SummaryTo investigate possible roles of T helper type 2 (Th2) cytokines in the anti-arthritic effects of a blood fluke, Schistosoma mansoni (Sm), for mouse collagen-induced arthritis (CIA), wild-type (WT), signal transducer and activator of transcription 6 (STAT6) knock-out (KO) and interleukin (IL)-10 KO mice were infected with Sm. Three weeks after infection, the mice were immunized with bovine type II collagen (IIC). Arthritis severity was monitored by scoring, measurement of paw thickness and the presence of ankylosis. Serum anti-IIC IgG levels, splenic cytokine production and cytokine gene expression in the popliteal lymph nodes (PLNs) were measured and compared among WT and gene-KO mice. Consistent with our previous findings, Sm infection reduced the arthritis severity in WT mice. Splenic production of IL-17A and tumor necrosis factor (TNF)-α was reduced by the infection. In contrast, Sm infection markedly exacerbated CIA in STAT6 KO mice. In the KO mice, IL-17A production was increased by the infection. Conversely, Sm infection did not affect the exacerbated arthritis in IL-10 KO mice, although IL-17A production was reduced by the helminth. Our results suggest that signaling via STAT6 (presumably IL-4 and/or IL-13) and IL-10 is required for the suppression of CIA by Sm infection, but through different mechanisms. STAT6 was essential for helminth-induced reduction of IL-17A, whereas regulation of the basal arthritis severity by IL-10 was needed in order for it to be sufficiently suppressed by the helminth.

PURPOSE To clarify the possible involvement of the type 2-initiating cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) in the pathophysiology of allergic conjunctivitis, we evaluated ragweed (RW)-induced experimental allergic conjunctivitis (EAC) models by using IL-25 knockout (KO), IL-33 KO, and TSLP receptor (TSLPR) KO mice. METHODS Interleukin-25 KO, IL-33 KO, TSLPR KO, and BALB/c wild-type mice were sensitized twice with RW in alum and then challenged with RW in eye drops. Clinical scores and eosinophil infiltration were evaluated. Expression levels of serum immunoglobulin E (IgE) and cytokines in the conjunctival tissues were quantified and immunohistochemical analysis was carried out. RESULTS Significant reductions in clinical scores and numbers of infiltrating eosinophils were observed in the RW-EAC model using IL-33 KO mice. There were no significant differences in clinical scores and numbers of infiltrating eosinophils among IL-25KO, TSLPR KO, and wild-type mice. Serum IgE concentration was upregulated after RW challenges, and there were no differences among the mouse genotypes. Expression levels of of il4, il5, il13, and ccl5 mRNA were diminished in the conjunctivae of the RW-EAC model using IL-33 KO mice compared to those in wild-type mice. Interleukin-33 expression was upregulated as early as 1 hour after RW eye-drop challenge. The number of infiltrating basophils in the conjunctivae of the RW-EAC model using IL-33 KO mice was diminished compared to that in wild-type mice. CONCLUSIONS Among the type 2-initiating cytokines, IL-33 may play a major role in conjunctival inflammation in an RW-EAC model.

Previously, we have shown that IL-21R−/− splenocytes ameliorate GVHD as compared with wild-type splenocytes. Here, we investigated whether or not IL-21R−/− splenocytes diminish the graft-versus-leukemia (GVL) effect. Surprisingly, IL-21R−/− splenocytes efficiently eliminate leukemic cells as well as wild-type splenocytes, suggesting the retention of GVL effects in the absence of IL-21 signaling. To compare the GVL effect between IL-21R−/− and wild-type cells, we titrated the number of splenocytes required for the elimination of leukemic cells and found that the threshold of GVL effect was obtained between 5 × 105 and 5 × 106 with both types of splenocytes. Cotransplantation with CD8-depleted splenocytes but not with purified CD8 T-cells resulted in a significant reduction in anti-leukemic effect of IL-21R−/− cells compared with wild-type cells, suggesting that the lack of IL-21 signaling primarily impairs CD4 T-cell rather than CD8 T-cell function and the comparable GVL effect with IL-21R−/− bulk splenocytes results from cooperative compensation by CD8 T-cells.

Recent advances of biotechnology have laid the groundwork for potent and specific molecular-targeting therapies including RNA interference. The largest remaining hurdle for widespread use of this technology in skin is an effective delivery system. Here, we demonstrate an effective topical delivery system using a cream formulation containing a small-interfering RNA (siRNA) that specifically targets osteopontin (OPN). OPN is a validated target in numerous inflammatory diseases, including rheumatoid arthritis (RA). The siRNA targeting OPN was incorporated into a cream formulation GeneCream that penetrates the stratum corneum, depositing siRNA in the epidermis, dermis, and to a lesser extent, subcutaneous tissue. In addition, when the OPN siRNA cream was topically applied to the skin of a collagen antibody-induced RA mouse model, the siRNA cream prevented the occurrence of severe, irreversible damage to bone and cartilage. Thus, the siRNA cream provides effective delivery of active OPN siRNA, suggesting this formulation may represent a platform technology for delivery of siRNAs for treating various disorders including RA.

Cysteinyl leukotrienes (CysLTs), the potent lipid inflammatory mediators, are elevated in many pathological conditions and implicated in various inflammatory diseases including asthma, however their role in airway epithelial cells modulation is not clearly understood. We have investigated the effects of a CysLT, Leukotriene D4 (LTD4) on human airway epithelial cells, and assessed its role and mode of action in these cells.Human small airway epithelial cells (SAECs) and A549 cells were incubated with different concentrations of LTD4 for different time intervals. Subsequently trypan blue dye exclusion assay, MTT assay, Western blotting, RT-PCR and immunofluorescence experiments were performed to examine the effects of LTD4 on proliferation and related molecular changes in the airway epithelial cells.The treatment of human airway epithelial cells with LTD4 resulted in a significant increase in cell proliferation and modulation in the expression of receptors, CysLT1R and CysLT2R in SAECs as well as A549 cells. In both types of cells, LTD4 increased the expression levels of PCNA and c-myc, and trans-activated EGF receptor and increased the activation of ERK1/2. When treated along with epidermal growth factor (EGF), LTD4 showed a marginal additive effect in ERK1/2 and EGFR phosphorylation compared to LTD4 alone in both types of airway epithelial cells.In conclusion, these results suggest that sustained presence of lipid inflammatory mediator LTD4 could induce human airway epithelial cell proliferation through ERK1/2 phosphorylation, either directly via CysLT1 receptor or by transactivating EGFR.

IL-17 is a proinflammatory cytokine that activates T cells and other immune cells to produce a variety of cytokines, chemokines, and cell adhesion molecules. This cytokine is augmented in the sera and/or tissues of patients with contact dermatitis, asthma, and rheumatoid arthritis. We previously demonstrated that IL-17 is involved in the development of autoimmune arthritis and contact, delayed, and airway hypersensitivity in mice. As the expression of IL-17 is also augmented in multiple sclerosis, we examined the involvement of this cytokine in these diseases using IL-17−/− murine disease models. We found that the development of experimental autoimmune encephalomyelitis (EAE), the rodent model of multiple sclerosis, was significantly suppressed in IL-17−/− mice; these animals exhibited delayed onset, reduced maximum severity scores, ameliorated histological changes, and early recovery. T cell sensitization against myelin oligodendrocyte glycoprotein was reduced in IL-17−/− mice upon sensitization. The major producer of IL-17 upon treatment with myelin digodendrocyte glycopritein was CD4+ T cells rather than CD8+ T cells, and adoptive transfer of IL-17−/− CD4+ T cells inefficiently induced EAE in recipient mice. Notably, IL-17-producing T cells were increased in IFN-γ−/− cells, while IFN-γ-producing cells were increased in IL-17−/− cells, suggesting that IL-17 and IFN-γ mutually regulate IFN-γ and IL-17 production. These observations indicate that IL-17 rather than IFN-γ plays a crucial role in the development of EAE.

ALS2/alsin is a member of guanine nucleotide exchange factors for the small GTPase Rab5 (Rab5GEFs), which act as modulators in endocytic pathway. Loss-of-function mutations in human ALS2 account for a number of juvenile recessive motor neuron diseases (MNDs). However, the normal physiological role of ALS2 in vivo and the molecular mechanisms underlying motor dysfunction are still unknown. To address these issues, we have generated mice homozygous for disruption of the Als2 gene. The Als2-null mice observed through 21 months of age demonstrated no obvious developmental, reproductive or motor abnormalities. However, immunohistochemical and electrophysiological analyses identified an age-dependent, slowly progressive loss of cerebellar Purkinje cells and disturbance of spinal motor neurons associated with astrocytosis and microglial cell activation, indicating a subclinical dysfunction of motor system in Als2-null mice. Further, quantitative epidermal growth factor (EGF)-uptake analysis identified significantly smaller-sized EGF-positive endosomes in Als2-null fibroblasts, suggesting an alteration of endosome/vesicle trafficking in the cells. Collectively, while loss of ALS2 does not produce a severe disease phenotype in mice, these Als2-null animals should provide a useful model with which to understand the interplay between endosomal dynamics and the long-term viability of large neurons such as Purkinje cells and spinal motor neurons.

IL-1 is a pro-inflammatory cytokine that plays an important role in inflammation and host responses to infection. We have previously shown that imbalances in the IL-1 and IL-1R antagonist (IL-1Ra) system cause the development of inflammatory diseases. To explore the role of the IL-1/IL-1Ra system in autoimmune disease, we analyzed myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in mice bearing targeted disruptions of the IL-1 alpha, IL-1 beta, IL-1 alpha and IL-1 beta (IL-1) or IL-1Ra genes. IL-1 alpha/beta double-deficient (IL-1(-/-)) mice exhibited significant resistance to EAE induction with a significant reduction in disease severity, while IL-1 alpha(-/-) or IL-1 beta(-/-) mice developed EAE in a manner similar to wild-type mice. IL-1Ra(-/-) mice also developed MOG-induced EAE normally with pertussis toxin (PTx) administration. In contrast to wild-type mice, however, these mice were highly susceptible to EAE induction in the absence of PTx administration. We found that both IFN-gamma and IL-17 production and proliferation were reduced in IL-1(-/-) T cells upon stimulation with MOG, while IFN-gamma, IL-17 and tumor necrosis factor-alpha production and proliferation were enhanced in IL-1Ra(-/-) T cells. These observations suggest that the IL-1/IL-1Ra system is crucial for auto-antigen-specific T cell induction and contributes to the development of EAE.

IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice spontaneously develop autoimmune arthritis. We demonstrate here that T cells are required for the induction of arthritis; T cell-deficient IL-1Ra(-/-) mice did not develop arthritis, and transfer of IL-1Ra(-/-) T cells induced arthritis in nu/nu mice. Development of arthritis was also markedly suppressed by TNF-alpha deficiency. We found that TNF-alpha induced OX40 expression on T cells and blocking the interaction between either CD40 and its ligand or OX40 and its ligand suppressed development of arthritis. These findings suggest that IL-1 receptor antagonist deficiency in T cells disrupts homeostasis of the immune system and that TNF-alpha plays an important role in activating T cells through induction of OX40.

Leukocyte cell-derived chemotaxin 2 (LECT2) was originally identified for its possible chemotactic activity against human neutrophils in vitro. It is a 16-kDa protein that is preferentially expressed in the liver. Its homologues have been widely identified in many vertebrates. Current evidence suggests that LECT2 may be a multifunctional protein like cytokines. However, the function of LECT2 in vivo remains unclear. To elucidate the role of this protein in vivo, we have generated LECT2-deficient (LECT2−/−) mice. We found that the proportion of NKT cells in the liver increased significantly in LECT2−/− mice, although those of conventional T cells, NK cells, and other cell types were comparable with those in wild-type mice. Consistent with increased hepatic NKT cell number, the production of IL-4 and IFN-γ was augmented in LECT2−/− mice upon stimulation with α-galactosylceramide, which specifically activates Vα14 NKT cells. In addition, NKT cell-mediated cytotoxic activity against syngeneic thymocytes increased in hepatic mononuclear cells obtained from LECT2−/− mice in vitro. Interestingly, the hepatic injury was exacerbated in LECT2−/− mice upon treatment with Con A, possibly because of the significantly higher expression of IL-4 and Fas ligand. These results suggest that LECT2 might regulate the homeostasis of NKT cells in the liver and might be involved in the pathogenesis of hepatitis.

Leukocyte cell-derived chemotaxin 2 (LECT2) was originally identified for its possible chemotactic activity against human neutrophils in vitro. It is a 16-kDa protein that is preferentially expressed in the liver. Its homologues have been widely identified in many vertebrates. Current evidence suggests that LECT2 may be a multifunctional protein like cytokines. However, the function of LECT2 in vivo remains unclear. To elucidate the role of this protein in vivo, we have generated LECT2-deficient (LECT2(-/-)) mice. We found that the proportion of NKT cells in the liver increased significantly in LECT2(-/-) mice, although those of conventional T cells, NK cells, and other cell types were comparable with those in wild-type mice. Consistent with increased hepatic NKT cell number, the production of IL-4 and IFN-gamma was augmented in LECT2(-/-) mice upon stimulation with alpha-galactosylceramide, which specifically activates Valpha14 NKT cells. In addition, NKT cell-mediated cytotoxic activity against syngeneic thymocytes increased in hepatic mononuclear cells obtained from LECT2(-/-) mice in vitro. Interestingly, the hepatic injury was exacerbated in LECT2(-/-) mice upon treatment with Con A, possibly because of the significantly higher expression of IL-4 and Fas ligand. These results suggest that LECT2 might regulate the homeostasis of NKT cells in the liver and might be involved in the pathogenesis of hepatitis.

Morphogenesis of the heart requires development of the endocardial cushion tissue that gives rise to the membranous septa and valves. Here we show that Meltrin beta/ADAM19, a novel metalloprotease-disintegrin, participates in the development of the endocardial cushion. Mice lacking Meltrin beta exhibit ventricular septal defect (VSD) and immature valves, and most of the animals die soon after birth. During development of the endocardial cushion, epithelial-mesenchymal transformation (EMT) of endocardial epithelial cells generates most of the cushion mesenchymes that constitute the main components of the septa and valves. Meltrin beta is expressed in both the epithelia and the mesenchymes of the endocardial cushion. In the absence of Meltrin beta, the cushion is small or thin in the septum-forming region and show poor remodeling of cardiac jelly components; both of these characteristics suggest impaired growth and differentiation of the endocardial cushion. When embryonic fibroblasts are cultured sparsely, Meltrin beta-lacking cells exhibit aberrant ectodomain shedding of type I Neuregulin, one of the ErbB ligands expressed in endocardial cells. These results suggest the necessity of proteolytic regulation of ErbB ligands by Meltrin beta for proper heart development.

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.

Contact hypersensitivity (CHS) is a typical inflammatory response against contact allergens. Inflammatory cytokines, including IL-1 and tumor necrosis factor (TNF)-alpha, are implicated in the reaction, although the precise roles of each cytokine have not been completely elucidated. In this report, we dissected the functional roles of IL-1 and TNF-alpha during CHS. CHS induced by 2,4,6-trinitorochlorobenzene as well as oxazolone was suppressed in both IL-1alpha/beta(-/-) and TNF-alpha(-/-) mice. Hapten-specific T cell activation, as examined by T cell proliferation, OX40 expression and IL-17 production, was reduced in IL-1alpha/beta(-/-) mice, but not in TNF-alpha(-/-) mice, suggesting that IL-1 but not TNF-alpha is required for hapten-specific T cell priming in the sensitization phase. On the other hand, TNF-alpha, induced by IL-1, was necessary for the induction of local inflammation during the elicitation phase. We also found that the expression of IFN-gamma-inducible protein 10 (IP-10) was augmented at the inflammatory site. Although IP-10 mRNA expression was abrogated in TNF-alpha(-/-) mice, both CHS development and TNF-alpha mRNA expression occurred normally in IFN-gamma(-/-) mice, indicating that the induction of IP-10 during CHS was primarily controlled by TNF-alpha. Interestingly, CHS was suppressed by treatment with anti-IP-10 mAb, suggesting a critical role for IP-10 in CHS. Reduced CHS in TNF-alpha(-/-) mice was reversed by IP-10 injection during the elicitation phase. Thus, it was shown that the roles for IL-1 and TNF-alpha are different, although both cytokines are crucial for the development of CHS.

Japan Abstract IL-1 is a proinflammatory cytokine that plays important roles in inflammation. However, the role of this cytokine under physiological conditions is not known completely. In this paper, we analyzed the role of IL-1 in maintaining body weight because IL-1 receptor antagonist–deficient (IL-1Ra (cid:2) / (cid:2) ) mice, in which excess IL-1 signaling may be induced, show a lean phenotype. Body fat accumulation was impaired in IL-1Ra (cid:2) / (cid:2) mice, but feeding behavior, expression of hypothalamic factors involved in feeding control, energy expenditure, and heat production were normal. When IL-1Ra (cid:2) / (cid:2) mice were treated with monosodium glutamate (MSG), which causes obesity in wild-type mice by ablating cells in the hypothalamic arcuate nucleus, they were resistant to obesity, indicating that excess IL-1 signaling antagonizes the effect of MSG-sensitive neuron deficiency. IL-1Ra (cid:2) / (cid:2) mice showed decreased weight gain when they were fed the same amount of food as wild-type mice, and lipid accumulation remained impaired even when they were fed a high-fat diet. Interestingly, serum insulin levels and lipase activity were low in IL-1Ra (cid:2) / (cid:2) mice, and the insulin levels were low in contrast to wild-type mice after MSG treatment. These observations suggest that IL-1 plays an important role in lipid metabolism by regulating insulin levels and lipase activity under physiological

Interleukin-17 is a T cell-derived proinflammatory cytokine. This cytokine is suspected to be involved in the development of rheumatoid arthritis (RA) because this cytokine expression is augmented in synovial tissues of RA patients. The pathogenic roles of IL-17 in the development of RA, however, still remain to be elucidated. In this study, effects of IL-17 deficiency on collagen-induced arthritis (CIA) model were examined using IL-17-deficient mice (IL-17(-/-) mice). We found that CIA was markedly suppressed in IL-17(-/-) mice. IL-17 was responsible for the priming of collagen-specific T cells and collagen-specific IgG2a production. Thus, these observations suggest that IL-17 plays a crucial role in the development of CIA by activating autoantigen-specific cellular and humoral immune responses.

NB-2, a member of the contactin subgroup in the immunoglobulin superfamily, is expressed specifically in the postnatal nervous system, reaching a maximum level at 3 weeks postnatal. NB-2 displays neurite outgrowth-promoting activity in vitro. To assess its function in the nervous system, we generated mutant mice in which a part of the NB-2 gene was ablated and replaced with the tau-LacZ gene. The general appearance of NB-2-deficient mice and their gross anatomical features were normal. The LacZ expression patterns in heterozygous mice revealed that NB-2 is preferentially expressed in the central auditory pathways. In the audiogenic seizure test, NB-2-deficient mice exhibited a lower incidence of wild running, but a higher mortality rate than the wild-type littermates. c-Fos immunohistochemistry demonstrated that neural excitability induced by the audiogenic seizure test in the NB-2-deficient mice was prominently attenuated in both the dorsal and external cortices of the inferior colliculus, where enhanced neural excitability was observed in the wild-type mice. In response to pure-tone stimulation after priming, NB-2-deficient mice exhibited a diffuse and low level of c-Fos expression in the central nucleus of the inferior colliculus, which was distinctly different from the band-like c-Fos expression corresponding to the tonotopic map in the wild-type littermates. Taken together, these results suggest that a lack of NB-2 causes impairment of the neuronal activity in the auditory system.

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.

Meltrin alpha (ADAM12) is a metalloprotease-disintegrin whose specific expression patterns during development suggest that it is involved in myogenesis and the development of other organs. To determine the roles Meltrin alpha plays in vivo, we generated Meltrin alpha-deficient mice by gene targeting. Although the number of homozygous embryos are close to the expected Mendelian ratio at embryonic days 17 to 18, ca. 30% of the null pups born die before weaning, mostly within 1 week of birth. The viable homozygous mutants appear normal and are fertile. Most of the muscles in the homozygous mutants appear normal, and regeneration in experimentally damaged skeletal muscle is unimpeded. In some Meltrin alpha-deficient pups, the interscapular brown adipose tissue is reduced, although the penetrance of this phenotype is low. Impaired formation of the neck and interscapular muscles is also seen in some homozygotes. These observations suggest Meltrin alpha may be involved in regulating adipogenesis and myogenesis through a linked developmental pathway. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a candidate substrate of Meltrin alpha, and we found that TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ectodomain shedding of HB-EGF is markedly reduced in embryonic fibroblasts prepared from Meltrin alpha-deficient mice. We also report here the chromosomal locations of Meltrin alpha in the mouse and rat.