Gene expression of the lysophosphatidic acid receptor 1 is a target of transforming growth factor beta.

The lysophosphatidic acid (LPA) receptor LPA1/Edg2 is the first identified LPA receptor. Although its wide tissue distribution and biological functions have been well studied, little is known about how LPA1 is transcriptionally regulated. In the current study, we showed that LPA1 is a physiological target of transforming growth factor beta (TGFß)-mediated repression. In both normal and neoplastic cells, TGFß inhibits LPA1 promoter activity, LPA1 mRNA expression and LPA1-dependent chemotaxis and tumor cell invasion. Knockdown of the TGFß intracellular effector Smad3 or Smad4 with lentivirally transduced short hairpin RNA relieved these inhibitory effects of TGFß. Interestingly, the LPA1 promoter contains two potential TGFß inhibitory elements (TIEs), each consisting of a Smad-binding site and an adjacent E2F4/5 element, structurally similar to the TIE found on the promoter of the well-defined TGFß target gene c-myc. Deletion and point mutation analyses indicate that the distal TIE located at 401 bp from the transcription initiation site, is required for TGFß repression of the LPA1 promoter. A DNA pull-down assay showed that the -401 TIE was capable of binding Samd3 and E2F4 in TGFß-treated cells. TGFß-induced binding of the Smad complex to the native -401 TIE sequence of the LPA1 gene promoter was further verified by chromatin immunoprecipitation assays. We therefore identified a novel role of TGFß in the control of LPA1 expression and LPA1-coupled biological functions, adding LPA1 to the list of TGFß-repressed target genes.

The microenvironment of human Peyer's patches inhibits the increase in CD38 expression associated with the germinal center reaction.

Analysis of B cells in the human tonsils identified CD38 expression as a hallmark of germinal center (GC) B cells. However, the signals responsible for the in vivo induction of CD38 have not been determined. The primary site for generation of memory and plasma cells in the gastrointestinal tract is the GCs of Peyer's patches (PP). PP and intestinal mucosa, but not tonsils or oral mucosa, express mucosal addressin cell adhesion molecule-1 (MAdCAM-1). The ligand for MAdCAM-1, integrin alpha(4)beta(7), is expressed on naive B cells and memory B cells that traffic to the gastrointestinal tract. In this study we determine that, unlike tonsil, human PP GC B cells do not express significant levels of CD38. PP B cells can be induced to express CD38 upon culture with CD40 ligand, anti-B cell receptor, and IFN-gamma. However, coculture of tonsil naive B cells with an Ab directed against integrin beta(7) inhibits IFN-gamma-induced CD38 hyperexpression. The absence of CD38 on PP GCs suggests that there are tissue-specific pathways of B cell development that differ between tonsil and PP. The differential expression pattern of MAdCAM-1, together with the observation that ligation of beta(7) can inhibit the induction of CD38 expression, suggests that ligation of alpha(4)beta(7) in vivo may contribute to a PP-specific GC phenotype.

Correct immunoglobulin alpha mRNA processing depends on specific sequence in the C alpha 3-alpha M intron.

The maturation of IgM-expressing B cells to IgM-secreting plasma cells is associated with both an increase in mu mRNA and the ratio of secreted to membrane forms of mu mRNA which differ at the 3' termini. In contrast, both in vitro and in vivo the secreted form of alpha mRNA is predominant at all stages in the development of a secretory IgA response. Previous studies demonstrated that preferential usage of the alpha s poly(A) site does not result from transcription termination and is independent of either the poly(A) sites or the 3' splice site associated with the exon encoding the membrane exon of IgA (alpha M). The present study demonstrates that a 349-bp region located 774 bp 3' to the alpha s poly(A) site is required for the preferential usage of the alpha s terminus. This region, which is the first isotype-specific cis-acting regulatory sequence not immediately adjacent to a secretory poly(A) site to be identified, contains regulatory elements that increase the efficiency of polyadenylation/cleavage. A ubiquitous, approximately 58-kDa RNA-binding protein interacts specifically with this regulatory region. These studies support the premise that cis-acting elements unique to each CH gene can impinge upon a common mechanism regulating Ig mRNA processing.

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.

Developmental regulation of immunoglobulin mRNA processing and the IgA response: establishing a paradigm.

IgA, which is protective at mucosal sites, is derived from memory B cells that develop in the organized lymphoid tissue of the gastrointestinal tract and subsequently mature to plasma cells in the lamina propria. Similarly to B cells expressing other isotypes, the maturation of IgA-expressing B cells is associated with both an increase in the steady-state level of immunoglobulin mRNA and the ratio of secreted to membrane forms of mRNA, which differ in 3' terminus. In contrast to B cells expressing other isotypes, at all stages in the development of an IgA response, the secreted form of alpha mRNA predominates. In this article, studies on the general features of IgA B cell development, mechanisms regulating 3' terminus usage of Ig mRNAs, and isotype-specific regulation of 3' terminus usage particularly in regard to alpha mRNA are discussed.

Predominant usage of the proximal poly(A) site in alpha mRNAs is not intrinsic to the 3' termini.

The maturation of IgM-expressing B cells to IgM-secreting plasma cells is associated with both an increase in mu mRNA and the ratio of secreted to membrane forms of mu mRNA. In contrast, previous studies demonstrated that in vitro the secreted form of alpha mRNA (alpha s mRNA) predominates regardless of the stage of B cell differentiation. The present study demonstrates that alpha s mRNA predominates in both B cells derived from the germinal centers of murine Peyer's patches and in the functional IgA memory population, suggesting that in vitro events accurately represent the generation of a secretory IgA response in vivo. Although the predominant usage of the alpha s poly(A) site is due to RNA processing, it does not depend on either the alpha s poly(A) site, the 3' splice site associated with the exon encoding the membrane exon of IgA (alphaM) or the alphaM poly(A) sites. Analysis of the sequence of the intron between the alpha s terminus and alphaM (alpha s-alphaM intron) demonstrates the existence of several potential regulatory elements. Furthermore, the effects of deletions within the alpha s-alphaM intron on 3' terminus usage demonstrate that the predominant usage of the proximal terminus is not strictly dependent on the length of the intron. Together with previous work, these observations support the idea that choice of 3' terminus for all Ig heavy chain genes is regulated by a similar mechanism, but specific sequences within a heavy chain gene can impinge upon that mechanism.

A transforming growth factor-beta regulable RNA-binding protein interacts specifically with germline Ig alpha transcripts.

Isotype switching is presaged by the transcriptional activation of the heavy chain class gene (CH) to which recombination will occur. As a result, mRNA or germline transcripts from the unrearranged gene accumulate in the cytoplasm. Previous studies demonstrated that transforming growth factor (TGF)-beta stimulated isotype switching to IgA in cultures of lipopolysaccharide (LPS)-stimulated murine B cells and increased the stability of C alpha mRNAs. The present study demonstrates that LPS-stimulated B cells express a 45 kDa protein, I alpha BP, that specifically binds to germline alpha transcripts. Following addition of TGF-beta, the binding activity of this protein is significantly reduced. The identification of a cytokine regulable RNA-binding protein that interacts with germline transcripts supports the idea that these transcripts are involved in recombination and raises the possibility that RNA-protein interactions play a role in regulating isotype switching.

Mechanism for transforming growth factor beta regulation of alpha mRNA in lipopolysaccharide-stimulated B cells.

Transforming growth factor (TGF)-beta has been shown to stimulate isotype switching to IgA in cultures of lipopolysaccharide (LPS)-stimulated B cells. The induction of isotype switching is associated with the appearance of novel germline alpha transcripts that cannot be found in cultures stimulated with LPS alone. TGF-beta also increases the steady state level of productive alpha mRNA. In order to further elucidate both the role of TGF-beta and germline transcripts in isotype switching to IgA in B cells, the mechanism responsible for the changes in alpha mRNA was investigated. The increase in alpha mRNA which does not occur until day 2 of culture continues until at least day 4. Nuclear run-on analysis demonstrated that TGF-beta does not significantly increase the rate of transcription of either germline or productive alpha mRNA after 12, 24, or 48 h of culture. However, by day 2 of culture TGF-beta increases the half-life of alpha mRNA. These findings support the idea that TGF-beta acts as a secondary signal to stimulate isotype switching to IgA in a population that has already received a signal that drives it toward IgA production. In addition these studies suggest that either the germline transcripts or processing of pre-germline alpha mRNA transcripts plays a role in targeting recombination.

Regulation of usage of membrane and secreted 3' termini of alpha mRNA differs from mu mRNA.

Previous studies demonstrated that the addition of transforming growth factor-beta (TGF-beta) to LPS-stimulated B cell cultures induced cells to express membrane IgA and to mature to IgA-secreting cells without a parallel change in usage of 3' termini by alpha mRNA. In these cultures, the secreted form of alpha mRNA was predominant even before expression of membrane IgA could be detected. In the present study, we demonstrate that the preferential usage of the secreted terminus of alpha mRNA in these cultures is not caused by transcription termination and reflects a difference in the regulation of choice of 3' terminus for alpha and mu mRNA. The addition of TGF-beta to LPS-stimulated cultures causes an increase in the steady state level of alpha mRNA using the secreted 3' terminus. In contrast, TGF-beta decreases the steady state level of mu mRNA and inhibits usage of the 3' terminus for the secreted form of mu, suggesting that the choice of 3' terminus for alpha and mu mRNA is regulated differently in LPS-stimulated cultures. To determine whether the difference in usage of 3' termini by alpha and mu mRNA was a property of the culture system or whether it reflected a difference in regulation, C alpha was transfected into cell lines representing different stages of B cell development. The secreted form of alpha mRNA predominates regardless of the ratio of membrane to secreted forms of the endogenous C mu gene. A similar dichotomy in 3' terminus usage occurred in a stable C alpha transfectant of the BCL1 lymphoma, suggesting that trans-acting factors are not limiting. Furthermore, as was the case with normal B cells, the predominance of the secreted form of the transfected C alpha genes was not due to transcription termination. These data demonstrate that usage of 3' terminus in alpha and mu mRNA is regulated differently.

Structure and expression of germline epsilon transcripts in human B cells induced by interleukin 4 to switch to IgE production.

Interleukin 4 (IL-4)-induced IgE production coincides with the appearance of the 2.2-kb productive epsilon-mRNA, but is preceded by synthesis of a 1.7-kb epsilon-RNA. Analysis of cDNA copies of the 5' end of this RNA indicated that the 1.7-kb epsilon-RNA is a germline epsilon immunoglobulin heavy chain transcript with an exon mapping 5' to the switch region. Transcription through switch regions has been implicated in the control of class switching. However, IL-4 or cloned CD4+ T cells were able to induce germline epsilon transcripts without inducing IgE synthesis, for which both signals were required. These results indicate that induction of human germline epsilon-RNA does not necessarily result in IgE synthesis, and that additional regulatory mechanisms are involved in class switching.

Molecular characterization of germ-line immunoglobulin A transcripts produced during transforming growth factor type beta-induced isotype switching.

The addition of transforming growth factor type beta to lipopolysaccharide-stimulated murine B-cell cultures enhances isotype switching to IgA and induces the appearance of two sizes of alpha mRNA transcripts. One of these is the same size as mRNA for secreted IgA but the other, which is 300-400 base pairs (bp) shorter, does not correlate in size with any form of productive alpha mRNA. Both sizes of transcript were shown to contain germ-line sequences 5' to the alpha switch region, suggesting that the longer transcripts included both germ-line and productive forms of alpha mRNA, whereas the shorter transcripts were only germ-line alpha mRNA. We isolated cDNA clones corresponding to the shorter, 1.3-kilobase (kb), transcript by using an anchored polymerase chain reaction and a specific primer for the constant region. Analyses of these cDNA clones show that the short transcript consists of a 126-bp exon located approximately 1.5 kb 5' to the alpha switch region spliced to the first exon of the alpha constant region locus. Furthermore, a minor fraction of the longer, approximately 1.7 kb, transcripts also contains this exon. These results demonstrate that transforming growth factor type beta-mediated isotype switching to IgA is preceded by transcriptional activation of the heavy-chain locus.

Mechanism for transforming growth factor beta and IL-2 enhancement of IgA expression in lipopolysaccharide-stimulated B cell cultures.

Transforming growth factor beta (TGF-beta), but not IL-2, causes LPS-stimulated surface (s)IgA- cells to express sIgA. Although there is a progression of sIgA- cells to sIgA+ cells and then to IgA-secreting cells, there is not a parallel change in ratio of membrane to secreted form of alpha-mRNA. In fact, the secreted form of alpha-mRNA is always the predominant form even before the expression of sIgA. However, at least some of the secreted alpha-mRNA transcripts are sterile. The increase in sIgA expression and the induction of sterile transcripts indicate that TGF-beta enhances H chain class switching to IgA as opposed to allowing the growth and maturation of cells precommitted to IgA secretion. The addition of IL-2 to cultures with TGF-beta results in a 5- to 10-fold increase in IgA secretion compared to cultures to which only TGF-beta was added. In these cultures IL-2 increases neither the proportion nor the total number of sIgA+ cells suggesting that IL-2 acts to increase IgA secretion. However, IL-2 does not cause a change in the ratio of secreted to membrane form of alpha-mRNA nor does it lead to an increase in the steady state level of alpha-mRNA comparable to the increase in secreted IgA. Thus, it appears that regulation of transcription of IgA as sIgA- cells proliferate and undergo H class switching and maturation does not follow the same sequence as is seen when sIgM+ cells proliferate and mature to Ig-secreting cells. Furthermore, the data suggest that maturation to high level secretion is controlled posttranscriptionally.

Transforming growth factor beta specifically enhances IgA production by lipopolysaccharide-stimulated murine B lymphocytes.

The addition of TGF-beta to cultures of LPS-stimulated murine B cells causes a approximately 10-fold enhancement of IgA production, yet causes a 10-fold decrease in total Ig production. IL-2 and, to a lesser extent, IL-5 synergize with TGF-beta to further enhance IgA production and partially reverse the inhibition of total Ig production. IgA constitutes only approximately 0.1% of total Ig in LPS-stimulated cultures, but that percentage rises to 15-25% in cultures to which TGF-beta and IL-2 are added. TGF-beta induces a substantial increase in IgA production from sIgA- B cells but inhibits IgA production by sIgA+ cells. This finding suggests that TGF-beta acts as an isotype-specific switch factor for IgA.

Cytokine regulation of immunoglobulin isotype switching and expression.

Several cytokines have been shown to regulate the expression of specific isotypes by affecting either the frequency of isotype switching or allowing maturation of precommited precursors. IL-4 enhances the production of IgG1 and is required for IgE production in vitro and in vivo. Evidence has been provided that Il-4 acts by enhancing switching to IgE, and perhaps IgG1. IFN-gamma was shown to stimulate the secretion of IgG2a in concentrations at which it inhibits IL-4 induced production of IgG1 and IgE. IL-5, on the other hand, appears to stimulate maturation. Recent evidence suggests that transforming growth factor-beta induces switching to IgA, despite the inhibitory activity on other isotypes.

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 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures.

Although it has been established that IL-4 enhances both IgG1 and IgE secretion in LPS-stimulated B cell cultures, these studies failed to determine whether IL-4 preferentially induces isotype switching or preferentially allows for the maturation of precommitted precursor cells. To distinguish between these possibilities, it is necessary to ascertain the effect of IL-4 on the isotypes secreted by individual precursor cells during clonal expansion. Therefore, clonal cultures of B cells stimulated with a Th2 helper cell line specific for rabbit Ig and rabbit anti-mouse IgM were established. The majority of B cells are capable of undergoing clonal expansion under these conditions. To vary the level of IL-4 present, either IL-4 or anti-IL-4 was added to cultures. In the presence of IL-4 there was an increase in the proportion of clones that secreted IgE and a decrease in the proportion of clones that secreted IgM. The addition of IL-4 to cultures also increased the amount of IgE secreted by individual clones. Thus, these experiments definitively prove that IL-4 causes specific heavy chain class switching to IgE in Th2-stimulated B cell cultures. In contrast, IL-4 does not affect the proportion of clones secreting IgG1, suggesting that other consequences of Th cell-B cell interactions play a role in the generation of an IgG1 response.

Increase of precursor frequency and clonal size of murine IgE-secreting cells by IL-4.

IL-4 is able to preferentially enhance murine IgE levels in the supernatant of LPS-stimulated T cell-depleted splenic B cell cultures. Clonal and quantitative analysis of this response revealed that this is due partly to a 14-fold increased IgE precursor frequency and partly to a three-fold increased clone size of IgE-secreting cells. IL-4 increased the precursor frequency and the clone size of IgM-secreting cells not more than twofold. Both the IgM and IgE response in LPS-stimulated B cells were completely inhibited by the addition of anti-IgM mAb (M41) to the cultures, indicating that the IgE-secreting clones developed as subclones from precursors that express IgM. These cells lacked expression of membrane-bound IgE up to day 5 of the culture. Application of feeder cells in these cultures resulted in an increased precursor frequency of IgE-secreting clones among LPS-reactive B cells that is due, partially, to IL-4 produced by the feeder cells.

Intraduodenal application of cholera holotoxin increases the potential of clones from Peyer's patch B cells of relevant and unrelated specificities to secrete IgG and IgA.

Although it has been established that cholera toxin is both an effective mucosal immunogen and adjuvant, the mechanism by which it acts has not been determined. The relative contributions of the pharmocologic and binding capacities of holotoxin have been assessed by comparing holotoxin, acid dissociated B subunit, and B subunit from the Texas Star variant, which is deficient in production of the A subunit, for their ability to generate toxin-specific precursors in vivo that give rise to clones secreting exclusively IgA in vitro. In this way we demonstrated that although B subunit is as immunogenic as the holotoxin, pharmacologic activity appears to play a role in the generation of IgA-committed precursors. In addition, intraduodenal (i.d.) application of holotoxin can act to alter the isotype display of previously primed B cells in Peyer's patches (PP) specific for a chemically unrelated hapten resulting in an increase in the proportion of their clones that secrete IgA or IgG and a decrease in the proportion that secrete IgM following antigen-dependent in vitro clonal expansion. Intraduodenal application of cholera toxin did not appear to nonspecifically increase the proportion of IgA-committed precursors as judged by staining for sIgA and mRNA alpha levels. However, following i.d. application of cholera toxin, an overall downward shift in the levels of sIgD and s kappa was observed in the PP B cell population. We suggest that the holotoxin can nonspecifically affect the isotype display of PP B cells by altering their responsiveness to the stimuli present in the in vitro cultures.