Cutting edge: IL-23 cross-regulates IL-12 production in T cell-dependent experimental colitis

Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.     

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.     

IL-10 transgenic mice present a defect in T cell development reminiscent of SCID patients.

To analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-alpha beta T cells as the absolute number of thymic dendritic, TCR-gamma delta and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.     

构建表达IL 2的大肠埃希菌对小鼠肠炎的影响

目的 以Escherichia coli Nissle 1917为基础建立一种与肠道菌群相关的新型白介素2(IL 2)递送方式,研究其对葡聚糖硫酸钠(DSS)诱导的实验性结肠炎的治疗作用。 方法 将小鼠随机分为4组(每组10只),以正常小鼠作为空白对照组,实验组小鼠用3%的DSS水诱导小鼠结肠炎模型,分别灌胃表达IL 2的菌株(E. coli 1917/IL 2)、空质粒转化的菌株(E. coli 1917/0)或PBS进行治疗5 d,定期评估各组小鼠的临床体征、疾病活动指数(DAI)、病理和免疫组织学变化。 结果 构建的益生工程菌E. coli 1917/IL 2可有效缓解DSS诱导的小鼠肠炎,小鼠DAI评分较低,体质量及结肠长度均高于对照组,肠黏膜组织中炎症细胞浸润较少。 结论 使用工程化益生大肠埃希菌编码免疫调节细胞因子的治疗策略为溃疡性结肠炎提供一种潜在的治疗方法。     

Treatment with IL-27 attenuates experimental colitis through the suppression of the development of IL-17-producing T helper cells

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory diseases characterized by inflammation and relapsing gastrointestinal disorders. Recent studies have shown that Th17 cells, which are well known as key mediators of chronic inflammation, have a pivotal role in onset and development of IBD in humans and mice, alike. In recent years, it has been reported that IL-27, which is an IL-12-related heterodimeric cytokine consisting of EBI3 and p28 subunits, act directly on naive T cells to suppress the differentiation of Th17 cells. However, effects of exogenous IL-27 on the IBD are not well elucidated. To clarify the suppressive effect of IL-27 treatment on IBD, we applied the flexible linking method to EBI3 and p28 subunits and generated a single-chain human IL-27 (scIL-27). scIL-27 inhibited xenogenic mouse Th17 cell differentiation in vitro, indicating that scIL-27 also acts in mouse immune systems. In a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced mouse acute colitis model, subcutaneous scIL-27 treatment significantly improved the colon length, extent of necrosis, and ulceration and thickened epithelium and several pathological scores in a dose-dependent manner. scIL-27 clearly suppressed several inflammatory cytokines, including IL-17, in inflamed colon, except for anti-inflammatory cytokine IL-10. The mesenteric lymph node cells from scIL-27-treated mice also exhibited a reduced inflammatory response and, furthermore, a lower population of Th17 cells than those of PBS-treated mice. Finally, we showed the therapeutic efficacy of scIL-27 on TNBS-induced colitis even after active colitis was established. These results suggest new possible therapeutic approaches for IBD, including disorders such as Crohn's disease and ulcerative colitis.     

Prevention of experimental colitis in SCID mice reconstituted with CD45RBhigh CD4+ T cells by blocking the CD40-CD154 interactions

Increased expression of CD40 and CD40 ligand (CD40L or CD154) has been found in inflamed mucosa of human inflammatory bowel disease (IBD), and interactions between these molecules seem to be involved in local cytokine production by macrophages. However, the precise role of CD40 signaling in the pathogenesis of IBD is still poorly understood. The aim of the present study was to investigate the in vivo relevance of CD40 signaling in experimental colitis in SCID mice reconstituted with syngeneic CD45RBhighCD4+ T cells. The results demonstrated that CD40+ and CD40L+ cells as well as their mRNA levels were significantly increased in inflamed mucosa. Administration of anti-CD40L neutralizing mAb over an 8-wk period starting immediately after CD45RBhighCD4+ T cell reconstitution completely prevented symptoms of wasting disease. Intestinal mucosal inflammation was effectively prevented, as revealed by abrogated leukocyte infiltration and decreased CD54 expression and strongly diminished mRNA levels of the proinflammatory cytokines IFN-gamma, TNF, and IL-12. When colitic SCID mice were treated with anti-CD40L starting at 5 wk after T cell transfer up to 8 wk, this delayed treatment still led to significant clinical and histological improvement and down-regulated proinflammatory cytokine secretion. These data suggest that the CD40-CD40L interactions are essential for the Th1 inflammatory responses in the bowel in this experimental model of colitis. Blockade of CD40 signaling may be beneficial to human IBD.     

Immunization of mice with T cell-dependent antigens promotes IL-6 and TNF-alpha production in muscle cells

IL-6 and TNF-alpha are proinflammatory cytokines involved in various inflammatory or non-inflammatory disorders characterized by muscle wasting. While infiltrating leukocytes are known to be the major source of these cytokines, it is unclear whether muscle cells also contribute to local inflammation. In this study, we first showed that cultured muscle cells and naive mouse muscle tissue were capable of producing IL-6 and TNF-alpha. We demonstrated an increased expression of IL-6 and TNF-alpha on muscle sections of C57BL/6J mice immunized with acetylcholine receptor (AChR) in the complete Freund's adjuvant (CFA) or with CFA only. In the presence of IL-6 or TNF-alpha, cultured AChR-expressing mouse (G-8) and human (TE671) skeletal muscle cells showed significantly decreased alpha-bungarotoxin-binding sites as measured by cellular ELISA. Moreover, IL-6- or TNF-alpha-treated muscle cells displayed a considerable increase in apoptotic cell ratios. Collectively, this study suggests a direct role for these two cytokines in muscle cell destruction and a possibility of muscle cell damage via an autocrine fashion.     

Up-regulation of IL-18BP,but not IL-18 mRNA in rat liver by LPS

Interleukin (IL)-18 is a pro-inflammatory cytokine that plays a critical role in inflammation leading to liver damage, through promotion of Fas-mediated apoptosis. Inhibition of IL-18 activity protects against LPS-induced lethality in mice and against liver damage induced by LPS after sensitisation of mice with Proprionibacterium acnes. A specific, potent, endogenous inhibitor of IL-18 (IL-18BP) has been identified in mice and humans, and IL-18BP mRNA is expressed constitutively in liver. The objectives of this study were to compare changes in IL-1beta and IL-18 mRNA expression in the liver of rats in response to peripheral injection of LPS, using real-time PCR, and also to investigate whether IL-18BP mRNA expression is affected by this treatment. LPS rapidly up-regulated IL-1beta mRNA expression, but IL-18 mRNA expression was unaffected by LPS treatment. Unlike IL-18, IL-18BP mRNA was up-regulated dramatically by approximately 12-fold above nai;ve levels, peaking 3 h after LPS injection. This ability of LPS to up-regulate expression of the endogenous IL-18 inhibitor may indicate a mechanism by which the inflammatory response to LPS is regulated.     

Systemic administration of IL-18 promotes diabetes development in young nonobese diabetic mice

IL-18 is now identified as a pleiotropic cytokine that acts as a cofactor for both Th1 and Th2 cell development. Type 1 diabetes is considered a Th1-type autoimmune disease, and to date, the suppressive effect of exogenous IL-18 on the development of diabetes has been reported in 10-wk-old nonobese diabetic (NOD) mice. In the present study we administered exogenous IL-18 systemically in 4-wk-old NOD mice using i.m. injection of the IL-18 expression plasmid DNA (pCAGGS-IL-18) with electroporation. Contrary to previous reports, the incidence of diabetes development was significantly increased in NOD mice injected with pCAGGS-IL-18 compared with that in control mice. Systemic and pancreatic cytokine profiles deviated to a Th1-dominant state, and the the frequency of glutamic acid decarboxylase-reactive IFN-gamma-producing CD4(+) cells was also high in the IL-18 group. Moreover, it was suggested that the promoting effect of IL-18 might be associated with increased peripheral IL-12, CD86, and pancreatic IFN-inducible protein-10 mRNA expression levels. In conclusion, we demonstrate here that IL-18 plays a promoting role as an enhancer of Th1-type immune responses in diabetes development early in the spontaneous disease process, which may contribute to elucidating the pathogenesis of type 1 diabetes.     

In situ hybridization analysis of the age-associated decline in IL-2 mRNA expressing murine T cells

Only a small decrease in the number of L3T4- cells was observed in the Con A-stimulated splenocyte cultures of old mice as compared to young, which cannot account for the threefold decrease in IL-2 production. Northern and dot blot analysis of RNA from splenocytes containing equivalent numbers of L3T4+ cells from young and old mice showed that cells from old mice express less IL-2 mRNA after mitogenic stimulation than cells from young mice. Direct analysis by in situ hybridization of stimulated splenocytes from young and old mice then showed approximately a threefold decrease in the percentage of IL-2 mRNA expressing cells in the spleens of old mice as compared to young (8.7 +/- 4.1% old; 28.7 +/- 11.7% young). The average level of expression of IL-2 mRNA was not significantly different between cells from young and old mice; however, there were approximately 40% fewer cells expressing an intermediate to high amount of IL-2 mRNA in old mice as compared to young (26.3% vs 41.8%). These data suggest that the decrease in IL-2 production with age is associated primarily with a decrease in the frequency of IL-2 mRNA-expressing cells in old mice, especially in those cells expressing intermediate to high levels of IL-2 mRNA.     

Transfer of severe experimental autoimmune encephalomyelitis by IL-12- and IL-18-potentiated T cells is estrogen sensitive

The aim of this study was to evaluate the roles of IL-18 and IL-12 in potentiating the encephalitogenic activity of T cell lines specific for myelin oligodendrocyte glycoprotein (MOG(35-55)). MOG-specific T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-12 or IL-18 alone transferred only mild experimental autoimmune encephalomyelitis (EAE) into a low percentage of recipients. However, T cells cocultured with both cytokines transferred aggressive clinical and histological EAE into all recipients. Coculture of T cells with IL-12 enhanced the secretion of IFN-gamma, but not TNF-alpha, whereas coculture with IL-18 enhanced the secretion of TNF-alpha, but not INF-gamma. However, coculture with both IL-18 and IL-12 induced high levels of both TNF-alpha and IFN-gamma. Additionally, IL-12 selectively enhanced mRNA expression of CCR5, whereas IL-18 selectively enhanced the expression of CCR4 and CCR7, and CCR4 and CCR5 were coexpressed on the surface of T cells cocultured with IL-12 and IL-18. Finally, estrogen treatment, previously found to inhibit both TNF-alpha and IFN-gamma production, completely abrogated all signs of passive EAE. These data demonstrate that optimal potentiation of encephalitogenic activity can be achieved by conditioning MOG-specific T cells with the combination of IL-12 and IL-18, which, respectively, induce the secretion of IFN-gamma/CCR5 and TNF-alpha/CCR4/CCR7, and that estrogen treatment, which is known to inhibit both proinflammatory cytokines, can completely ablate this aggressive form of passive EAE.     

T cells enhance production of IL-18 by monocytes in response to an intracellular pathogen

We studied the effect of T cells on IL-18 production by human monocytes in response to Mycobacterium tuberculosis. Addition of activated T cells markedly enhanced IL-18 production by monocytes exposed to M. tuberculosis. This effect was mediated by a soluble factor and did not require cell-to-cell contact. The effect of activated T cells was mimicked by recombinant IFN-gamma and was abrogated by neutralizing Abs to IFN-gamma. IFN-gamma also enhanced the capacity of alveolar macrophages to produce IL-18 in response to M. tuberculosis, suggesting that this mechanism also operates in the lung during mycobacterial infection. IFN-gamma increased IL-18 production by increasing cleavage of pro-IL-18 to mature IL-18, as it enhanced caspase-1 activity but did not increase IL-18 mRNA expression. These findings suggest that activated T cells can contribute to the initial immune response by augmenting IL-18 production by monocytes in response to an intracellular pathogen.     

Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

Role of gamma delta T cells in the inflammatory response of experimental colitis mice

We examined the severity of experimental colitis induced by dextran sulfate sodium (DSS) using immunologically manipulated mice. C57BL/6 mice showed more severe colitis than BALB/c mice, but mice of both strains recovered fully from the disease after the removal of DSS from their drinking water. The infiltrated cells at the lesions were mainly granulocytes in normal littermates. However, C.B-17 scid, IL-7Ralpha deficient, and TCR-Cbetadelta double-deficient mice showed severe colitis and did not recover from the disease even after the removal of DSS. It was found that the infiltrated cells at the lesions in the lethal strains were monocytes. Although both TCR-Cdelta(-/-) and TCR-Cbeta(-/-) mice showed severe colitis phenotypes, infiltration in the former is monocyte-dominant while that in the latter is granulocyte-dominant. Thus the type of cells that infiltrate at the lesions of DSS-induced experimental colitis may be controlled by functional T cell subsets. Immunohistological and RT-PCR analyses of the inflamed colon revealed that the murine homologue of human GROalpha released by some cells under the control of gammadeltaT cells is a possible candidate determining the severity of DSS-induced experimental colitis.     

IL-7 exacerbates chronic colitis with expansion of memory IL-7Rhigh CD4+ mucosal T cells in mice

We have previously demonstrated that mucosal CD4(+) T cells expressing high levels of IL-7 receptor (IL-7R(high)) are pathogenic cells responsible for chronic colitis. Here we investigate whether IL-7 is directly involved in the expansion of IL-7R(high) memory CD4(+) mucosal T cells and the exacerbation of colitis. We first showed that CD4(+) lamina propria lymphocytes (LPLs) from wild-type, T cell receptor-alpha-deficient (TCR-alpha(-/-)), and recombinase-activating gene (RAG)-2(-/-)-transferred mice with or without colitis showed phenotypes of memory cells, but only CD4(+) LPLs from colitic mice showed IL-7R(high). In vitro stimulation by IL-7, but not by IL-15 and thymic stromal lymphopoietin, enhanced significant proliferative responses and survival of colitic CD4(+), but not normal CD4(+) LPLs. Importantly, in vivo administration of IL-7 mice accelerated the expansion of IL-7R(high) memory CD4(+) LPLs and thereby exacerbated chronic colitis in RAG-2(-/-) mice transferred with CD4(+) LPLs from colitic TCR-alpha(-/-) mice. Conversely, the administration of anti-IL-7R monoclonal antibody significantly inhibited the development of TCR-alpha(-/-) colitis with decreased expansion of CD4(+) LPLs. Collectively, the present data indicate that IL-7 is essential for the expansion of pathogenic memory CD4(+) T cells under pathological conditions. Therefore, therapeutic approaches targeting the IL-7R pathway may be feasible in the treatment of human inflammatory bowel disease.     

IFN-gamma-dependent and -independent mechanisms in adverse effects caused by concomitant administration of IL-18 and IL-12

IL-18 is a new type of inflammatory cytokine similar to but distinct from IL-12 and IL-1beta. One intriguing property of IL-18 is synergism with IL-12 in many respects. In this study we examined the in vivo synergistic effects of IL-18/IL-12 in mice and found lethal toxicity accompanying an elevated IFN-gamma level in the serum. Since treatment with IL-18 alone did not have any apparent toxicity, and treatment with IL-12 alone showed only limited toxicity in our system, the synergy between the two cytokines was all the more remarkable. The major symptoms of the toxicity were weight loss, diarrhea, hemorrhagic colitis, splenomegaly, fatty liver, and atrophic thymus, most of which are similarly found in endotoxin-induced septic shock. However, in contrast to septic shock, TNF-alpha was not induced. The involvement of IFN-gamma in the toxicity was further studied in detail. Treatment of athymic nude mice with anti-asialo-GM1 did not reduce the toxicity, whereas anti-IFN-gamma treatment of wild-type mice alleviated it. When IFN-gamma-deficient mice were treated with IL-18/IL-12, the majority of them showed mortality and toxicity with severe pulmonary edema. These results indicate that IL-18/IL-12 treatment induces severe adverse effects through not only IFN-gamma-dependent mechanisms but also IFN-gamma-independent processes.     

Long-term xenogeneic chimeras. Full differentiation of rat T and B cells in SCID mice

To test whether T and B cell differentiation can proceed across species barriers, rat fetal liver (FL) cells were used to reconstitute SCID mice. Provided that the hosts were conditioned with light irradiation, i.v. injection of FL cells caused near-complete repopulation with rat-derived lymphohematopoietic cells, including myeloid and erythroid cells, Ia+ cells of the macrophage/dendritic cell lineages, and mature T and B cells. In keeping with the known hypersensitivity of SCID cells to irradiation, host hematopoietic cells in the chimeras were almost undetectable, even with hosts exposed to as low as 250 rad. In the case of T cells, the distribution of immature and mature cells in the thymus of rat FL----SCID chimeras closely resembled the normal rat thymus in terms of architecture and expression of CD4, CD8, and alpha beta-TCR molecules. Thymopoiesis was followed by the appearance of large numbers of typical rat CD4+ and CD8+ cells in spleen and lymph nodes. These organs also contained substantial numbers of rat B (mu+) cells. The data thus indicate that the xenogeneic environment of SCID mice is fully capable of sustained de novo differentiation of rat T and B cells.     

Characterization of middle T antigen expressed by using an adenovirus expression system.

The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60c-src. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60c-src remained unassociated. Immunoaffinity purification resulted in approximately 100 micrograms of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74- and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.