In vitro production of cytokines by peripheral blood mononuclear cells from hydatid patients.

The role of cytokines in human hydatidosis (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In cell cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P < or 0.005). Spontaneous and mitogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-gamma production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-gamma concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-gamma production, but high IgG4 responders had IFN-gamma levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-gamma production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.     

Antigen dose-dependent differences in IgE antibody production are not due to polarization towards Th1 and Th2 cell subsets

The quality of the humoral immune response against protein antigens in CBA/J mice is dependent on the antigen dose used for immunization: low doses induce high titers of IgE antibodies, whereas high doses promote the production of IgG2a antibodies but inhibit IgE formation. To investigate whether the reciprocal regulation of antibody production is possibly due to a differential activation of Th1 and Th2 cell populations in the two immunization groups, the cytokine pattern of spleen cells from both groups, cultured with antigen in vitro, was analyzed by measurement of intracellular and secreted cytokine levels. The data presented show that in vitro restimulated spleen cells from mice primed with low as well as with high doses of antigen produce predominantly the Th2 cytokines IL-4 and IL-10 but reduced levels of IL-12. The release of IFN-γ is only slightly enhanced compared to unstimulated control cultures. The results indicate that CD4⁺ T cells in both groups belong mainly to the Th2 cell subset. This finding is contradictory to the general allegation that the antigen dose is decisive for the polarization of Th1 versus Th2 immune responses and shows that the antigen dose-dependent regulation of IgE antibody production is not due to differential polarization towards Th1 and Th2 cells.     

Oral cow milk challenge abolishes antigen-specific interferon-γ production in the peripheral blood of children with atopic dermatitis and cow milk allergy

Background A major feature of atopic dermatitis (AD) is the propensity to generate IgE to environmental antigens. Despite extensive information on IgE dysregulation in AD, the nature of immune responses to ingested allergens is poorly characterized. Objective To determine the clinical and immunological responses to food in AD patients. Methods To characterize the type and timing of clinical reactions to oral cow milk, 83 AD patients aged 2 to 60 months were subjected to double-blind placebo-controlled food challenge (DBPCFC). IFN-γ and IL-4 production by their peripheral blood mononuclear cells (PBMC) was determined before and after DBPCFC. Results Of 50 patients positive to DBPCFC, 46% manifested exanthematous-type immediate-onset reactions and 54% eczematous-type late-onset reactions. In either group, the production of lL-4 and IFN-γ by Concanavalin A-stimulated FBMC was comparable before and after DBPCFC. For immediate-reacting patients, the median IFN-γ production by milk-stimulated PBMC was 11.5 (4.2–17.2) pg/mL as against 2.3 (0.2–5.7) pg/mL by unstimulated PBMC, P = 0.0008 before DBPCFC, and 4.6 (2.8–10.3) pg/mL vs 4.2 (1.7–9.0) pg/mL, P = 0.40, correspondingly after DBPCFC. Conclusion Before DBPCFC, immediate-reacting but not late-reacting patients were found to be capable of allergen-specific IFN-γ production in vitro, indicating the heterogeneity in AD patients. After DBPCFC, the IFN-γ generation abolished, indicating the effect of oral allergen exposure on IFN-γ-producing responses of AD patients.     

CCK-8对KLH免疫小鼠脾细胞Th1/Th2平衡的影响

目的： 探讨八肽胆囊收缩素（CCK-8）对Th1/Th2平衡的调节作用。方法: 给予BALB/c小鼠钥孔戚血蓝蛋白（KLH）免疫同时体内给予不同剂量的CCK-8,酶联免疫吸附试验(ELISA)检测其脾细胞培养上清中Th1型细胞因子γ-干扰素(IFN-γ)、白细胞介素-2（IL-2）和Th2型细胞因子白细胞介素-4（IL-4）、白细胞介素-5（IL-5）水平,逆转录聚合酶链式反应(RT-PCR)法检测脾细胞中IFN-γ、IL-2、IL-4、IL-5 mRNA表达;ELISA法检测血清中Th1型抗KLH抗体IgG2a和Th2型抗KLH抗体IgG1水平。结果: ①KLH免疫使小鼠脾细胞分泌Th1/Th2型细胞因子水平明显增高,mRNA表达增高,KLH免疫同时给予CCK-8可使脾细胞培养上清中IFN-γ、IL-2含量进一步增加和IFN-γ、IL-2mRNA表达增高,而使IL-4、IL-5含量降低,IL-4、IL-5 mRNA表达减低和降低IL-4/IFN-γ比值。②KLH免疫小鼠血清中IgG2a、IgG1发生不同程度增高,CCK-8可使其血清中IgG1水平减低而使IgG2a水平增高。结论: CCK-8可促进KLH免疫小鼠体内Th1反应,使Th2优势反应向Th1方向转变。     

Adjuvant effect of zinc oxide on Th2 but not Th1 immune responses in mice

Background and aim: We investigated the effect of zinc oxide (ZnO) on Th1 and Th2 immune responses in mice.

Material and methods: Mice were intraperitoneally administered with ovalbumin (OVA) with or without varying doses of ZnO (day 0). On day 21, anti-OVA IgG, IgG2a, IgG1, and IgE antibodies in sera, OVA-specific proliferative responses of spleen cells, and production of Th1 cytokines including IFN-γ as well as Th2 cytokines such as IL-4 and IL-5 were measured.

Results: The results showed that administration of OVA with ZnO was followed by greater increases in anti-OVA IgG and the antigen-specific splenocyte proliferation compared to that of OVA alone. The production of anti-OVA IgG1 and IgE and secretion of IL-4 and IL-5 were markedly enhanced by ZnO. The enhancing effect of ZnO on these Th2 responses was as strong as aluminium hydroxide (Alum) that was widely used as an adjuvant. In contrast, treatment with OVA plus ZnO failed to affect production of anti-OVA IgG2a as well as IFN-γ. It was also observed that ZnO had a stimulating effect on the secretion of the proinflammatory cytokine IL-17 from a new lineage of effector Th cells.

Conclusion: These results suggest that ZnO appears to have an adjuvant effect on the immune system, especially Th2 but not Th1 immune responses.     

Evidence of a T helper type 2 activation in human schistosomiasis

The lymphocyte proliferative response and cytokine production to S. mansoni antigen in vitro were evaluated in 22 schistosomiasis patients living in an area endemic for this disease. The majority of patients (86%) showed no lymphocyte proliferative response and none of them showed interferon-γ (IFN-γ) production, following in vitro stimulation with soluble adult worm antigen preparation (SWAP). In contrast, interleukin (IL)-5 (2038 ± 1757 pg/ml) and IL-10 (867 ± 762 pg/ml) were detected in peripheral blood mononuclear cell (PBMC) cultures stimulated with SWAP. Moreover, mRNA for IL-4 was detected in SWAP-stimulated PBMC from 4 of 6 patients evaluated. Restoration of lymphoproliferative response was achieved in 4 of 6 patients by adding anti-IL-10 monoclonal antibody (mAb) to PBMC cultures [mean stimulation index (SI) in the presence of antigen = 2.7 ± 2.9; SI in the presence of antigen plus anti-IL-10, 21 ± 16]. Restoration of IFN-γ production by addition of anti-IL-10 mAb was achieved in 4 of 12 patients evaluated (248, 350, 687 and 710 pg/ml). Moreover, the addition of IL-10 to PBMC cultures of 3 schistosomiasis patients and 2 cured subjects who had high lymphoproliferative responses to SWAP resulted in the suppression of these responses by 90%, and completely suppressed IFN-γ production in one of the subjects, whose PBMC produced IFN-γ after stimulation with SWAP. The presence of IL-4 mRNA, high levels of IL-5, and the absence of IFN-γ in PBMC culture supernatants from infected patients, supports the conclusion that patients living in an endemic area of schistosomiasis express a predominant T helper type 2 response. The high levels of IL-10 and the ability of neutralizing anti-IL-10 mAb to restore T cell responses indicate that this cytokine plays an important role in the modulation of T cell responses in schistosomiasis.     

Selective stimulation of T helper 2 cytokine responses by the anti-psoriasis agent monomethylfumarate

Type 2 cytokines are thought to have a protective role in psoriasis vulgaris by dampening the activity of T helper 1 (Th1) lymphocytes. The aim of the present study was to determine the effect of monomethylfumarate (MMF), the most active metabolite of the new anti-psoriatic drug Fumaderm®, on the production of cytokines and the development of Th subsets. MMF was found to enhance interleukin (IL)-4 and IL-5 production by CD2/CD8 monoclonal antibody-stimulated peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Maximal effects of MMF were found at a concentration of 200 μM and resulted in tenfold enhanced levels of IL-4 and IL-5 production. MMF did not affect the levels of IL-2 production, interferon (IFN)-γ production or proliferative T cell responses in these cultures. Similar effects of MMF were observed in cultures of purified peripheral blood T cells indicating that this compound can act directly on T cells. MMF did not influence cytokine production by purified CD4⁺CD45RA⁺ (unprimed) T cells, but greatly enhanced IL-4 and IL-5 production without affecting IFN-γ production by purified CD4⁺CD45R0⁺ (primed) T cells. Furthermore, MMF also augmented IL-4 and IL-5 production in established Th1/Th0 clones that were stimulated with CD2/CD28 monoclonal antibody. Finally, when PBMC were challenged with Mycobacterium tuberculosis that typically induces Th1 recall responses with strong IFN-γ secretion, MMF again appeared to induce high levels of IL-4 and IL-5 secretion while IFN-γ production was unaffected. These results may be relevant for the development of therapeutic regimens designed to correct inappropriate Th1 subset development in immunopathologic conditions.     

Role of interleukin-2 and interferon-γ in inducing production of IgG subclasses in lymphocytes of human newborns

Unlike lymphocytes from adults, lymphocytes from cord blood of neonates cannot synthesize immunoglobulin G (IgG) in response to pokeweed mitogen (PWM). By using this mitogen in concert with interferon-γ (IFN-γ), interleukin-2 (IL-2), or interleukin-6 (IL-6), we studied the induction of IgG subclass molecules in lymphocytes of human neonates. IFN-γ induced a limited, but substantial, enhancement of IgG2 production by neonatal lymphocytes. IL-2 dose dependently increased the production of each neonatal IgG subclass, whereas IL-6 did not. However, in adult lymphocytes, and under specific conditions, IL-6 or IL-2 each increased the production of all four IgG subclasses. Early in the culture IFN-γ synergized with IL-2 during the latter or whole culture period to enhance cord blood IgG2 levels. This finding contrasted with the adult IgG2 synthesis synergistically up-regulated by IFN-γ and IL-6. IL-2 caused a graded increase in immunoglobulin production in neonatal lymphocytes with IgG3 being the highest and IgG2 the lowest, thus corresponding to the differential increase of serum levels of IgG3/IgG1 and IgG4/IgG2 early in childhood. Results suggest that IL-2, but not IL-6, is critical to the development of human IgG subclass production.     

In interleukin-4-deficient mice,alum not only generates T helper 1 responses equivalent to Freund's complete adjuvant,but continues to induce T helper 2 cytokine production

The role of interleukin (IL)-4 in the activity of two frequently used vaccine adjuvants, Freund's complete adjuvant (FCA) and the aluminum hydroxide gels (alum), was studied using the standard antigen ovalbumin (OVA) in IL-4 genedisrupted mice (IL-4 -/-). In the absence of adjuvant, there was an overall reduction in antibody production to OVA in IL-4 −/− mice and significantly greater amounts of interferon (IFN)-γ were produced following restimulation of splenocytes with antigen in vitro compared with immunocompetent controls (IL-4 +/+). FCA and alum boosted the immune response to OVA in both IL-4 −/− and IL-4 +/+ mice. In IL-4 +/+ mice, while FCA stimulated a wide-spectrum immunoglobulin response, including both Th1-associated IgG2a and Th2-associated IgG1, alum enhanced only Th2 antibody production and no OVA-specific IgG2a could be detected. In IL-4-deficient mice, however, not only was IgG2a production increased in all adjuvant-treated groups, but alum was as potent at stimulating this antibody subclass as FCA. Similarly, increased production in vitro by splenocytes of the Th1 cytokine IFN-γ, equivalent to that produced after inoculation with FCA/OVA, was only detected in IL-4 −/− mice inoculated with alum/OVA. There was no IgE production in IL-4 −/− mice and OVA-specific IgG1 production, although still at significant levels, was reduced compared with wild-type mice irrespective of the adjuvant used. However, although production of the Th2 cytokine IL-5 was totally inhibited in IL-4-deficient mice inoculated with FCA/OVA, there was no significant difference in IL-5 production between the two strains when alum was used as adjuvant.     

呼吸道合胞病毒毛细支气管炎患儿IL4/IFNγ水平变化及意义

为研究Th1/Th2类细胞因子 (CK )在呼吸道合胞病毒毛细支气管炎 (RSV毛支 )发病中的作用 ,本文采用酶联免疫吸附试验 ,对 2 0例RSV毛支患儿和 15例健康婴幼儿外周血单个核细胞 (PBMC )经植物血凝素 (PHA )刺激培养后 ,测定上清液中某些细胞因子 ,并同时检测患儿血浆总IgE水平。实验结果表明 :(1)经PHA刺激培养后 ,RSV毛支组上清液中IL 4,IFN γ水平及IL 4/IFN γ比值与对照组比较 ,差异有显著性 (P <0 0 1、 0 0 5、 0 0 1)。RSV毛支组血浆总IgE水平较对照组明显升高 (P <0 0 1) ;(2 )相关分析 :培养上清液中IL 4水平 ,IL 4/IFN γ比值与血浆总IgE水平间呈显著正相关 ,IFN γ与IgE间呈显著负相关。此外IL 4与IFN γ间呈显著负相关。结果提示 ,RSV毛支患儿存在Th1/Th2功能紊乱 ,主要表现为Th2细胞功能亢进 ;IL 4、IFN γ参与了RSV感染时的免疫病理机制。     

Differential regulation of human T cell cytokine patterns and IgE and IgG4 responses by conformational antigen variants

Bee venom phospholipase A₂ (PLA) represents the major allergen and antigen in allergic and non-allergic individuals sensitized to bee sting. We have studied specific activation of peripheral T cells by different structural and conformational variants of PLA and secretion of cytokines regulating IgE and IgG4 antibody (Ab) formation. PLA molecules expressing the correctly folded tertiary structure, which show high affinity to membrane phospholipids and were recognized by Ab from bee sting allergic patients, induced high IL-4, IL-5 and IL-13 production in peripheral blood mononuclear cell cultures. In contrast, non-refolded recombinant PLA (rPLA) and reduced and alkylated native PLA (nPLA) induced more IFN-γ and IL-2 and higher proliferative responses. Differences in proliferation and cytokine patterns among correctly folded and non-refolded PLA resulted from conformation-dependent involvement of different antigen-presenting cell (APC) types. Antigen (Ag)-presenting B cells recognized PLA only in its natural conformation, stimulated Th2 type cytokines and induced IgE Ab. Non-refolded PLA was recognized, processed and presented exclusively by monocytes and induced a Th1 dominant cytokine profile leading to IgG4 production by B cells. The possibility that production of particular cytokine patterns and Ig isotype was influenced by the enzymatic activity of PLA was excluded by using enzymatically inactive H34Q point-mutated, refolded rPLA. These findings demonstrate the decisive role of specific Ag recognition by different APC, depending on structural features, membrane phospholipid binding property and the existence of conformational B cell epitopes, in the differential regulation of memory IgE and IgG4 Ab. Furthermore, they show that a change from IgE-mediated allergy to normal immunity against a major allergen can be induced by rPLA variants that are not recognized by specific Ab and B cells but still carry the T cell epitopes. These features may enable new applications for safer immunotherapy.     

Influence of both TCR repertoire and severity of the atopic status on the cytokine secretion profile of Parietaria officinalis -specific T cells

Peripheral blood mononuclear cells (PBMC) from both nonatopic and Parietaria officinalis -sensitive donors proliferated in response to the allergen Par o 1 and developed into Par o 1-specific T cell lines and clones, which also showed reactivity for Par o 1-derived peptides. Virtually all Par o 1-specific T cell lines and large numbers of Par o 1-specific T cell clones proliferated in response to two Par o 1 nonapeptides (p92 and p96), which probably contain immunodominant epitopes of the Par o 1 allergen. Both p92- and p96-specific T cell clones showed the ability to produce IFN-γ, but p92-specific T cell clones produced significantly lower amounts of IL-4 and IL-5 than p96-specific T cell clones, indicating that distinct epitopes, able to elicit functionally different T helper cell responses, may coexist in Par o 1. How ever, p92-specific T cell clones derived from atopic subjects with high IgE serum levels (high IgE producers) secreted significantly higher amounts of IL-4 and IL-5 than corresponding T cell clones generated from non atopic subjects or patients with low IgE serum levels (low IgE producers), whereas p96-specific T cell clones secreted high IL-4 and IL-5 concentrations irrespective of whether they derived from high or low IgE producers. The addition of IL- 4 and anti-IL-12 mAb to bulk culture significantly up-regulated the development of p92-specific T cells into IL-4-producing cells, whereas the addition of IL-12 and anti-IL-4 mAb shifted the differentiation of p96-specific T cells towards IFN-γ-producing cells. Taken together, these results suggest that the cytokine profile of allergen-specific T cells is influenced by both the T cell receptor repertoire and the severity of atopic status and can be modulated, at least in vitro, by stimulation with the specific peptide in the presence, or after removal, of appropriate cytokines.     

内源性IL-12决定人PBMC产生干扰素γ的水平

目的：IFN－γ是由被有丝分裂原或抗原所激活的T细胞和NK细胞所产生,它具有广泛的免疫调节活性,现认为IL－12（外源性）是诱导IFN－γ产生的强诱导剂,并可促进静息CD4^ T细胞朝向Th1表型分化,即诱导细胞免疫。目的是为了解PBMC产生的内源性IL－12是否在体外可诱导IFN－γ的产生及通过何机制诱导细胞免疫。方法：用抗CD3抗体、PHA、抗CD3抗体加抗CD28抗体和抗原（MLC）来检测被刺激的PBMC细胞的IFN－γ的产生。同时也用IL－12和IL－12Rβ1的中和抗体来抑制IFN－γ的产生。结果：活的人PBMC中IFN－γ分泌依赖于内源性IL－12的产生,而且激活的T细胞可诱导APC细胞产生IL－2,此过程是通过T细胞表面的CD40L和APC的CD40相互作用而实现。结论：这些结果显示,内源性IL－12在正常罕主抗细胞内抗原的感染反应中起重要作用,在某些形式的自身免疫性疾病和移植排斥反应的免疫病理发生中也起中心作用。     

慢性乙肝患者树突状细胞对Th1/Th2分化的影响

探讨慢性乙肝患者树突状细胞(dendritic cells,DC)对CD4+Th细胞亚群分化的影响。分离慢性乙肝患者外周血单个核细胞(PBMC),以rhIL-4(50 ng/ml)、rhGM-CSF(10 ng/ml)和rhTNF-α(100 u/ml)诱导培养DC。以流式细胞仪检测DC表面CD1a、CD83、CD80、CD86、HLA-DR分子表达情况。MTT法检测DC刺激同种异体淋巴细胞增殖能力。免疫磁珠分离外周血CD4+T细胞亚群,PMA+Ionomycin刺激后胞内荧光染色,流式细胞仪检测辅助性T细胞(helper T cell,Th)内特征性细胞因子IFN-γ/IL-4以判断Th1/Th2分化。ELISA法检测DC或Th细胞培养上清中IL-6、IL-12、IFN-γ和IL-4的含量。结果:慢性乙肝患者的DC表达CD1a、CD83、CD80、CD86、HLA-DR分子水平明显低于正常人(P<0.01);培养至第7天,慢性乙肝患者DC分泌的IL-12水平低于正常人(P<0.01),而分泌的IL-6水平增高(P<0.05)。与正常人相比,慢性乙肝患者外周血中Th1细胞占CD4+T细胞的百分比较低(P<0.01),其Th细胞培养上清中IFN-γ的量也较低(P<0.01)。患者DC与同种异体的健康人Th细胞共培养,刺激Th1型细胞因子IFN-γ产生的能力低于正常人(P<0.01)。慢性乙肝患者体内DC功能的异常可能导致了外周血Th1细胞分化不足。     

Selective suppression of antigen-specific Th2 cells by continuous micro-dose oral tolerance

The effect on antigen (Ag)-specific Th2 response as well as IgE production of continuous oral administration of micro-doses of Ag was investigated. Transgenic (Tg) mice carrying the α β-T cell receptor (TCR) genes specific for ovalbumin (OVA) peptide fragment 323 – 339 were continuously fed with micro-doses of OVA (100 μg/day) for 14 days. Mice were first immunized by OVA in alum and pertussis toxin 7 days before the oral feeding and given a second immunization 1 day after the oral treatment. This feeding regimen tolerized Th2 but not Th1 responses as shown by decrease of Ag-driven cell proliferation and cytokine secretion of IL- 4 but not of IL-2 or IFN-γ as well as by the absence of Ag-specific antibody production of IgE and IgG1, but not of IgG2a or total IgG. Numbers of clonotype-specific TCR-high CD4-positive T cells in peripheral lymphoid tissues markedly decreased in the orally treated group but not in the control group. However, total numbers of CD4-positive T cells in thymus, spleen and lymph nodes were not affected by the oral treatment, indicating that tolerance induction in Th2 cells was mainly due to the down-regulation of TCR and not clonal deletion. The population of antigen-presenting cells expressing B7-2 (CD86) Ag on the surface was decreased in the spleen of the mice which underwent the feeding regimen. The present results suggest that Ag-specific low responsiveness in Th2 cells, which resulted in suppres sion of the Ag-specific IgE production, can be achieved by continuous feeding with microdoses of Ag.     

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-γ production by T helper 1 cells

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-γ (IFN-γ), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell-mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, Northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-γ bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 bymurine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral blood mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-γ production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.     

Anti-IL-12 antibody prevents the development and progression of collagen-induced arthritis in IFN-γ receptor-deficient mice

In several models of inflammation, including collagen-induced arthritis (CIA), the disease-promoting effect of IL-12 has been attributed to its well-known ability to produce IFN-γ. However, IFN-γ receptor knockout (IFN-γ R KO) mice of the DBA/1 strain have been reported to be more susceptible to CIA than corresponding wild-type mice, indicating the existence of an IFN-γ-mediated protective pathway in this model. In the present study the development of CIA was found to be completely prevented by pretreatment with a neutralizing anti-IL-12 antibody, not only in wild-type, but significantly also in IFN-γ R KO mice. In both strains of mice, the protective effect of anti-IL-12 was associated with lower production of anti-collagen type II antibodies. In vivo stimulation with anti-CD3 antibody in arthritic IFN-γ R KO mice resulted in production of higher levels of circulating IFN-γ, TNF and IL-2 than in corresponding control mice that had not received the arthritis-inducing immunization. This was not the case in arthritis-developing wild-type mice. Furthermore, the protective effect of anti-IL-12 antibody in mutant, but not in wild-type mice, was associated with lower circulating IFN-γ, TNF and IL-2 and higher IL-4 and IL-5 cytokine levels following an anti-CD3 challenge. The data indicate that IL-12 promotes the development of arthritis independently of its ability to induce or favor production of IFN-γ. In fact, any IFN-γ produced in the course of the disease process rather exerts a protective effect. Furthermore, our study suggests that, in the absence of a functional IFN-γ system, endogenous IL-12 exerts its disease-promoting effect by favoring production of other Th1-associated cytokines (IL-2 and TNF), by inhibiting development of IL-4- and IL-5-producing T cells and by stimulating production of anti-collagen autoantibodies.     

Percutaneous sensitization with allergens through barrier-disrupted skin elicits a Th2-dominant cytokine response

We investigated whether percutaneous sensitization with different allergens through barrier-disrupted skin regulates the balance of Th1/Th2 cytokine expression. When mice were sensitized with the typical hapten picryl chloride (PiCl) by a single topical application to intact skin, there was an up-regulation in the lymph nodes (LN) of mRNA expression for the Th1 cytokines IL-2 or IFN-γ, and for the Th2 cytokine IL-4. In contrast, sensitization with PiCl after barrier disruption of the skin down-regulated the expression of mRNA for IFN-γ in a tape-stripping number-dependent manner without changing the expression of mRNA for IL-4. When mice were sensitized with house dust mite antigens (MA) by a single topical application to barrier-disrupted abdominal skin, there was a tape-stripping number-dependent up-regulation in the LN of mRNA expression for IL-4 but not for IL-2 or IFN-γ. In the LN, mRNA for the IL-4-inducible immunoglobulins IgE and IgG1, but not for the IFN-γ-inducible IgG2a, were up-regulated after sensitization with MA, while all three immunoglobulin mRNA were augmented after PiCl sensitization through intact skin. Antigenic elicitation by a topical application of PiCl in aural skin of mice sensitized through intact skin consistently increased the expression of mRNA for all three cytokines in the challenged skin, whereas elicitation in mice sensitized through barrier-disrupted skin decreased the expression of mRNA for IL-2 and IFN-γ, but not for IL-4. Antigenic elicitation by subcutaneous injection of MA in aural skin consistently increased the expression of mRNA for IL-4, but not for IL-2 or IFN-γ in the challenged skin. Infiltration of eosinophils in the dermis was more prominent following elicitation with MA in mice sensitized through barrier disruption than with PiCl in mice sensitized through intact skin. These findings suggest that the percutaneous entry of environmental allergens through barrier-disrupted skin is strongly associated with the induction of Th2-dominant immunological responses, as is seen in atopic dermatitis.     

IFN-γ上调Th17/IL-17应答介导衣原体呼吸道感染免疫保护

目的 探讨IFN-γ在小鼠沙眼衣原体感染中对Th17/IL-17应答的调节作用.方法 利用沙眼衣原体鼠肺炎株小鼠呼吸道感染模型,用抗鼠IFN-γ单克隆抗体吸入中和肺组织IFN-γ,对照组给予同等剂量的独特型抗体IgG2a,于感染后7 d处死小鼠.免疫酶法检测小鼠肺组织衣原体生长;利用RT-PCR技术检测衣原体感染小鼠肺组织中Th17相关因子IL-17及其上游因子IL-23 mRNA的表达;细胞内细胞因子染色检测衣原体感染小鼠脾脏IL-17-CD4+T细胞的扩增.结果 与对照组相比,IFN-γ抗体中和小鼠有严重的疾病状态,包括明显的体重下降、肺组织更高的衣原体负荷和肺组织更严重的病理损伤;肺组织IL-17和IL-23 mRNA的表达水平显著降低;脾脏IL-17-CD4+T细胞百分率也显著降低.结论 小鼠衣原体感染中,IFN-γ通过上调Th17/IL-17应答起保护作用.

Abstract：

Objective To investigate the regulation of IFN-γ to Th17 response in Chlamydia muridarum (Cm) lung infection in mice. Methods A murine model of pneumonia induced by intranasal inoculation of Cm was used for this study. Anti-mouse IFN-γ McAbs were used to neutralize endogenous IFN-γfollowing Cm lung infection. Control group received the same dose of isotype antibody (IgG2a). Mice were sacrificed at day 7 postinfection. Chlamydial growth in the lung was assessed by immunoenzyme technique.IL-17 and IL-23 mRNA expression in the lung was assayed by RT-PCR and the proliferation of IL-17 + CD4 +T cells in the spleen was assayed by intracellular cytokine staining. Results IFN-γ-neutralized mice exhibited serious disease course, include greater body weight loss, higher organism growth and much more severe pathological changes in the lung compared with control mice. The mRNA expression of IL-17 and IL-23 in the lung and the proliferation of IL-17 + CD4 + T cells in the spleen significantly decreased in the IL-17- neutralized mice. Conclusion IFN-γ was protective in Cm lung infection through up-regulating the antigen specific Th17 responses.