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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.

SummaryInterleukin 4 (11,4)-induced IgE production coincides with the appearance of the 2.2-kb productive e-mRNA, but is preceded by synthesis of a 1 .7-kb e-RNA. Analysis of cDNA copies of the 5' end of this RNA indicated that the 1.7-kb E-RNA is a germline e 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, IL4 or cloned CD4+ T cells were able to induce germline e transcripts without inducing IgE synthesis, for which both signals were required. These results indicate that induction of human germline e-RNA does not necessarily result in IgE synthesis, and that additional regulatory mechanisms are involved in class switching.

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.

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Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide array of biologic effects through its interaction with a family of five G protein-coupled receptors. Cytokines and growth factors interact with this signaling pathway in a variety of ways, including both activation and regulation of the expression of the enzymes that regulate synthesis and degradation of S1P. Not only do many growth factors and cytokines stimulate S1P production, leading to transactivation of S1P receptors, ligation of S1P receptors by S1P can also transactivate growth factor tyrosine kinase receptors and stimulate growth factor and cytokine signaling cascades. This review discusses the mechanisms involved in cross-talk between S1P, cytokines, and growth factors and the impact of that cross-talk on cell signaling and cell biology. The biological effects of sphingosine-1-phosphate (S1P) overlap with those of a wide array of cytokines and growth factors. There is increasing evidence that this overlap in functions relates to both the ability of growth factors and cytokines to transactivate S1P signaling cascades as well as the ability of S1P to transactivate growth factor and cytokine signaling cascades. This mutual pathway cross-talk affects cell growth, differentiation, and movement and is integral to a variety of normal processes as well as disease processes, including inflammation and cancer. This review will focus on the ways in which S1P and growth factor or cytokine signaling pathways interact. S1P S1P is a potent bioactive lipid that regulates processes that are essential to cellular homeostasis, including cell viability, differentiation, and motility (1-3). Most of its bestcharacterized actions are mediated through a family of five G protein-coupled receptors, (GPCRs) S1P 1-5 (4, 5). The S1P receptors couple to a variety of G proteins, allowing them to mediate many different biologic responses (6, 7).One mechanism for cross-talk between cytokine or growth factor pathways and S1P signaling involves regulation of S1P metabolism. Sphingolipid metabolism was the focus of a recent excellent review (3) and will not be discussed in detail here. Cellular levels of S1P are regulated by the activities of the enzymes that are responsible for its synthesis and degradation. In mammalian cells, two sphingosine kinases, SphK1 and SphK2, catalyze the phosphorylation of sphingosine to generate S1P (8). Although both isozymes phosphorylate sphingosine, they differ in their enzymatic properties, cellular location, and biological roles (9). SphK1 is generally regarded as providing pro-survival signals, whereas SphK2, at least when overexpressed, induces apoptosis in several cell types (10). S1P is degraded to sphingosine by specific S1P phosphatases (11) or cleaved to palmitaldehyde and phosphoethanolamine by S1P lyase (12). Thus, it is clear that S1P levels can potentially be altered by stimuli that affect activity or expression of any of these enzymes. However, the majority of cytokines and growth factors that coopt regu...

Transforming growth factor ␤ (TGF␤) plays a dual role in oncogenesis, acting as both a tumor suppressor and a tumor promoter. These disparate processes of suppression and promotion are mediated primarily by Smad and non-Smad signaling, respectively. A central issue in understanding the role of TGF␤ in the progression of epithelial cancers is the elucidation of the mechanisms underlying activation of non-Smad signaling cascades. Because the potent lipid mediator sphingosine-1-phosphate (S1P) has been shown to transactivate the TGF␤ receptor and activate Smad3, we examined its role in TGF␤ activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and promotion of migration and invasion of esophageal cancer cells. Both S1P and TGF␤ activate ERK1/2, but only TGF␤ activates Smad3. Both ligands promoted ERK1/2-dependent migration and invasion. Furthermore, TGF␤ rapidly increased S1P, which was required for TGF␤-induced ERK1/2 activation, as well as migration and invasion, since downregulation of sphingosine kinases, the enzymes that produce S1P, inhibited these responses. Finally, our data demonstrate that TGF␤ activation of ERK1/2, as well as induction of migration and invasion, is mediated at least in part by ligation of the S1P receptor, S1PR 2 . Thus, these studies provide the first evidence that TGF␤ activation of sphingosine kinases and formation of S1P contribute to non-Smad signaling and could be important for progression of esophageal cancer.