Effect of transforming growth factor (TGF)-beta 1 on IgA isotype expression. TGF-beta 1 induces a small increase in sIgA+ B cells regardless of the method of B cell activation.

Transforming growth factor-beta (TGF-beta) has been reported to play an important role in IgA isotype expression when B cells are stimulated with LPS. The goal of the present study was to determine whether TGF-beta has similar effects on IgA isotype expression under more physiologic conditions utilizing a variety of B cell activation systems. As previously reported, in the LPS system TGF-beta caused a small, but significant, absolute increase in surface IgA (sIgA) expression and a very definite increase in IgA secretion; these effects were enhanced by IL-2 and IL-5. In the case of B cell stimulation with another B cell stimulant, the thymus-independent type II mitogen, anti-IgD-dextran, TGF-beta led to a similar small increase in sIgA expression, but caused suppression of IgA secretion. Using the Th2 cell clones CDC35 and D10 to stimulate resting B cells in a cognate and non-cognate T cell-dependent fashion, respectively, TGF-beta again increased sIgA expression to a similar small extent. TGF-beta at low doses (0.1 ng/ml) did not increase IgA secretion significantly and, at higher doses (1.0 ng/ml) caused significant suppression of IgA secretion. Addition of various cytokines (IL-2, -4, -5, D10sup) other than TGF-beta to stimulated B cells did not increase sIgA expression, but did give rise to increased amounts of IgA secretion, especially when activated D10 T cells were used as the B cell stimulant. Finally, the addition of an antibody against TGF-beta to cultures containing TGF-beta on day 2 led to partial or complete reversal of the inhibitory effects of TGF-beta on IgA secretion. In conclusion, TGF-beta causes a consistent, but small increase in sIgA+ B cells, in cultures of B cells stimulated by a variety of T cell-dependent or independent stimuli. In contrast, TGF-beta either promotes or inhibits B cell survival and IgA secretion, depending on the method of B cell activation. These results are most consistent with the view that TGF-beta provides only a partial or incomplete IgA switch signal but that additional factors are involved in IgA isotype switching and differentiation.     

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.     

The roles of IL-4, TGF-beta and LPS in IgA switching.

CH12.LX B cells have been used as a lymphoma model of MHC restricted, antigen-dependent B cell differentiation. These B cells express surface IgM and secrete IgM. Most recently we have demonstrated that CH12.LX is a model of cytokine driven IgA differentiation. Recently, transforming growth factor beta (TGF-beta) has been shown to be a probable switch factor for IgA in LPS-stimulated mouse lymphocytes, therefore we chose CH12.LX B cells to study the effect of IL-4, TGF-beta and LPS in IgA isotype switching. Adding TGF-beta to the monoclonal cell line CH12.LX results in induction of mIgA expression but no enhancement of IgA secretion similar to the effect of IL-4. The addition of LPS serves as a non-specific stimulus to enhance the secretion of the expressed immunoglobulin, but has no IgA specific activity of its own. IL-4 and TGF-beta together are synergistic for mIgA expression. Pretreatment studies show that TGF-beta added after IL-4 is the same as TGF-beta alone whereas the converse is the same as adding both cytokines together. TGF-beta acts by increasing the steady state levels of alpha message, whereas northern analysis indicates that IL-4 does not induce alpha message the way TGF-beta does. These data confirm that TGF-beta by itself is an isotype switch factor for IgA. In addition, IL-4 and TGF-beta cause mIgA expression through different mechanisms. CH12.LX B cells serve as a valuable model to study the role of multiple signals required for mIgA expression and IgA secretion.     

Transforming growth factor-beta directs IgA switching in human B cells.

Transforming growth factor (TGF)-beta added to cultures of highly purified human splenic B cells induced high levels of IgA synthesis in the presence of PWM and activated cloned CD4+ T cells. TGF-beta had no effect on IgM or IgG production. The induction of IgA synthesis by TGF-beta reflected IgA switching, because a strong induction of IgA production was also observed, when sIgA- B cells were cocultured with cloned activated CD4+ T cells in the presence of pokeweed mitogen. Resting CD4+ T cell clones or activated CD8+, TCR-gamma delta + CD4-,CD8- T cell clones failed to provide the co-stimulatory signal that in addition to TGF-beta and pokeweed mitogen was required for induction of IgA switching and IgA synthesis. mAb against CD4 or class II MHC molecules inhibited TGF-beta induced IgA synthesis, indicating that CD4-class II MHC interactions are required for productive T-B cell contacts resulting in IgA production. In contrast, anti-LFA-1, anti-CD2, and anti-class I MHC mAb were ineffective. TGF-beta failed to induce IgA synthesis by sIgA+ B cells under these culture conditions. Interestingly, induction of IgA production by sIgA- B cells required neutralization of TGF-beta activity by addition of the anti-TGF-beta mAb 1D11.1G 24 h after onset of the cultures. IgA production was prevented when the anti-TGF-beta mAb was added at the start of the cultures, indicating the specificity of the reaction. IgA synthesis was completely suppressed when TGF-beta was present during the total culture period of 11 days. These findings indicate that TGF-beta can act as a specific switch factor for IgA, provided it is only present at early stages of the cultures.     

The role of TGF-beta in growth, differentiation, and maturation of B lymphocytes

Transforming growth factor-beta (TGF-beta) affects B cells at all stages in development. It appears to be involved in lymphopoiesis and is required for the development of plasma cells secreting all secondary isotypes. Its ability to inhibit proliferation and stimulate apoptosis suggest that it may be involved both in germinal center development and regulation of B-cell proliferation at sites of high antigen load such as the gastrointestinal tract. Although TGF-beta appears to be required for the generation of B cells secreting secondary isotypes, it inhibits secretion of IgM and IgA from cells expressing those isotypes. In this regard, TGF-beta may alter the level of RNA processing factors either directly or indirectly by inhibiting progression through the cell cycle. One of the best characterized effects of TGF-beta is its ability to stimulate isotype switching to IgA in both mouse and man. There is some controversy concerning its mechanism of action in this process, but its critical role is without question. The controversy may stem in part from an inability to separate the effects of endogenous and exogenous TGF-beta in the multiple models of isotype switching. The influence of endogenous TGF-beta is perhaps best exemplified by analysis of production of the different classes of IgG in mouse strains producing different levels of TGF-beta.     

Transforming growth factor beta 1 increases IgA isotype switching at the clonal level

Transforming growth factor beta 1 (TGF beta 1) has important effects on expression of the IgA isotype. TGF beta 1 alone, or in combination with IL-5 or IL-2 increases IgA secretion by populations of LPS-activated surface IgA negative (sIgA-) spleen B cells, while concurrently decreasing IgM and IgG secretion. The present study demonstrates the activity of TGF beta 1 as an IgA isotype switch factor at the clonal level. Stimulation of LPS-activated sIgA- spleen B cell populations with TGF beta 1, or a combination of TGF beta 1 and IL-2, resulted in a significant increase in total numbers of IgA secreting cells, and this increase ultimately was paralleled by an increase in total IgA secretion. Using limiting dilution analysis, TGF beta 1 was shown to increase the frequency of IgA secreting B cell clones, by approximately 20-fold. This was not accompanied by increased numbers of IgA secreting cells/clone. In contrast, IL-2 does not have activity as an IgA switch factor, but does increase IgA production by B cells already committed to secrete that isotype. Cell cycle inhibitors such as thymidine and hydroxyurea also selectively increased numbers of IgA secreting cells and total IgA secretion among populations of LPS-activated sIgA- spleen B cells. This suggests the IgA enhancing activity of TGF beta 1 may, in part, be related to its ability to inhibit cell growth.     

A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit

Previous studies have shown that the isotype of an antibody response is selected, in part, by the inhibition of isotype-specific suppression. The antisuppressor model predicts that isotype selection is initiated through an interaction between Ag, Ig, and a T cell-derived factor within 6 h of immunization. This report characterizes some of these molecules and their contribution to isotype regulation. Cultures of murine spleen cells stimulated with the T cell-dependent Ag SRBC led to Ag-specific IgG and IgA responses that could be suppressed and then antisuppressed by a molecular complex produced by mixing purified serum Ig with the supernatant of Ag-pulsed macrophages co-cultured with T cells. The supernatants from separate cultures of Ag-pulsed macrophages and rIL-1 alpha stimulated CD4+ T cells, could be pooled and mixed with Ig to produce functional antisuppressive complexes thereby allowing the factors from the different cell types to be studied separately. Adsorption of the co-culture or the rIL-1 alpha stimulated T cell supernatants against monoclonal IgG or IgA, removed IgG and IgA binding factors, respectively, and abrogated the ability to enhance the corresponding isotype. The adherent material could be recovered and used to reconstitute enhancement by the supernatants depleted of the binding factors. When affinity purified IgG or IgA was used as the source of Ig within the antisuppressive complexes, the enhancement of the antibody response was limited to the isotype of the regulatory Ig used to form the complex. Thus, manipulation of the antisuppressive molecules has a predictable effect on isotype selection. Release of isotype-specific binding factors by CD4+ cells by rIL-1 alpha supports the hypothesis that T cell circuits play a role in initiating isotype regulation.     

Regulation of mucosal B cell immunoglobulin secretion by intestinal epithelial cell-derived cytokines

Intestinal epithelial cells (IEC) secrete a variety of cytokines and, because of their close proximity to B cells in the lamina propria, may affect local antibody production via these cytokines. However, studies have not yet addressed which and to what extent these IEC-derived cytokines may affect B cell antibody production. In this study, rat mesenteric lymph node B cells were cultured with culture supernatants from the rat IEC-6 intestinal epithelial cell line to determine their effect on immunoglobulin (Ig) secretion. Unstimulated IEC-6 cells were found to secrete sufficient levels of IL-6 to enhance IgA, IgG and IgM secretion by unstimulated B cells. However, culture of lipopolysaccharide (LPS)-stimulated B cells with the unstimulated IEC-6 supernatant resulted in an enhancement of IgA secretion while IgM secretion was significantly suppressed. Depletion of the IEC-6 supernatant using cytokine specific antibodies revealed that both interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) were responsible for the enhanced IgA secretion while TGF-beta suppressed IgM secretion. More importantly, culture supernatants from LPS stimulated IEC-6 cells contained enhanced levels of IL-6 which enhanced both IgG and IgA production and partially overcame the suppressive effect of TGF-beta on IgM secretion. These results suggest that intestinal epithelial cells may secrete IL-6 and TGF-beta to regulate local B cell antibody secretion and their effect may be highly dependent upon the activation state of the epithelial cells.     

The measurement of specific cell: cell interactions by dual-parameter flow cytometry

The Fc receptor-mediated aggregation of antibody-coated spleen cells with cells from the P388D1 mouse macrophage line was followed using a novel flow cytometric technique. P388D1 and spleen cells were directly labeled with green-emitting (fluorescein isothiocyanate) and red-emitting (substituted rhodamine isothiocyanate) fluorophores, respectively. They were mixed, incubated in suspension at 4 degrees C, and analyzed for aggregation with a dual laser flow cytometer. Unconjugated cells appeared as particles which were either red or green, while conjugates were detected as particles which were both red and green. Using this assay procedure, 5 X 10(4) cells were analyzed in 2-3 min for the percentages of conjugates, free spleen cells, and free P388D1 cells. Intercellular aggregation required both antibody on the spleen cells and free Fc receptors on the P388D1 cells; nonspecific aggregates accounted for 1% or less of the total particles analyzed. Measurements of the fluorescence distributions within conjugates indicated that the majority of conjugates contained a single P388D1 cell bound to 1-3 spleen cells, and that only heterophilic aggregation occurred. The flow cytometric technique described here should be applicable for the measurement of the initial events of intercellular aggregation in other systems as well.     

IL-21-induced isotype switching to IgG and IgA by human naive B cells is differentially regulated by IL-4

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-beta. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1(+) cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA(+) cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1(+) cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.     

Enhanced IgA class switching in marginal zone and B1 B cells relative to follicular/B2 B cells

Mouse splenic marginal zone (MZ) B cells and B1 B cells enriched in the peritoneal cavity respond preferentially to T cell-independent Ags compared with follicular (FO)/B2 B cells. Despite the differential responses of B cell subsets to various stimuli, and despite the need for multiple stimuli to induce IgA class switching, the relative contribution of B cell subpopulations to IgA production is unknown. By culturing purified B cell populations, we find that MZ and peritoneal B1 cells switch more readily to IgA than do splenic FO or peritoneal B2 cells in BLyS/LPS/TGF-beta. Addition of IL-4, IL-5, and anti-IgD dextran to the cultures enhances IgA switching in FO/B2 and MZ B cells to a similar frequency, but this treatment suppresses IgA class switching in B1 cells. Thus, IgA switching differs among purified B cell subsets, suggesting that individual B cell populations could contribute differentially to IgA expression in vivo, depending on available stimuli.     

Accessory cell function in a Con A response: role of Ia and interleukin 1

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.     

Transforming growth factor-beta 1 (TGF-beta 1) is a bifunctional immune regulator for mucosal IgA responses

Porcine transforming growth factor-beta 1 (TGF-beta 1) exerts a unique bifunctional immunoregulatory effect on IgA responses in murine mesenteric and Peyer's patches lymphocyte cultures. TGF-beta 1 is a potent costimulator of LPS-induced IgA responses. The enhancement was observed at TGF-beta 1 at 0.1 to 10 ng/ml in both early and late phases of IgA responses from Day 5 to Day 14 in cultures. On the contrary, TGF-beta 1 exerts a profound immunosuppressive effect on IL-5-induced IgA synthesis. TGF-beta 1 is a natural cytokine produced by intestinal epithelial cells and may account for the polyclonal production and secretion of IgA at the mucosal surface and ensure the integrity of the primary host defense by mucosa-associated lymphoid tissues (MALT).     

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.     

Interleukin 5 and interleukin 4 produced by Peyer's patch T cells selectively enhance immunoglobulin A expression

Considerable evidence suggests that the high frequency of B cells committed to the IgA isotype in Peyer's patches is regulated by T lymphocytes. To understand more accurately the mechanism of this immunoregulation, an autoreactive T cell line from Peyer's patches was generated by culturing L3T4+ Peyer's patches T cells with syngeneic B cell blasts. The resulting T cell line, designated PT-1, and a clone derived from this line, PT-1.14, stimulated immunoglobulin secretion in spleen B cells with a preferential enhancement of IgA and IgG1 isotypes. Supernatant derived from concanavalin A-stimulated PT-1 or PT-1.14 cells could also enhance IgA secretion if spleen B cells were preactivated with lipopolysaccharide. Peyer's patches T cell supernatant did not contain IgA-specific binding factors. PT-1 supernatant scored positive in lymphokine assays for interleukin (IL)-2, IL-4 (B cell stimulatory factor 1), IL-5 (B cell growth factor II), and interferon-gamma, whereas PT-1.14 supernatant was positive for IL-4 and IL-5 and negative for IL-2 and interferon-gamma. Only IL-5 enhanced IgA secretion in lipopolysaccharide-activated B cells and this response was increased two- to three-fold by IL-4. These results suggest that the type 2 T helper subset which produces both IL-5 and IL-4 plays a primary role in regulating IgA expression.     

Lymphocyte activation by the Fc region of immunoglobulin. I. Role of prostaglandins in the down regulation of Fc fragment-induced polyclonal antibody production

Fc fragment-, subfragment-, and p23-induced polyclonal antibody production are regulated by endogenous and exogenous PGE. Addition of the PG synthetase inhibitor indomethacin (IM) to murine spleen cell cultures resulted in a significant increase in the amount of Ig secreted. Moreover, addition of exogenous PGE to culture resulted in a marked suppression of IgM and IgG secretion. Splenic adherent macrophages and P388D1 cells release PGE upon stimulation with Fc fragments, subfragments, and p23. The inclusion of IM or aspirin in culture was found to abrogate the ability of Fc fragments to induce PGE release from adherent cells. These results suggest a role for PG in immune complex mediated regulation of immune responses.