CD8+ T lymphocytes regulating Th2 pathology escape neonatal tolerization

Transplantation tolerance induced by neonatal injection of semiallogeneic spleen cells is associated in several strain combinations with a pathological syndrome caused by Th2 differentiation of donor-specific CD4(+) T lymphocytes. We investigated the role of host CD8(+) T cells in the regulation of this Th2 pathology. IgE serum levels and eosinophilia significantly increased in BALB/c mice neonatally injected with (A/J x BALB/c)F(1) spleen cells when CD8(+) T cells were depleted by administration of anti-CD8 mAb or when beta(2)-microglobulin-deficient mice were used as recipients. In parallel, increased serum levels of IL-5 and IL-13 were measured in blood of tolerant CD8(+) T cell-deficient mice. Whereas neonatally injected mice were unable to generate anti-donor cytotoxic effectors, their CD8(+) T cells were as efficient as control CD8(+) T cells in reducing the severity of Th2 pathology and in restoring donor-specific cytotoxicity in vitro after in vivo transfer in beta(2)-microglobulin-deficient mice. Likewise, CD8(+) T cells from control and tolerant mice equally down-regulated the production of Th2 cytokines by donor-specific CD4(+) T cells in vitro. The regulatory activity of CD8(+) T cells depended on their secretion of IFN-gamma for the control of IL-5 production but not for IL-4 or IL-13. Finally, we found that CD8(+) T cells from 3-day-old mice were already able to down-regulate IL-4, IL-5, and IL-13 production by CD4(+) T cells. We conclude that regulatory CD8(+) T cells controlling Th2 responses are functional in early life and escape neonatal tolerization.     

IL-2 suppression of IL-12p70 by a recombinant HSV-1 expressing IL-2 induces T-cell auto-reactivity and CNS demyelination

To evaluate the role of cellular infiltrates in CNS demyelination in immunocompetent mice, we have used a model of multiple sclerosis (MS) in which different strains of mice are infected with a recombinant HSV-1 expressing IL-2. Histologic examination of the mice infected with HSV-IL-2 demonstrates that natural killer cells, dendritic cells, B cells, and CD25 (IL-2rα) do not play any role in the HSV-IL-2-induced demyelination. T cell depletion, T cell knockout and T cell adoptive transfer experiments suggest that both CD8(+) and CD4(+) T cells contribute to HSV-IL-2-induced CNS demyelination with CD8(+) T cells being the primary inducers. In the adoptive transfer studies, all of the transferred T cells irrespective of their CD25 status at the time of transfer were positive for expression of FoxP3 and depletion of FoxP3 blocked CNS demyelination by HSV-IL-2. The expression levels of IL-12p35 relative to IL-12p40 differed in BM-derived macrophages infected with HSV-IL-2 from those infected with wild-type HSV-1. HSV-IL-2-induced demyelination was blocked by injecting HSV-IL-2-infected mice with IL-12p70 DNA. This study demonstrates that suppression of the IL-12p70 function of macrophages by IL-2 causes T cells to become auto-aggressive. Interruption of this immunoregulatory axis results in demyelination of the optic nerve, the spinal cord and the brain by autoreactive T cells in the HSV-IL-2 mouse model of MS.     

Antibodies to L-periaxin in sera of patients with peripheral neuropathy produce experimental sensory nerve conduction deficits

L-Periaxin is a PDZ-domain protein localized to the plasma membrane of myelinating Schwann cells and plays a key role in the stabilization of mature myelin in peripheral nerves. Mutations in L-periaxin have recently been described in some patients with demyelinating peripheral neuropathy, suggesting that disruption of L-periaxin function may result in nerve injury. In this study, we report the presence of autoantibodies to L-periaxin in sera from two of 12 patients with diabetes mellitus (type 2)-associated neuropathy and three of 17 patients with IgG monoclonal gammopathy of undetermined significance (MGUS) neuropathy, an autoimmune peripheral nerve disorder. By comparison, anti-L-periaxin antibodies were not present in sera from nine patients with IgM MGUS neuropathy or in sera from 10 healthy control subjects. The effect of anti-L-periaxin serum antibody on peripheral nerve function was tested in vivo by intraneural injection. Sera containing anti-L-periaxin antibody, but not sera from age-matched control subjects, injected into the endoneurium of rat sciatic nerve significantly (p < 0.005, n = 3) attenuated sensory-evoked compound muscle action potential (CMAP) amplitudes in the absence of temporal dispersion. In contrast, motor-evoked CMAP amplitudes and latencies were not affected by intraneural injection of sera containing anti-L-periaxin antibody. Light and electron microscopy of anti-L-periaxin serum-injected nerves showed morphologic evidence of demyelination and axon enlargement. Depleting sera of anti-L-periaxin antibodies neutralized the serum-mediated effects on nerve function and nerve morphology. Together, these data support anti-L-periaxin antibody as the pathologic agent in these serum samples. We suggest that anti-L-periaxin antibodies, when present in sera of patients with IgG MGUS- or diabetes-associated peripheral neuropathy, may elicit sensory nerve conduction deficits.     

Monocyte-derived dendritic cells generated after a short-term culture with IFN-alpha and granulocyte-macrophage colony-stimulating factor stimulate a potent Epstein-Barr virus-specific CD8+ T cell response

Cellular immune responses are crucial for the control of EBV-associated lymphoproliferative diseases. To induce an anti-EBV cell-mediated immunity, we have used dendritic cells (DCs) generated by a 3-day culture of human CD14(+) monocytes in the presence of GM-CSF and type I IFN (IFN-DCs) and pulsed with peptides corresponding to CTL EBV epitopes. The functional activity of IFN-DCs was compared with that of APCs differentiated by culturing monocytes for 3 days with GM-CSF and IL-4 and indicated as IL-4-DCs. Stimulation of PBLs from EBV-seropositive donors with EBV peptide-pulsed autologous IFN-DCs resulted in a stronger expansion of specific T lymphocytes producing IFN-gamma with respect to stimulation with peptide-loaded IL-4-DCs, as assessed by ELISPOT assays. When purified CD8(+) T cells were cocultured with EBV peptide-pulsed IFN-DCs or IL-4-DCs, significantly higher levels of specific cytotoxic activity were observed in CD8(+) T cell cultures stimulated with IFN-DCs. Injection of peptide-pulsed IFN-DCs into SCID mice transplanted with autologous PBLs led to the recovery of a significantly greater number of EBV-specific human CD8(+) T cells from the spleen and the peritoneal cavity with respect to that recovered from mice injected with peptide-pulsed IL-4-DCs. Moreover, a significant delay in lymphoma development was observed when peptide-pulsed IFN-DCs were injected into SCID mice reconstituted with PBMCs endowed with a high capability of lymphoma induction, whereas injection of unpulsed IFN-DCs was ineffective. Our results indicate that IFN-DCs efficiently promote in vitro and in vivo the expansion of CD8(+) T lymphocytes acting as cytotoxic effectors against EBV-transformed cells.     

Dendritic cells genetically engineered to express IL-10 induce long-lasting antigen-specific tolerance in experimental asthma

Dendritic cells (DCs) are professional APCs that have a unique capacity to initiate primary immune responses, including tolerogenic responses. We have genetically engineered bone marrow-derived DCs to express the immunosuppressive cytokine IL-10 and tested the ability of these cells to control experimental asthma. A single intratracheal injection of OVA-pulsed IL-10-transduced DCs (OVA-IL-10-DCs) to naive mice before OVA sensitization and challenge prevented all of the cardinal features of airway allergy, namely, eosinophilic airway inflammation, airway hyperreactivity, and production of mucus, Ag-specific Igs, and IL-4. OVA-IL-10-DCs also reversed established experimental asthma and had long-lasting and Ag-specific effects. We furthermore showed, by using IL-10-deficient mice, that host IL-10 is required for mediating the immunomodulatory effects of OVA-IL-10-DCs and demonstrated a significant increase in the percentage of OVA-specific CD4(+)CD25(+)Foxp3(+)IL-10(+) regulatory T cells in the mediastinal lymph nodes of OVA-IL-10-DC-injected mice. Finally, adoptive transfer of CD4(+) mediastinal lymph node T cells from mice injected with OVA-IL-10-DCs protected OVA-sensitized recipients from airway eosinophilia upon OVA provocation. Our study describes a promising strategy to induce long-lasting Ag-specific tolerance in airway allergy.     

Kit (W-sh) mice develop earlier and more severe experimental autoimmune encephalomyelitis due to absence of immune suppression

Mast cells (MCs) have been thought to play a pathogenic role in the development of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, an immunoregulatory function of these cells has recently been suggested. We investigated the role of MCs in EAE using the W(-sh) mouse strain, which is MC deficient. W(-sh) mice developed earlier and more severe clinical and pathological disease with extensive demyelination and inflammation in the CNS. The inflammatory cells were mainly composed of CD4(+) T cells, monocyte/macrophages, neutrophils, and dendritic cells. Compared with wild-type mice, MC-deficient mice exhibited an increased level of MCP-1/CCR2 and CD44 expression on CD4(+) T cells in addition to decreased production of regulatory T cells, IL-4, IL-5, IL-27, and IL-10. We also found that levels of IL-17, IFN-γ, and GM-CSF were significantly increased in peripheral lymphocytes from immunized W(-sh) mice compared with those in peripheral lymphocytes from wild-type mice. Reconstitution of W(-sh) mice downregulated susceptibility to EAE, which correlated with MC recruitment and regulatory T cell activation in the CNS. These findings indicate that responsiveness is not required in the pathogenesis of inflammatory demyelination in the CNS and that, in the absence of MCs, increased MCP-1, CCR2, IL-17, IFN-γ, CD44, and other inflammatory molecules may be responsible for increased severity of EAE.     

IL-7 Is essential for the development and the persistence of chronic colitis

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4(+) T cells, its potential to be sustained pathogenic CD4(+) T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4(+)CD45RB(high) T cells or colitogenic lamina propria (LP) CD4(+) memory T cells into immunodeficient IL-7(+/+) x RAG-1(-/-) and IL-7(-/-) x RAG-1(-/-) mice. Although IL-7(+/+) x RAG-1(-/-) recipients transferred with CD4(+)CD45RB(high) splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7(-/-) x RAG-1(-/-) recipients did not. Furthermore, IL-7(-/-) x RAG-1(-/-), but not IL-7(+/+) x RAG-1(-/-), mice transferred with LP CD4(+)CD44(high)CD62L(-)IL-7Ralpha(high) effector-memory T cells (T(EM)) isolated from colitic CD4(+)CD45RB(high)-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4(+) T(EM) cells was observed in the in IL-7(-/-) x RAG-1(-/-) mice to a similar extent of those in IL-7(+/+) x RAG-1(-/-) mice, Bcl-2 expression was significantly down-modulated in the transferred CD4(+) T cells in IL-7(-/-) x RAG-1(-/-) mice compared with those in IL-7(+/+) x RAG-1(-/-) mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4(+) T(EM) cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.     

Gene therapy of rat malignant gliomas using neural stem cells expressing IL-12

Primary malignant brain tumors have a poor prognosis. This report investigates the potential for gene therapy of experimental brain tumors using neural stem cells (NSCs) expressing IL-12. In this study NSCs were isolated from the hippocampi of 3-5-month human embryos and used for lipofectamine mediated transfer of the IL-12 gene. Positive clones of anti-G418 were obtained and were proliferated in culture and expression of IL-12 was demonstrated by RT-PCR. For the in vivo studies three groups of rats were used and stereotactic injections were made into the striatum. In the first group C6 tumor cells were injected, in the second C6 cells and hNSCs. IL-12, and in the third C6 cells on Day 0 followed by hNSCs.IL-12 on day 5. The growth of the resulting tumors was monitored by magnetic resonance imaging (MRI) and after sacrifice by immunohistochemistry. Rats injected with C6 cells and hNSCs.IL-12 had a significantly prolonged survival. Injections of hNSCs.IL-12 were also made into established gliomas. The survival time was also significantly prolonged compared to controls. MR imaging demonstrated that there was initial growth of tumor followed by shrinkage and then disappearance. After sacrifice, tumor areas were studied by histochemistry. NSCs were often seen intermingled with tumor cells, particularly when they had been injected into established tumors; they were also present at the boundaries of the tumor mass. The immunohistochemical analysis showed that these infiltrates were mostly constituted by CD4(+) and CD8(+) T-lymphocytes, the CD8(+) being more numerous than the CD4(+). These findings indicated that NSCs engineered to release IL-12 could have a strong antitumor effect. Neural stem/precursor cells could be useful vectors in genetic approaches to brain tumors.     

Contribution of antigen-primed CD8+ T cells to the development of airway hyperresponsiveness and inflammation is associated with IL-13

The role of Th2/CD4 T cells, which secrete IL-4, IL-5, and IL-13, in allergic disease is well established; however, the role of CD8(+) T cells (allergen-induced airway hyperresponsiveness (AHR) and inflammation) is less clear. This study was conducted to define the role of Ag-primed CD8(+) T cells in the development of these allergen-induced responses. CD8-deficient (CD8(-/-)) mice and wild-type mice were sensitized to OVA by i.p. injection and then challenged with OVA via the airways. Compared with wild-type mice, CD8(-/-) mice developed significantly lower airway responsiveness to inhaled methacholine and lung eosinophilia, and exhibited decreased IL-13 production both in vivo, in the bronchoalveolar lavage (BAL) fluid, and in vitro, following Ag stimulation of peribronchial lymph node (PBLN) cells in culture. Reconstitution of sensitized and challenged CD8(-/-) mice with allergen-sensitized CD8(+) T cells fully restored the development of AHR, BAL eosinophilia, and IL-13 levels in BAL and in culture supernatants from PBLN cells. In contrast, transfer of naive CD8(+) T cells or allergen-sensitized CD8(+) T cells from IL-13-deficient donor mice failed to do so. Intracellular cytokine staining of lung as well as PBLN T cells revealed that CD8(+) T cells were a source of IL-13. These data suggest that Ag-primed CD8(+) T cells are required for the full development of AHR and airway inflammation, which appears to be associated with IL-13 production from these primed T cells.     

B7 interactions with CD28 and CTLA-4 control tolerance or induction of mucosal inflammation in chronic experimental colitis

CD28-B7 interaction plays a critical costimulatory role in inducing T cell activation, while CTLA-4-B7 interaction provides a negative signal that is essential in immune homeostasis. Transfer of CD45RB(high)CD4(+) T cells from syngeneic mice induces transmural colon inflammation in SCID recipients. This adoptive transfer model was used to investigate the contribution of B7-CD28/CTLA-4 interactions to the control of intestinal inflammation. CD45RB(high)CD4(+) cells from CD28(-/-) mice failed to induce mucosal inflammation in SCID recipients. Administration of anti-B7.1 (but not anti-B7.2) after transfer of wild-type CD45RB(high)CD4(+) cells also prevented wasting disease with colitis, abrogated leukocyte infiltration, and reduced production of proinflammatory cytokines IL-2 and IFN-gamma by lamina propria CD4(+) cells. In contrast, anti-CTLA-4 treatment led to deterioration of disease, to more severe inflammation, and to enhanced production of proinflammatory cytokines. Of note, CD25(+)CD4(+) cells from CD28(-/-) mice similar to those from the wild-type mice were efficient to prevent intestinal mucosal inflammation induced by the wild-type CD45RB(high) cells. The inhibitory functions of these regulatory T cells were effectively blocked by anti-CTLA-4. These data show that the B7-CD28 costimulatory pathway is required for induction of effector T cells and for intestinal mucosal inflammation, while the regulatory T cells function in a CD28-independent way. CTLA-4 signaling plays a key role in maintaining mucosal lymphocyte tolerance, most likely by activating the regulatory T cells.     

In vivo delivery of interleukin-6 using vaccinia virus: effects on T lymphocytes in nude mice

We have constructed a recombinant vaccinia virus (VV) expressing the human interleukin-6 (IL-6) gene, VV(IL-6). After injection of VV(IL-6) i.v. into Balb/c mice, circulating IL-6 was detected during 3 days with the peak activity on day 4, indicating that VV injection is an effective method to deliver lymphokines in vivo. We have further examined the effects of IL-6 in vivo in immunodeficient mice. Nude mice were injected i.v. with VV(IL-6). Ten days after the injection, mice were sacrificed and spleen cells were obtained. Spleen cells from VV(IL-6) injected mice proliferated remarkably in response to IL-2, while spleen cells from mice injected with unrelated VV manifested no particular proliferation in response to lymphokines. When spleen cells were further cultured in vitro for 5 days in the presence of Concanavalin-A stimulated rat spleen cell supernatant (Con-A factor), CD4 or CD8 positive cells were detected in the VV (IL-6) injected group, while few positive cells were detected in the control groups. These results suggest that IL-6 stimulates nude mice spleen cells in vivo, to a stage where they are able to proliferate in response to IL-2, or to differentiate into CD4 or CD8 positive cells in presence of rat Con-A factor.     

TH(1)- and TH(2)-TYPE cytokine expression by activated t lymphocytes from the lung and spleen during the inflammatory response to respiratory syncytial virus

RSV is an important cause of lower respiratory tract illness in infants and the elderly worldwide. The components involved in immunity and those that contribute to inflammation of RSV-induced disease are not clearly understood. To address the relationship between activation antigen and cytokine expression, intracellular levels of IL-2, IL-4, IL-5 and IFN-gamma were determined for CD3, CD44, CD49d, CD54, CD62L and CD102 lymphocytes from the bronchoalveolar lavage and spleen. To examine activation at the DNA level, lymphocytes expressing IL-2, IL-4, IL-5 or IFN-gamma were analysed for G2+M DNA content or phosphatidylserine expression (apoptosis). Trafficking of lymphocytes to the BAL was detected at day 5 p.i., peaked day 7 p.i., and predominately involved CD54(+)and CD102(+)lymphocytes expressing high levels of IL-2, IL-4, IL-5 and IFN-gamma. Lymphocytes expressing CD44(+), CD49d(+)and CD62L(lo)were also observed, however they expressed these cytokines to a lesser extent. DNA analysis of lymphocytes expressing IL-2 or IFN-gamma revealed higher G2'M levels compared to lymphocytes expressing IL-4 or IL-5, suggesting greater activation of Th(1)-type lymphocytes in the lung. These data demonstrate that RSV-induced pulmonary inflammation involves extensive cellular activation and cytokine expression, particularly by CD54(+)and CD102(+)lymphocytes in the lung.     

Pulmonary CCL18 recruits human regulatory T cells

CCL18 is both a constitutively expressed and an inducible chemokine, whose role in the inflammatory reaction is poorly known. The aim of this study was to evaluate whether CCL18 has the capacity to attract human T cells with a regulatory function (regulatory T cells [Treg]). Results from chemotaxis assays performed on different types of Treg showed that CD4(+)CD25(+)CD127(low) cells, but neither T regulatory type 1 clones nor Treg differentiated in vitro with anti-CD3/CD46 mAbs, were recruited by CCL18 in a dose-dependent manner. CCL18-recruited memory CD4(+) T cells were enriched in CD25(high), CD25(+)CD127(low), latency-associated peptide/TGF-β1, and CCR4-expressing T cells, whereas there was no enrichment in Foxp3(+) cells as compared with controls. Stimulated CCL18-recruited memory T cells produced significantly increased amounts of the regulatory cytokines IL-10 and TGF-β1, as well as IL-4, but not IFN-γ and IL-17. Cell surface CCL18 binding was found predominantly on IL-10(+) (26.3 ± 5.8%) and on a few latency-associated peptide/TGF-β1(+) (18.1 ± 1.9%) and IL-4(+) (14.5 ± 2.9%) memory T cells. In an in vivo model of SCID mice grafted with human skin and reconstituted with autologous PBMCs, the intradermal injection of CCL18 led to the cutaneous recruitment of CD4(+), CD25(+), and IL-10(+) cells, but not Foxp3(+) cells. Furthermore, CCL18-recruited memory T cells inhibited the proliferation of CD4(+)CD25(-) effector T cells through an IL-10-dependent mechanism. These data suggest that CCL18 may contribute to maintaining tolerance and/or suppressing deleterious inflammation by attracting memory Tregs into tissues, particularly in the lung, where it is highly and constitutively expressed.     

Role of regulator of G protein signaling 16 in inflammation-induced T lymphocyte migration and activation

Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Galpha subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4(+) and CD8(+) cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4(+) lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4(+)CCR3(+) cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.     

Expression of interleukin-4 by recombinant respiratory syncytial virus is associated with accelerated inflammation and a nonfunctional cytotoxic T-lymphocyte response following primary infection but not following challenge with wild-type virus

The outcome of a viral infection or of immunization with a vaccine can be influenced by the local cytokine environment. In studies of experimental vaccines against respiratory syncytial virus (RSV), an increased stimulation of Th2 (T helper 2) lymphocytes was associated with increased immunopathology upon subsequent RSV infection. For this study, we investigated the effect of increased local expression of the Th2 cytokine interleukin-4 (IL-4) from the genome of a recombinant RSV following primary infection and after a challenge with wild-type (wt) RSV. Mice infected with RSV/IL-4 exhibited an accelerated pulmonary inflammatory response compared to those infected with wt RSV, although the wt RSV group caught up by day 8. In the first few days postinfection, RSV/IL-4 was associated with a small but significant acceleration in the expansion of pulmonary T lymphocytes specific for an RSV CD8(+) cytotoxic T-lymphocyte (CTL) epitope presented as a major histocompatibility complex class I tetramer. However, by day 7 the response of tetramer-positive T lymphocytes in the wt RSV group caught up and exceeded that of the RSV/IL-4 group. At all times, the CTL response of the RSV/IL-4 group was deficient in the production of gamma interferon and was nonfunctional for in vitro cell killing. The accelerated inflammatory response coincided with an accelerated accumulation and activation of pulmonary dendritic cells early in infection, but thereafter the dendritic cells were deficient in the expression of B7-1, which governs the acquisition of cytolytic activity by CTL. Following a challenge with wt RSV, there was an increase in Th2 cytokines in the animals that had previously been infected with RSV/IL-4 compared to those previously infected with wt RSV, but the CD8(+) CTL response and the amount of pulmonary inflammation were not significantly different. Thus, a strong Th2 environment during primary pulmonary immunization with live RSV resulted in early inflammation and a largely nonfunctional primary CTL response but had a minimal effect on the secondary response.     

IL-6单克隆抗体对自身免疫性心肌炎的保护作用及其机制

目的：观察白介素-6（interleukin,IL-6）单克隆抗体（IL-6 mAb）治疗Lewis大鼠自身免疫心肌炎（EAM）的疗效。探讨IL-6与辅助性T细胞17（Th17）、调节性T细胞（Treg）在EAM发病中的机制。方法：将34只8-10周龄Lewis大鼠随机分为正常对照组（n=6）,EAM组（n=12）,IL-6mAb干预组（n=16）。对EAM组和干预组注射心肌肌凝蛋白,干预组于免疫注射后第1、7至第20天腹腔注射IL-6 mAb1nlg,分别于急性峰值期（第21天）、慢性持续期（第84天）取材,观察心肌炎症浸润、纤维化、细胞凋亡以判断IL-6mAb疗效。检测脾脏TH17、Treg细胞数量和功能,比较各组血清中IL-6、IL-10、IL-17和转化生长因子．β（TGF-β）的浓度,实时定量PCR测定外周血STAT3、RORγt、Foxp3mRNA水平,对EAM源性脾细胞进行体外IL-6mAb刺激,并用ELISA法测定IL-10、IL-17和TGF-β的浓度。结果：炎症积分、纤维化积分、凋亡指数IL-6mAb干预组较EAM组明显下降（P〈0．01）。急性峰值期（21d组）EAM组TH17和Treg细胞数量上调,干预组则受明显抑制（P〈0．01）;21d干预组血清IL-6、IL-10、IL-17和TGF-β的浓度较EAM组明显下降（P〈0．01）;21d干预组外周血STAT3、RORγt、Foxp3mRNA水平下降（P〈0．01）;体外IL-6mAb刺激EAM源性脾细胞,IL-10、IL-17和TGF-β表达明显增加。结论：IL6mAb对EAM有明显的保护作用,IL6mAb通过抑制Th17、Treg细胞的数量和功能,实现对EAM的保护作用。     

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.     

Role of mesenchymal stem cells on cornea wound healing induced by acute alkali burn

The aim of this study was to investigate the effects of subconjunctivally administered mesenchymal stem cells (MSCs) on corneal wound healing in the acute stage of an alkali burn. A corneal alkali burn model was generated by placing a piece of 3-mm diameter filter paper soaked in NaOH on the right eye of 48 Sprague-Dawley female rats. 24 rats were administered a subconjunctival injection of a suspension of 2×10(6) MSCs in 0.1 ml phosphate-buffered saline (PBS) on day 0 and day 3 after the corneal alkali burn. The other 24 rats were administered a subconjunctival injection of an equal amount of PBS as a control. Deficiencies of the corneal epithelium and the area of corneal neovascularization (CNV) were evaluated on days 3 and 7 after the corneal alkali burn. Infiltrated CD68(+) cells were detected by immunofluorescence staining. The mRNA expression levels of macrophage inflammatory protein-1 alpha (MIP-1α), tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) were analyzed using real-time polymerase chain reaction (real-time PCR). In addition, VEGF protein levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). MSCs significantly enhanced the recovery of the corneal epithelium and decreased the CNV area compared with the control group. On day 7, the quantity of infiltrated CD68(+) cells was significantly lower in the MSC group and the mRNA levels of MIP-1α, TNF-α, and VEGF and the protein levels of VEGF were also down-regulated. However, the expression of MCP-1 was not different between the two groups. Our results suggest that subconjunctival injection of MSCs significantly accelerates corneal wound healing, attenuates inflammation and reduces CNV in alkaline-burned corneas; these effects were found to be related to a reduction of infiltrated CD68(+) cells and the down-regulation of MIP-1α, TNF-α and VEGF.     

IL-17, produced by lymphocytes and neutrophils,is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4(+) and CD8(+) cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4(+) cells, but not spleen CD8(+) or airway neutrophils. We have shown that IL-17, produced mainly by CD4(+) cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.