Preferential enhancement of B cell IgA secretion by intestinal epithelial cell-derived cytokines and interleukin-2

Intestinal epithelial cells (IEC) are known to secrete a number of important cytokines. Recently, we determined that IEC-derived IL-6 and TGF-β could enhance IgA secretion and suppress IgM secretion by isolated mucosal B cells. However, since the IEC-derived cytokines must function in the context of locally produced T cell cytokines, the effect of IEC-and T cell-derived cytokines on mucosal B cell immunoglobulin secretion was determined. Using 4 day culture supernatants (IEC-SN) from the rat IEC-6 intestinal epithelial cell line and lipopolysaccharide (LPS) stimulated Peyer's patch or mesenteric lymph node B cells, the IEC-SN was found to act with IL-2 to greatly enhance IgA secretion but limit or suppress IgM secretion as compared to cultures of LPS stimulated B cells alone. However, neither IL-4, IL-5, nor IFN-γ affected IgA secretion with the IEC-SN. Deletion of the IEC-SN with specific anti-cytokine antibodies suggested that IEC-derived TGF-β and IL-6 were both responsible for the enhancing effect along with IL-2 on IgA secretion, whereas IEC-derived TGF-β alone limited or suppressed IgM secretion. These results suggest that cytokines derived from local IEC and T cells may create an environment which may contribute to the preferential enhancement of IgA secretion seen in mucosal tissues.     

Preferential enhancement of B cell IgA secretion by intestinal epithelial cell-derived cytokines and interleukin-2

Intestinal epithelial cells (IEC) are known to secrete a number of important cytokines. Recently, we determined that IEC-derived IL-6 and TGF-β could enhance IgA secretion and suppress IgM secretion by isolated mucosal B cells. However, since the IEC-derived cytokines must function in the context of locally produced T cell cytokines, the effect of IEC-and T cell-derived cytokines on mucosal B cell immunoglobulin secretion was determined. Using 4 day culture supernatants (IEC-SN) from the rat IEC-6 intestinal epithelial cell line and lipopolysaccharide (LPS) stimulated Peyer's patch or mesenteric lymph node B cells, the IEC-SN was found to act with IL-2 to greatly enhance IgA secretion but limit or suppress IgM secretion as compared to cultures of LPS stimulated B cells alone. However, neither IL-4, IL-5, nor IFN-γ affected IgA secretion with the IEC-SN. Deletion of the IEC-SN with specific anti-cytokine antibodies suggested that IEC-derived TGF-β and IL-6 were both responsible for the enhancing effect along with IL-2 on IgA secretion, whereas IEC-derived TGF-β alone limited or suppressed IgM secretion. These results suggest that cytokines derived from local IEC and T cells may create an environment which may contribute to the preferential enhancement of IgA secretion seen in mucosal tissues.     

IL-4 enhances IEC-6 intestinal epithelial cell proliferation yet has no effect on IL-6 secretion

Intestinal epithelial cells (IEC) form an important line of defence at the intestinal mucosa by providing a barrier to lumenal contents and also by their ability to secrete various inflammatory cytokines. Recently, several T cell-derived cytokines have been shown to regulate specific IEC functions. In this study, the effect of IL-4 on IEC proliferation and secretion of the inflammatory cytokine IL-6 was investigated using the non-transformed rat IEC-6 intestinal epithelial cell line. Recombinant rat (rr)IL-4 was found to enhance IEC-6 cell proliferation over 4 days of culture, and this enhancement was dose-dependent. Further studies using specific antibodies confirmed that IL-4 induced the effect and that the effect was not mediated by autocrine-produced transforming growth factor-alpha. However, IL-4 did not induce IL-6 secretion by the IEC-6 cells, nor did it alter IL-1β-induced IL-6 secretion. These results indicate that T cells may be capable of regulating IEC proliferation via the secretion of IL-4 without altering the capacity of the IEC to function in the inflammatory response by secreting IL-6.     

A synergistic relationship between TNF-alpha, IL-1 beta, and TGF-beta 1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line.

Intestinal epithelial cells are known to secrete a variety of cytokines and may play a role in the immune response at the intestinal mucosa. However, the regulatory mechanisms that govern the secretion of these cytokines are largely unknown. In this report, we have focused on the cytokine interactions that regulate interleukin (IL)-6 secretion by the non-transformed rat small intestinal epithelial cell line IEC-6. Tumour necrosis factor-alpha (TNF-alpha) was found to enhance both IL-6 mRNA expression and protein secretion by the IEC-6 cells. Furthermore, TNF-alpha acted in synergy with either transforming growth factor-beta 1 (TGF-beta 1) or IL-1 beta to greatly enhance IEC-6 cell IL-6 secretion. Although the IEC-6 cells are known to produce TGF-beta, autocrine-secreted TGF-beta was found to have no effect on the elevated IL-6 secretion induced by both TNF-alpha plus IL-1 beta. However, the addition of activated TGF-beta 1 to IEC-6 cultures stimulated with both TNF-alpha and IL-1 beta resulted in greatly elevated levels of IL-6 secretion. Therefore, activated TGF-beta 1 can augment IL-6 secretion stimulated by TNF-alpha and IL-1 beta, either alone or in combination, suggesting that intestinal epithelial cell IL-6 secretion may be under the control of a cytokine network at the intestinal mucosa.     

IgA production by peritoneal cavity B cells is IL-6 independent: Implications for intestinal IgA response

We have shown previously both in vitro and in vivo that IL-6 is an important factor for the development of IgA-producing B cells. However, despite the lack of this cytokine in mice with targeted disruption of the interleukin (IL)-6 gene (gene knockout mice), a substantial number of IgA-producing plasma cells occur in their intestinal mucosa. The experiments reported here indicate that there is a population of IgA-producing B cell precursors originating from the peritoneal cavity, distinguished from conventional Peyer's patch-derived precursors by their expression of CD5, and that IgA secretion by these cells is IL-6-independent. Further, there is an increase in CD5 expression among brightly staining IgA-producing cells obtained from the intestinal lamina propria of IL-6 gene-disrupted mice compared to normal controls. These data suggest an explanation for the persistence of IgA-producing plasma cells in the intestinal mucosa of IL-6-depleted mice and indicate the importance of IL-6 for development of conventional precursors of IgA-producing B cells, but not those derived from the peritoneal cavity pool.     

Enhancing effect of cholera toxin on interleukin-6 secretion by IEC-6 intestinal epithelial cells: mode of action and augmenting effect of inflammatory cytokines.

Oral administration of cholera toxin (CT) induces a strong mucosal immune response to CT as well as having a potent adjuvant effect. Since one of the first cell types to encounter CT during cholera infection or after oral administration is the epithelial cell, we studied the effect of CT on interleukin-6 (IL-6) secretion by the rat intestinal epithelial cell line IEC-6. CT was found to rapidly enhance IL-6 secretion and IL-6 gene expression by these cells. The addition of dibutyryl cyclic AMP (cAMP) to cultures of IEC-6 cells had little effect on IL-6 secretion, yet mRNA levels were elevated, suggesting that the response may have been regulated by cAMP. Purified B subunit of CT did not significantly enhance IL-6 secretion or mRNA expression. CT and transforming growth factor beta 1 synergistically enhanced IL-6 secretion in IEC-6 cells. The addition of CT with either IL-1 beta or tumor necrosis factor alpha gave even greater synergistic enhancement of IL-6 secretion, and dibutyryl cAMP could mimic CT's synergy with IL-1 beta. These results indicate that the intestinal epithelial cell is capable of secreting high levels of IL-6 after encountering CT, especially in the presence of inflammatory cytokines. This high level of IL-6 secretion could be a very important component of the mucosal immune response to CT and may also account for a portion of the adjuvant effect of CT.     

Expression of IL-5 receptors on B cells: Role in mucosal B cell responses

IL-5 plays an important role in mucosal B cell responses. An in vitro model of IgA B cell differentiation, CH12LX, has been used to demonstrate that IL-5R are expressed on both IgM+ and IgA+ B cells and that IL-5 will enhance secretion of both IgM and IgA by CH12LX B cells, depending on the surface isotype of individual cells. Further, the enhanced secretion of Ig in response to IL-5 is associated with increased steady-state levels of Ig mRNA. Finally, normal Peyer's patch B cells are also capable of expressing IL-5R in response to appropriate stimuli (i.e. anti-IgM- or anti-IgD-dextran). Thus, mucosal B cell responses to IL-5 are brought about by interaction of IL-5 with specific receptors expressed on the surface of those cells.     

Interferon-gamma stimulates the secretion of IL-1, but not of IL-6, by glomerular mesangial cells.

IL-1 activity in culture supernatant and cell lysate from rat mesangial cells stimulated with interferon-gamma (IFN-gamma) was measured by a thymocyte proliferation assay. While IFN-gamma alone had no effect on the secretion or the intracellular pool of IL-1, the enhancement by IFN-gamma of IL-1 secretion in response to lipopolysaccharide (LPS) was observed. The stimulatory effect of culture supernatant on thymocyte proliferation was abrogated by preincubation with the anti-IL-1 antibody. At least 4-h incubation with IFN-gamma and LPS was required to detect enhancing effect of IFN-gamma. The addition of as little as 1 U/ml IFN-gamma significantly increased IL-1 secretion in the presence of 10 micrograms/ml LPS. The IL-6 activity in culture supernatants was determined by measurement of thymidine uptake in mouse IL-6-dependent cell line (MH60.BSF2). Mesangial cells secreted IL-6 in culture supernatant without additional stimuli and LPS distinctly increased it as described previously. However, in contrast to IL-1 production, no effect of IFN-gamma on IL-6 secretion was observed in the presence or absence of LPS. Moreover, we determined whether enhanced IL-1 release is associated with Ia expression on mesangial cells. IFN-gamma alone and the combination with LPS induced marked expression of Ia antigen, whereas LPS alone did not. We conclude that IFN-gamma stimulates the production of IL-1, but not IL-6, by mesangial cells and suggest an important role of IFN-gamma in the pathogenesis of glomerulonephritis by regulating the mesangial production of IL-1 and the accessory cell function of mesangial cells.     

Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells

A number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood–retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1β (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE-19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.     

IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-beta1 and IL-4

Regulatory cytokines mediate the participation of oral mucosal epithelial cells (OMEC) in local immune responses. The aim of this study was to characterize the isoforms of IL-1 receptor antagonist (IL-1ra) in cultured human primary OMECs and to compare its production with that of IL-1 alpha (IL-1alpha) and IL-1 beta (IL-1beta). Western blot analysis showed that IL-1ra was 22 kDa in size hence slightly smaller than monocyte IL-1ra (25 kDa). A minor form of 20 kDa was also found in unstimulated cell culture lysates. In culture supernatants, IL-1 bioactivity increased after IL-1ra neutralization, indicating that the baseline production of IL-1ra is biologically relevant. Immunohistochemistry showed a relation between IL-1ra and involucrin expressions, suggesting that intracytoplasmic IL-1ra may be involved in cell terminal differentiation. In unstimulated culture lysates, there was far more IL-1ra than IL-1alpha and IL-1beta. TGF-beta1 markedly increased the IL-1ra/IL-1beta ratio from 93.6 : 1 to 300 : 1. IL-4, which is generally described as an anti-inflammatory cytokine, increased IL-1 but not IL-1ra production. TNF-alpha increased intracellular production of the three IL-1 members. IL-1ra levels were lower in supernatants than in lysates of cultured cells. Our results show that human OMECs constitutively produce significant amounts of a biologically active form of IL-1ra. TGF-beta1 mu(p)-regulation points to a positive amplification loop and IL-4 to a down-regulation loop, both including Th2 cells and OMECs. They may be important in oral tolerance and IgA production, respectively.     

Glomerular epithelial cells transform to myofibroblasts: early but not late removal of TGF-beta1 reverses transformation

Studies were carried out to determine whether epithelial-mesenchymal transformation (EMT), well described in renal tubular epithelial cells, also occurs in glomerular epithelial cells and whether it is reversible. To this effect, cultured glomerular epithelial cells were incubated with TGF-beta(1) and their transformation into myofibroblasts was studied. At 4 days, the cells altered their phenotype, as shown by a change in shape, an increase in intracellular staining for alpha-smooth muscle actin (alpha-SMA), a decrease in membrane staining for cytokeratin, and an increase in matrix deposition. Changing the medium after 4 days by excluding TGF-beta(1) and adding fetal bovine serum (FBS) [as a source of epidermal growth factor (EGF) and other growth factors] caused the cells to revert to their original epithelial phenotype. By contrast, when the medium was changed in the same manner after 8 days of exposure to TGF-beta(1), the cells did not revert but remained myofibroblastic. Staining the cells for expression of EGF receptor before and after exposure to TGF-beta(1) caused this receptor, originally present on the plasma membrane, to become partly intracellular after 4 days of TGF-beta(1) exposure and completely intracellular after 8 days of TGF-beta(1) exposure. Kidney sections from 2 models of renal mass reduction were stained. Loss of the epithelial marker (podocalyxin) staining and the acquisition of alpha-SMA staining was observed in the glomeruli. It is concluded that EMT takes place in glomerular epithelial cells in vivo and in vitro. In cultured glomerular epithelial cells, the process can be reversed by early, but not late intervention. It seems that TGF-beta(1) exposure progressively downregulates the EGF receptor on the membrane, rendering the cell refractory to EGF signals critical for maintaining the epithelial phenotype.     

Cytokine-induced differentiation of IgA B cells: studies using an IgA expressing B-cell lymphoma.

Cytokines such as interleukin-5 (IL-5) and transforming growth factor beta 1 (TGF beta 1) increase IgA production by heterogeneous populations of lipopolysaccharide (LPS)-activated murine B cells. We have used IgA expressing murine B-lymphoma cells CH12.LX.C4.4F10 (4F10) to define the activity of these and other cytokines on IgA secretion at the single-cell level, membrane IgA expression, IgA polymerization and cell growth. IL-5 as well as LPS significantly increases IgA secretion of 4F10 cells, whereas TGF beta 1, a cytokine known to stimulate isotype switching to IgA among surface IgM-bearing B cells, inhibits IgA secretion. When tested alone, IL-1 beta, IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) do not significantly alter IgA secretion. However, there is a synergistic increase in IgA secretion when 4F10 cells are co-stimulated with IL-5 and IL-4, while IFN-gamma inhibits IL-5-stimulated up-regulation of IgA secretion. In parallel with increased IgA secretion after cytokine stimulation, 4F10 cells display less membrane IgA. Increased J-chain steady-state mRNA levels after IL-5 or LPS stimulation are paralleled by increased mRNA levels for secreted IgA, but are not accompanied by alterations in the ratio of monomeric to polymeric IgA. IL-5 and LPS initially stimulated but later inhibited 4F10 cell proliferation suggesting an inverse relationship between proliferation and differentiation in this cell line. 4F10 cells are a useful model for the characterization of discrete aspects of IgA B-cell differentiation, since the secretory and membrane Ig and proliferative responses of this IgA B-cell line to cytokines and LPS appear to parallel those of freshly isolated murine B cells.     

八肽胆囊收缩素对大鼠滑膜细胞株RSC-364分泌IL-6的影响

探讨八肽胆囊收缩素（CCK-8）对大鼠滑膜细胞株RSC-364分泌IL-6的调控作用。应用ELISA法观察不同浓度梯度的CCK-8对在TNF-α或IL-1β诱导下的RSC-364分泌IL-6水平的影响。结果显示：10^-6mol/L,10^-8mol/L浓度的CCK-8可分别促进TNF-α或IL-1β诱导24h后RSC-364细胞株分泌IL-6,而应用CCK受体拮抗剂丙谷胺则可明显抑制这种刺激效应,提示CCK-8可调节滑膜细胞分泌IL-6,在类风湿性关节炎的发病机制中可能起潜在的调控作用。     

视黄酸对细胞因子诱导的人肺泡Ⅱ型上皮细胞分泌C3及B因子的影响

目的:探讨视黄酸(RA)对细胞因子诱导的人肺泡Ⅱ型上皮细胞A549分泌C3及B因子的影响。方法:用ELISA检测TNFα和IL1β诱导的A549细胞培养上清中C3及B因子的水平。用RTPCR分析C3及B因子mRNA的表达。结果:TNFα和IL1β诱导A549细胞分泌C3及B因子具有时间和剂量依赖性。IL6诱导A549细胞分泌C3和B因子的水平是未处理组的4.7、1.4倍,IFNγ诱导A549细胞分泌C3和B因子的水平是未处理组的2.1、1.7倍。RA本身对A549细胞分泌C3及B因子没有影响,但可显著增强TNFα和IL1β诱导的A549细胞分泌C3和B因子及其mRNA的表达,以及IL6和IFNγ诱导的B因子合成。结论:RA可上调TNFα、IL1β、IL6和IFNγ诱导的A549细胞分泌C3及B因子,调节肺局部组织的免疫防御反应,为临床上应用RA和细胞因子防治肺部疾病提供了理论依据。     

CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance

CD4(+) T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non-pathogenic stimuli, and further understanding of the precise mechanisms by which these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR-transgenic bone marrow into irradiated recipients in which it has been possible to track antigen-specific CD4(+) cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut-associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter a state of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA-4 either using anti-CTLA-4 mAb or by generating chimeras using DO11.10xCTLA-4(-/-) mice as donors we have clearly shown that antigen-specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10xIL-10(-/-) cells, indicating that secretion of this cytokine by antigen-specific cells is not required.     

Non-classical MHC class I molecules on intestinal epithelial cells: mediators of mucosal crosstalk

Summary: The mucosal immune environment consists of a complex combination of lymphoid cells, non‐lymphoid cells, and lumenal bacteria. Signals from lumenal bacteria are constantly transmitted to the underlying tissues across the intestinal epithelial barrier. Intestinal epithelial cells (IECs) can sense these signals, integrate them, and interpret them for lamina propria lymphoid populations. One mechanism by which these signals are communicated is by the expression of non‐classical major histocompatibility complex (MHC) class I molecules by IECs. Epithelial cells can express a surprising variety of non‐classical MHC class I molecules. In some cases, IECs can act as non‐professional antigen‐presenting cells utilizing the expression of such non‐classical MHC class I molecules to directly present bacterial antigens. In other cases, the expression of non‐classical MHC class I molecules may act as a co‐stimulatory molecule or adhesion molecule that can modify the mucosal immune response. Finally, the expression of these molecules on IECs can lead to a broad array of responses ranging from tolerance to inflammation. Overall, the IEC, via the expression of non‐classical MHC class I molecules, is a central mediator of the constant crosstalk between the intestinal lumen and the mucosal immune system.     

IL-2对中子照射后肠上皮细胞生长和凋亡的影响及其机制

目的:观察中子照射对体外培养的IEC-6细胞生长的影响及IL-2对其损伤后增殖和恢复的作用,并进一步探讨IL-2调节受照射肠上皮细胞生长的相关机制。方法:单独用IL-2(1×105U/L)或同时施加JAK1激酶阻断剂(A77-1726)处理受4Gy中子照射的IEC-6细胞,并于照后10、15、30min和1、3、6、12、24、48及72h,用MTT比色法和流式细胞术检测受照射后IEC-6细胞的增殖活力和死亡方式的改变。以免疫细胞化学染色和Western blot检测IEC-6细胞上IL-2Rβ的表达和JAK1活化情况。结果:4Gy中子照射后24h,IEC-6细胞的增殖活力明显降低;而IL-2处理组该细胞的增殖活力有显著提高(P<0.05)。受中子照射的IEC-6细胞经IL-2作用24h,其凋亡率明显降低(P<0.05),而坏死率变化不明显。以IL-2刺激中子照射的IEC-6细胞后,于10及15min可见JAK1发生明显磷酸化活化,24h时IL-2Rβ的表达明显增多。同时应用A77-1726和IL-2处理受中子照射的IEC-6细胞后,其增殖活力明显低于单纯IL-2处理组。结论:IL-2可促进受中子照射的IEC-6细胞增殖,具有抗辐射作用。IL-2Rβ和JAK1活化参与了IL-2对中子损伤的IEC-6细胞生长的调控。