells after 4 days of in vitro stimulation, induced the secretion of IgM, IgG3, IgG1, IgG2a, and IgA. Effector T cells were significantly better helpers of the response of small dense B cells, inducing Ig at lower numbers and inducing at optimal numbers 2- to 3-fold more Ig production than fresh T cells. The predominant isotype secreted was IgM. Supernatants derived from fresh T cell cultures contained moderate levels of IL-2, whereas those from effector cultures contained significant levels of IL-6 and IFN-gamma in addition to IL-2. The involvement of soluble factors in the B cell response was demonstrated by the ability of antibodies to the cytokines IL-2, IL-4, and IL-6 to each block Ig secretion. Antibodies to IL-5 and IFN-gamma had no effect on the T cell-induced response. Kinetic studies suggested that IL-4 acted during the initial stages of the response, whereas the inability of anti-IL-6 to block B cell proliferation suggested that IL-6 was involved in part in promoting differentiation of the B cells. The relative contributions of cognate (MHC-restricted) and bystander (MHC-unrestricted) T-B cell contact vs cytokine (non-contact)-mediated responses were assessed in a transwell culture system. The majority of the IgM, IgG3, IgG1, and IgG2a response induced by both fresh and effector T cells was dependent on cognate interaction with small, high density B cells. In contrast, a small proportion of these isotypes and most of the IgA secreted resulted from the action of IL-6 on large, presumably preactivated, B cells. The IgA response did not require cell contact or vary when fresh and effector cells were the helpers. The contribution of bystander contact in the overall antibody response to both T cell populations was minimal.(ABSTRACT TRUNCATED AT 400 WORDS)