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.     

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 localization of the antibody response in milk or bile depends on the nature of the antigen

Immunization in the Peyer's patches of rats with horse spleen ferritin or Escherichia coli 06 carrying type 1 pili resulted in an IgA antibody response detected in milk and bile and an IgG and IgM antibody response in serum, milk, and bile. The IgA antibody response to type 1 pili was as a mean 5.0-fold higher in milk than in bile. In contrast IgA antibody activity to 06 LPS was as a mean 6.3-fold higher in bile than in milk. The IgA antibodies to ferritin were randomly distributed between milk and bile. The IgG and IgM antibody activity to all three antigens studied were higher in the milk than in the bile. The secretory antibody response could be transferred from immunized rats to unimmunized rats with mesenteric lymph node cells (MLN) taken from donor rats 4 days after immunization in the Peyer's patches. IgA antibodies to pili and ferritin appeared solely in the milk of the recipients, whereas IgA antibodies to the 06 LPS only appeared in the bile. The ratios serum:milk and serum:bile for the IgG and IgM antibodies indicated an antigen-specific direction of homing with local production of these two isotypes primarily in the mammary gland. Antibody-forming cells of the IgA class could not be detected in the MLN on the day the cells were transferred. It is concluded that the difference seen in antibody distribution between milk and bile is not due to dissemination of antigen, but instead a result of different homing or expansion at the mucosal-glandular site dependent on the antigen specificity of the migrating cells.     

Intraperitoneal delivery of cholera toxin B subunit enhances systemic and mucosal antibody responses

Although cholera toxin (CT) is a potent mucosal adjuvant, its activity in systemic immunity is relatively undocumented. In the present study, we investigated its adjuvant effect on systemic and mucosal antibody responses following intraperitoneal immunization of mice with BSA. CT increased levels of anti-BSA specific IgG1, IgM, and IgA antibodies in the peritoneum and serum, as well as IgA and IgG1 antibodies in the intestinal fluids. The B subunit of CT (CTB) was as potent as CT itself, with potency comparable to that of incomplete Freund's adjuvant. CTB also increased the number of BSA-specific Ig secreting cells in the spleen and mesenteric lymph node, and stimulated expression of B7.2 but not of MHC class II molecules on peritoneal macrophages, particularly in the presence of IFN-gamma. Our results imply that intraperitoneally administered CTB enhances systemic and mucosal antibody responses, in part at least via effects on macrophages.     

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.     

Influences of orally administered lactoferrin on IFN-gamma and IL-10 production by intestinal intraepithelial lymphocytes and mesenteric lymph-node cells.

Intestinal mucosal immunity plays an important role in mucosal and systemic immune responses. We investigated the influences of orally administered bovine lactoferrin (LF) on cytokine production by intestinal intraepithelial lymphocytes (IEL) and mesenteric lymph-node (MLN) cells, especially T cells. Bovine LF or bovine serum albumin (control) was administered to mice once daily for 3 d. After 24 h from the last administration, IEL of the jejunum and ileum and MLN cells were isolated. These cells were cultured with and without the anti-T-cell-receptor antibody, and then the culture supernatants were assayed for cytokines with ELISA. Oral LF did not affect the ratio of T-cell subpopulations in IEL and MLN; however, LF enhanced both interferon (IFN)-gamma and interleukin (IL)-10 production by unstimulated IEL and by IEL stimulated with the alphabeta T-cell receptor but not with the gammadelta T-cell receptor. LF also enhanced both IFN-gamma and IL-10 production by stimulated and unstimulated MLN cells. The production level of IFN-gamma by MLN cells was correlated with that of IL-10. These results suggest that oral LF enhances the production of both Th1-type and Th2/Tr-type cytokines in the small intestine of healthy animals.     

Influence of bedding type on mucosal immune responses

The mucosal immune system interacts with the external environment. In the study reported here, we found that bedding materials can influence the intestinal immune responses of mice. We observed that mice housed on wood, compared with cotton bedding, had increased numbers of Peyer's patches (PP) visible under a dissecting microscope. In addition, culture of lymphoid organs revealed increased production of total and virus-specific IgA by PP and mesenteric lymph node (MLN) lymphocytes from mice housed on wood, compared with cotton bedding. However, bedding type did not influence serum virus-specific antibody responses. These observations indicate that bedding type influences the intestinal immune system and suggest that this issue should be considered by mucosal immunologists and personnel at animal care facilities.     

Role of mesenteric lymph nodes and aging in secretory IgA production in mice

Although it is known that Peyer's patches are the major inductive site for S-IgA production and B1 cells contribute to half of the IgA plasma cells detected in the gut lamina propria, the type of contribution of mesenteric lymph nodes to the process is still unclear. Cytokines such as TGF-beta, IL-10, IL-4, IL-5, and IL-6, are required to promote IgA class switching and IgA synthesis. Aging-related alterations in T and B cells and in cytokine production are already known. Some reports have also proposed that S-IgA production might be altered in aged animals. Herein, we investigated the role of MLN and aging in S-IgA production. Two- to 18-month-old BALB/c mice were used to evaluate aging-related alterations and MLN were removed to study its role in S-IgA production. We found that MLN are important, although not essential for S-IgA production. In addition, we showed that production of IgA-related cytokines are well preserved in MLN but not in PP of aged mice and that S-IgA levels are not affected by aging. Our results suggest that MLN may play a complementary role in S-IgA production mostly in aged animals.     

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.