Suppressor of cytokine signalling 1 (SOCS1) is a physiological regulator of the asthma response

Background The molecular determinants of the severity and persistence of allergic asthma remain poorly understood. Suppressor of cytokine signalling 1 (SOCS1) is a negative regulator of IL‐4‐dependent pathways in vitro and might therefore control T‐helper type 2 (Th2) immunity associated traits, such as IgE levels, mucin production, IL‐5 and IL‐13 induction, and eosinophilic mucosal inflammation, which are implicated in allergic asthma. Objective To investigate the role of SOCS1 in regulating Th2‐associated disease traits in a murine sub‐chronic aeroallergen‐driven asthma model. Methods Following sensitization and challenge with ovalbumin (OVA), bronchoalveolar lavage and serum were collected from mice lacking the Socs1 gene on an IFN‐γ null background (Socs1^?/?Ifnγ^?/?). The composition of infiltrating cells in the lung, serum IgE and IgG1 levels and cytokine levels were analysed. Results Serum IgE levels and infiltrating eosinophils were considerably increased in the lungs of OVA‐treated Socs1^?/?Ifnγ^?/? mice compared with Ifnγ^?/? and C57BL/6 controls. Expression of the Th2 cytokines, IL‐4, IL‐5 and IL‐13 was increased in CD4⁺ cells and lung tissue from OVA‐treated Socs1^?/?Ifnγ^?/? mice. IgE, IL‐5 levels and infiltrating eosinophils were also elevated in saline‐treated Socs1^?/?Ifnγ^?/? mice, suggesting that in the absence of SOCS1, mice are already biased towards a Th2 response. It is at present unclear whether the elevated cytokine levels are sufficient to result in the exacerbated Th2 response to OVA challenge or whether enhanced intra‐cellular signalling also contributes. Surprisingly, of the various IL‐4/IL‐13 responsive genes tested, only Arginase I appeared to be modestly up‐regulated in the lungs of OVA‐treated Socs1^?/?Ifnγ^?/? mice, suggesting that regulation by SOCS1 occurs primarily in haematopoietic cells and not in the airway epithelium. Conclusions Together these results indicate that SOCS1 is an important regulator of the Th2 response.     

Interleukin-33 amplifies IgE synthesis and triggers mast cell degranulation via interleukin-4 in naïve mice

Background

The regulation and function of IgE in healthy individuals and in antigen‐naïve animals is not well understood. IL‐33 administration increases serum IgE in mice with unknown mechanism. We tested the hypothesis that IL‐33 provides an antigen‐independent stimulus for IgE production and mast cell degranulation.

Methods

IL‐33 was administered to naïve wild‐type (WT), nude and ST2^?/?, IL‐4^?/?, IL4Rα^?/? and T‐or B‐cell‐specific IL‐4Rα^?/? mice. IgEand cytokines were quantified by ELISA. T‐ and B‐lymphocyte numbers and CD40L expression were determined by flow cytometry. Anaphylaxis was measured by temperature, mast cell degranulation and histamine release.

Results

IL‐33 enhanced IgE production in naïve WT, T‐IL‐4Rα^?/? but not in ST2^?/?, IL‐4^?/?, IL‐4Rα^?/? or B‐cell‐specific IL‐4Rα^?/? mice, demonstrating IL‐33 specificity and IL‐4 dependency. Moreover, IL‐4 was required for IL‐33‐induced B‐cell proliferation and T‐cell CD40L expression, which promotes IgE production. IL‐33‐induced IL‐4 production was mainly from innate cells including mast cells and eosinophils. IL‐33 increased mast cell surface IgE and triggered degranulation and systemic anaphylaxis in allergen‐naïve WT but not in IL‐4Rα^?/? mice.

Conclusion

IL‐33 amplifies IgE synthesis and triggers anaphylaxis in naïve mice via IL‐4, independent of allergen. IL‐33 may play an important role in nonatopic allergy and idiopathic anaphylaxis.

     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Staphylococcus aureus enterotoxin B facilitates allergic sensitization in experimental asthma

Background Staphylococcus aureus Enterotoxin B (SEB) has immunomodulatory effects in allergic airway disease. The potential contribution of SEB to the sensitization process to allergens remains obscure. Objective In order to study the effects of staphylococcal‐derived toxins on the sensitization to ovalbumin (OVA) and induction of allergic airway inflammation, we have combined the nasal application of OVA with different toxins. Methods Nasal applications of OVA and saline, SEA, SEB, toxic shock syndrome toxin (TSST)‐1, protein A or lipopolysaccharide (LPS) were performed on alternate days from day 0 till 12. On day 14, mice were killed for the evaluation of OVA‐specific IgE, cytokine production by mediastinal lymph node (MLN) cells and bronchial hyperreactivity (BHR) to inhaled metacholine. The effect of SEB on dendritic cell (DC) migration and maturation, and on T cell proliferation was evaluated. Results Concomitant endonasal application of OVA and SEB resulted in OVA‐specific IgE production, whereas this was not found with SEA, TSST‐1, protein A, LPS or OVA alone. Increased DC maturation and migration to the draining lymph nodes were observed in OVA/SEB mice, as well as an increased T cell proliferation. Bronchial inflammation with an influx of eosinophils and lymphocytes was demonstrated in OVA/SEB mice, together with hyperresponsiveness and the production of IL‐4, IL‐5, IL‐10 and IL‐13 by MLN stimulated with OVA. Conclusions Our data demonstrate that SEB facilitates sensitization to OVA and consecutive bronchial inflammation with features of allergic asthma. This is likely due to augmentation of DC migration and maturation, as well as the allergen‐specific T cell proliferation upon concomitant OVA and SEB application. Cite this as: W. Huvenne, I. Callebaut, M. Plantinga, J. A. J. Vanoirbeek, O. Krysko, D. M. A. Bullens, P.Gevaert, P. Van Cauwenberge, B. N. Lambrecht, J. L. Ceuppens, C. Bachert and P. W. Hellings, Clinical & Experimental Allergy, 2010 (40) 1079–1080.     

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose‐dependent regulation of IgE formation. Methods Wild‐type mice as well as T helper (Th)1‐deficient IL‐12p40^?/? and IFN‐γ^?/? mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen‐dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT‐PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL‐4, IL‐5 and IL‐13, but no induction of IFN‐γ production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen‐ and isotype‐specific manner. Neither CD4⁺ nor CD8⁺ T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4^?CD8^? double‐negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4^?CD8^? dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.     

DC within the pregnant mouse uterus influence growth and functional properties of uterine NK cells

The vascular addressins mucosal addressin cell adhesion molecule‐1, P‐selectin and ICAM‐1 permit α₄β₇‐integrin‐expressing DC, especially those of the myeloid lineage (CD11c⁺CD11b⁺ DC), to access the pregnant mouse uterus. Injection of blocking monoclonal antibodies against mucosal addressin cell adhesion molecule‐1 in P‐selectin^?/? mice or experimental approaches with β7‐integrin^?/? or ICAM‐1^?/? mice revealed that limited access or absence of CD11c⁺CD11b⁺ DC at the maternal/fetal interface negatively affects the frequency, size and functional properties of uterine NK (uNK) cells. Adoptive transfer of DC obtained from WT mice into β7‐integrin^?/? mice abrogates these effects and emphasizes the importance of DC in uNK cell differentiation. Interestingly, those implantation sites lacking CD11c⁺CD11b⁺ DC are characterized by decreased IL‐15 and IL‐12 mRNA and/or protein levels. Chronic administration of IL‐15 in these mice gives rise to uNK cell numbers and size comparable to those of WT mice, whereas additional injection of IL‐12 positively affects the IFN‐γ expression of uNK cells. Real‐time RT‐PCR and protein arrays performed with isolated uterine DC underline the role of DC as a source of IL‐15 and IL‐12 in the pregnant mouse uterus.     

15‐lipoxygenase metabolites play an important role in the development of a T‐helper type 1 allergic inflammation induced by double‐stranded RNA

Background We recently demonstrated that the T‐helper type 1 (Th1) immune response plays an important role in the development of non‐eosinophilic inflammation induced by airway exposure of an allergen plus double‐stranded RNA (dsRNA). However, the role of lipoxygenase (LO) metabolites in the development of Th1 inflammation is poorly understood. Objective To evaluate the role of LO metabolites in the development of Th1 inflammation induced by sensitization with an allergen plus dsRNA. Methods A Th2‐allergic inflammation mouse model was created by an intraperitoneal injection of lipopolysaccharide‐depleted ovalbumin (OVA, 75 μg) and alum (2 mg) twice, and the Th1 model was created by intranasal application of OVA (75 μg) and synthetic dsRNA [10 μg of poly(I : C)] four times, followed by an intranasal challenge with 50 μg of OVA four times. The role of LO metabolites was evaluated using two approaches: a transgenic approach using 5‐LO^?/? and 15‐LO^?/? mice, and a pharmacological approach using inhibitors of cysteinyl leucotriene receptor‐1 (cysLTR1), LTB4 receptor (BLT1), and 15‐LO. Results We found that the Th1‐allergic inflammation induced by OVA+dsRNA sensitization was similar between 5‐LO^?/? and wild‐type (WT) control mice, although Th2 inflammation induced by sensitization with OVA+alum was reduced in the former group. In addition, dsRNA‐induced Th1 allergic inflammation, which is associated with down‐regulation of 15‐hydroxyeicosateraenoic acids production, was not affected by treatment with cysLTR1 or BLT1 inhibitors, whereas it was significantly lower in 12/15‐LO^?/? mice compared with WT control mice. Moreover, dsRNA‐induced allergic inflammation and the recruitment of T cells following an allergen challenge were significantly inhibited by treatment with a specific 15‐LO inhibitor (PD146176). Conclusion 15‐LO metabolites appear to be important mediators in the development of Th1‐allergic inflammation induced by sensitization with an allergen plus dsRNA. Our findings suggest that the 15‐LO pathway is a novel therapeutic target for the treatment of virus‐associated asthma characterized by Th1 inflammation.     

IL-10 contributes to the suppressive function of tumour-associated myeloid cells and enhances myeloid cell accumulation in tumours

Studies have revealed that tumour‐associated myeloid cells (TAMC) are one of the major sources of IL‐10 in tumour‐bearing mice. However, the significance of TAMC‐derived IL‐10 in tumour immunity is poorly understood. Here, we show that IL‐10 blockade or IL‐10 deficiency reduces the capacity of TAMC in suppressing the proliferation of P1A‐specific CD8 T cells. In the spleen, IL‐10‐deficient and wild‐type (WT) mice bearing large tumour burdens have similar TAMC populations. The tumours from IL‐10‐deficient mice, however, have reduced numbers of TAMC compared with tumours from their WT counterparts. IL‐10^?/?RAG‐2^?/? mice also had reduced numbers of TAMC compared with tumours from IL‐10^+/+RAG‐2^?/? mice; therefore, the reduction in TAMC in IL‐10‐deficient tumours was not because of adaptive immune response in tumours. Adoptively transferred tumour antigen–specific CD8 T cells expanded more efficiently within tumours in IL‐10^?/?RAG‐2^?/? mice than in tumours from IL‐10^+/+RAG‐2^?/? mice. Cytotoxic T lymphocyte adoptive transfer therapy prevented tumour evasion in IL‐10^?/?RAG‐2^?/? mice more efficiently than in IL‐10^+/+RAG‐2^?/? mice. Thus, IL‐10 enhances the accumulation of myeloid cells in tumours, and TAMC‐derived IL‐10 suppresses the activation and expansion of tumour antigen–specific T cells.     

Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3

Background Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers. Objective The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity. Methods Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL‐Per a 3), on proliferation and T‐helper type 1 (Th1)/Th2 cytokine production of human CD4⁺ T cells in co‐culture with allergen‐pulsed monocyte‐derived autologous dendritic cells (DC) as well as the leukotriene release of basophils. Results In P. americana‐sensitized and non‐sensitized donors the HL‐Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN‐γ production than the hexamers. While in non‐sensitized donors IL‐4 and IL‐5 as well as IL‐10 production were also increased after stimulation with monomeric HL‐Per a 3‐pulsed DC, Th2 cytokine and IL‐10 production were only enhanced in P. americana‐sensitized donors using hexameric HL‐Per a 3‐pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers. Conclusion Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE‐mediated allergic reaction and the Th2‐inducing capacity are diminished while the Th1‐inducing capacity is retained.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.     

Attenuated Bordetella pertussis vaccine strain BPZE1 modulates allergen‐induced immunity and prevents allergic pulmonary pathology in a murine model

Background Virulent Bordetella pertussis, the causative agent of whooping cough, exacerbates allergic airway inflammation in a murine model of ovalbumin (OVA) sensitization. A live genetically attenuated B. pertussis mucosal vaccine, BPZE1, has been developed that evokes full protection against virulent challenge in mice but the effect of this attenuated strain on the development of allergic responses is unknown. Objective To assess the influence of attenuated B. pertussis BPZE1 on OVA priming in a murine model of allergic airway inflammation. Methods Mice were challenged with virulent or attenuated strains of B. pertussis, and sensitized to allergen (OVA) at the peak of bacterial carriage. Subsequently, airway pathology, local inflammation and OVA‐specific immunity were examined. Results In contrast to virulent B. pertussis, live BPZE1 did not exacerbate but reduced the airway pathology associated with allergen sensitization. BPZE1 immunization before allergen sensitization did not have an adjuvant effect on allergen specific IgE but resulted in a statistically significant decrease in airway inflammation in tissue and bronchoalveolar lavage fluid. BPZE1 significantly reduced the levels of OVA‐driven IL‐4, IL‐5 and IL‐13 but induced a significant increase in IFN‐γ in response to OVA re‐stimulation. Conclusions These data demonstrate that, unlike virulent strains, the candidate attenuated B. pertussis vaccine BPZE1 does not exacerbate allergen‐driven airway pathology. BPZE1 may represent an attractive T‐helper type 1 promoting vaccine candidate for eradication of whooping cough that is unlikely to promote atopic disease.     

Antigen‐specific airway IL‐33 production depends on FcγR‐mediated incorporation of the antigen by alveolar macrophages in sensitized mice

Although interleukin (IL)‐33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen‐specific IL‐33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra‐tracheal administration of ovalbumin (OVA) evoked increases in IL‐33 and IL‐33 mRNA in the lungs of both non‐sensitized and OVA‐sensitized mice, and the increases in the sensitized mice were significantly higher than in the non‐sensitized mice. However, intra‐tracheal administration of bovine serum albumin did not increase the IL‐33 level in the OVA‐sensitized mice. Depletion of neither mast cells/basophils nor CD4⁺ cells abolished the OVA‐induced IL‐33 production in sensitized mice, suggesting that the antigen recognition leading to the IL‐33 production was not related with either antigen‐specific IgE‐bearing mast cells/basophils or memory CD4⁺ Th2 cells. When a fluorogenic substrate‐labelled OVA (DQ‐OVA) was intra‐tracheally administered, the lung cells of sensitized mice incorporated more DQ‐OVA than those of non‐sensitized mice. The lung cells incorporating DQ‐OVA included B‐cells and alveolar macrophages. The allergic IL‐33 production was significantly reduced by treatment with anti‐FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL‐33 production, whereas depletion of B‐cells by anti‐CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen‐specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL‐33 production in sensitized mice. The mechanisms underlying the antigen‐specific IL‐33 production may aid in development of new pharmacotherapies.     

Immunoglobulin E antibodies enhance pulmonary inflammation induced by inhalation of a chemical hapten

Background Occupational exposure to chemicals is an important cause of asthma. Recent studies indicate that IgE antibodies enhance sensitization to chemicals in the skin. Objective We investigated whether IgE might similarly promote the development of airway inflammation following inhalation of a contact sensitizer. Methods A model of chemical‐induced asthma is described in which introduction of the low‐molecular‐weight compound, trinitrobenzene sulphonic acid (TNBS), via the respiratory tract was used for both sensitization and challenge. The role of IgE antibodies in the immune response to inhaled TNBS in this model was assessed by comparing the responses of wild‐type (WT) and IgE‐deficient (IgE^?/?) mice on the BALB/c background. Reconstitution of circulating IgE levels by intravenous injection of IgE antibodies into IgE^?/? mice before sensitization was performed to confirm the role of IgE in any differences observed between the responses of WT and IgE^?/? mice. Results Intranasal challenge of TNBS‐sensitized (but not sham‐sensitized control mice) induced intense pulmonary inflammation. Macrophages, eosinophils and lymphocytes, including T, B, natural killer and natural killer T cells, were recruited to the airway and the animals displayed bronchial hyperresponsiveness (BHR) to methacholine. Serum levels of murine mast cell protease‐1 (mMCP‐1) were elevated suggesting mast cell activation. In contrast, the development of airway inflammation, recruitment of lymphocytes, induction of BHR and production of mMCP‐1 were all significantly attenuated in IgE‐deficient mice. Reconstitution of IgE^?/? mice with IgE (of unrelated antigen specificity) before sensitization partially restored these features of asthma. Conclusion Our data indicate that IgE antibodies non‐specifically enhance the development of airway inflammation induced by exposure to chemical antigens.     

MicroRNA‐155 Deficiency Suppresses Th17 Cell Differentiation and Improves Locomotor Recovery after Spinal Cord Injury

Spinal cord injury (SCI) is considered to be primarily associated with loss of motor function and leads to activate diverse cellular mechanisms in the central nervous system to attempt to repair the damaged spinal cord tissue. Mir‐155 has been reported to be involved in both innate and adaptive immune responses. But the role of Mir‐155 in spinal cord injury is still unknown. In our current study, Mir‐155 deficiency displays increased myelin sparring and enhanced SC repair process. The number of T cells, B cells and neutrophils are all significantly lower in Mir‐155^?/? group than that in WT group after SCI. IL‐17A‐producing cells and the expression of IL‐17A are markedly lower in Mir‐155^?/? mice than that in WT mice. We also found higher production of IL‐17 by WT CD4⁺ T cells than Mir‐155^?/? CD4⁺ T cells in vitro. In our further DC‐T cell coculture system, Mir‐155 deficiency in DCs results in significantly less IL‐17 production from T cells. Furthermore, the inhibited Th17 differentiation induced by Mir‐155 deficiency is partly dependent on increased expression of SOCS1. In conclusion, our present work provides evidence to support the concept that Mir‐155 deficiency suppresses Th17 cell differentiation and improves locomotor recovery after SCI.     

EGF receptor activation during allergic sensitization affects IL‐6‐induced T‐cell influx to airways in a rat model of asthma

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA‐specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4⁺ T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL‐6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA‐specific IgE and IL‐4 mRNA expression in CD4⁺ T cells were not affected by AG1478. BALF from OVA‐sensitized/challenged rats induced CD4⁺ T‐cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL‐6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4⁺ T cells to airways, mainly mediated through IL‐6.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Induction of systemic tolerance in normal but not in transgenic mice through continuous feeding of ovalbumin

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)‐2 and interferon (IFN)‐γ] and Th2 (IL‐4 and IL‐10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T‐cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)‐β secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF‐β as a regulatory cytokine in oral tolerance in the murine model.