Human eosinophils constitutively express multiple Th1, Th2, and immunoregulatory cytokines that are secreted rapidly and differentially

Eosinophils are innate immune leukocytes implicated in the initiation and maintenance of type 2 immune responses, including asthma and allergy. The ability to store and rapidly secrete preformed cytokines distinguishes eosinophils from most lymphocytes, which must synthesize cytokine proteins prior to secretion and may be a factor in the apparent Th2 bias of eosinophils. Multiple studies confirm that human eosinophils from atopic or hypereosinophilic donors can secrete over 30 cytokines with a varying and often opposing immune-polarizing potential. However, it remains unclear whether all of these cytokines are constitutively preformed and available for rapid secretion from eosinophils in the circulation of healthy individuals or are restricted to eosinophils from atopic donors. Likewise, the relative concentrations of cytokines stored within eosinophils have not been studied. Here, we demonstrate that human blood eosinophils are not singularly outfitted with Th2-associated cytokines but rather, constitutively store a cache of cytokines with nominal Th1, Th2, and regulatory capacities, including IL-4, IL-13, IL-6, IL-10, IL-12, IFN-gamma, and TNF-alpha. We demonstrate further rapid and differential release of each cytokine in response to specific stimuli. As agonists, strong Th1 and inflammatory cytokines elicited release of Th2-promoting IL-4 but not Th1-inducing IL-12. Moreover, a large quantity of IFN-gamma was secreted in response to Th1, Th2, and inflammatory stimuli. Delineations of the multifarious nature of preformed eosinophil cytokines and the varied stimulus-dependent profiles of rapid cytokine secretion provide insights into the functions of human eosinophils in mediating inflammation and initiation of specific immunity.     

Th1型和Th2型细胞因子对人中性粒细胞吞噬结核杆菌活性的影响

目的 建立用流式细胞术检测人外周血中性粒细胞(PMNs)吞噬结核分枝杆菌(Mtb)的方法 ,并探讨Th1和Th2型细胞因子对PMNs吞噬Mtb活性的影响.方法 运用抗酸染色、激光共聚焦显微镜观察人PMNs吞噬Mtb,并用流式细胞术检测人PMNs对FTTC标记Mtb的吞噬活性.外周血预先分别与IL-2、IFN-γ、GM-CSF和IL-4等细胞因子孵育,再加FTTC标记Mtb后,用流式细胞术检测PMNs对Mtb吞噬率,并与对照组比较吞噬率的变化.结果 抗酸染色和激光共聚焦显微镜均能观测到人PMNs吞噬Mtb.用流式细胞术检测健康人外周血PMNs对Mtb的吞噬率在5 min时为47%,15～20 min达到平台期,为66%～72%.外周血预先加IL-2或IFN-γ作用后,PMNs对Mtb的吞噬率可分别增加76.7%和75.2%;而预先加IL-4作用后,吞噬率降低31.7%.结论 IL-2和IFN-γ对PMNs吞噬Mtb功能有增强作用,而IL-4有降低作用,表明Th1型和Th2型细胞因子参与调节PMNs抗结核杆菌感染的免疫作用.     

Th1型和Th2型细胞因子对人中性粒细胞吞噬结核杆菌活性的影响

Objective To establish a flowcytometry method for detecting phagocysis of Mycobacterium tuberculosis(Mth) by the human peripheral blood neutrophils( PMNs), and to explore the effects of Thl and Th2 cytokine on phagocytotic activity of PMNs. Methods By using acid-fast staining the phagocytosis of Mtb by human neutrophil was observed by microscopy, and the phagocytosis of FTTC labeled Mtb by human neutrophils was detected under confocal microscope. The whole fresh peripheral blood of healthy adults was incubated with FTTC labeled Mtb and the phagocytosis were measured by flow cytometry. The altered phagocytosis of FTTC-Mtb by neutrophils pretreated with IL-2, IFN-γ, GM-CSF or IL-4 were measured. Results Human peripheral blood neutrophils activity of Mtb phagocytosis was observed by acid-fast staining and confocal microscope. The percentage of phagocytosis of Mtb was dependent on the time of incubation with Mtb. The percentages were 47% at 5 min and reached plateau about 66% ～72% at 15 min to 20 min.Pretreatment with different concentrations of IL-2 or IFN-γincreased the phagocytosis of Mtb by 76.7% and 75.2%, respectively; but pretreatment with IL-4 decreased the phagocytosis by 31.7%. Conclusion IL-2and IFN-γcan increase phagocytosis of Mth by neutrophils; while IL-4 can reduced neutrophil activity of phagocytosis of Mtb by human neutrophils, and demonstrate that Th1/Th2 type cytokins involve in regulating the acitvities of neutrophils anti-Mtb infection.     

Th1型和Th2型细胞因子对人中性粒细胞吞噬结核杆菌活性的影响

Objective To establish a flowcytometry method for detecting phagocysis of Mycobacterium tuberculosis(Mth) by the human peripheral blood neutrophils( PMNs), and to explore the effects of Thl and Th2 cytokine on phagocytotic activity of PMNs. Methods By using acid-fast staining the phagocytosis of Mtb by human neutrophil was observed by microscopy, and the phagocytosis of FTTC labeled Mtb by human neutrophils was detected under confocal microscope. The whole fresh peripheral blood of healthy adults was incubated with FTTC labeled Mtb and the phagocytosis were measured by flow cytometry. The altered phagocytosis of FTTC-Mtb by neutrophils pretreated with IL-2, IFN-γ, GM-CSF or IL-4 were measured. Results Human peripheral blood neutrophils activity of Mtb phagocytosis was observed by acid-fast staining and confocal microscope. The percentage of phagocytosis of Mtb was dependent on the time of incubation with Mtb. The percentages were 47% at 5 min and reached plateau about 66% ～72% at 15 min to 20 min.Pretreatment with different concentrations of IL-2 or IFN-γincreased the phagocytosis of Mtb by 76.7% and 75.2%, respectively; but pretreatment with IL-4 decreased the phagocytosis by 31.7%. Conclusion IL-2and IFN-γcan increase phagocytosis of Mth by neutrophils; while IL-4 can reduced neutrophil activity of phagocytosis of Mtb by human neutrophils, and demonstrate that Th1/Th2 type cytokins involve in regulating the acitvities of neutrophils anti-Mtb infection.     

The paradigm of Th1 and Th2 cytokines

In the past few years, considerable evidence has accumulated to suggest the existence of functionally polarized responses by the CD4⁺ T helper (Th)—and the CD8⁺ T cytotoxic (Tc)—cell subsets that depend on the cytokines they produce. The Th1 and Th2 cellular immune response provide a useful model for explaining not only the different types of protection, but also the pathogenic mechanisms of several immunopathological disorders. The factors responsible for the polarization of specific immune response into a predominant Th1 or Th2 profile have been extensively investigated in mice and humans. Evidence has accumulated from animal models to suggest that Th1type lymphokines are involved in the genesis of organ-specific autoimmune diseases, such as experimental autoimmune uveitis, experimental allergic encephalomyelitis, or insulin-dependent diabetes mellitus. Accordingly, data so far available in human diseases favor a prevalent Th1 lymphokine profile in target organs of patients with organ-specific autoimmunity. By contrast, Th2-cell predominance was found in the skin of patients with chronic graft-versus host disease, progressive systemic sclerosis, systemic lupus erythematosus, and allergic diseases. The Th1/Th2 concept suggests that modulation of relative contribution of Th1 or Th2-type cytokines regulate the balance between protection and immunopathology, as well as the development and/or the severity of some immunologie disorders. In this review, we have discussed the paradigm of Th1 and Th2 cytokines in relation to autoimmunity and allergy.     

Simultaneous analysis of eight human Th1/Th2 cytokines using microarrays

The adaptive immune system induces T cells to change from a naive phenotype to a Th1/Th2 phenotype each of which produce characteristic types of cytokines. Knowledge of whether a specific immune response is Th1 or Th2 is a useful indicator for diseases with basis in immune function disorder. An assay that can rapidly analyze multiple cytokines indicative of these two cell types from small sample quantities can be an extremely useful research and diagnostic tool. Silanized glass slides were printed with multiple arrays of capture antibodies to detect eight different cytokines involved in the Th1/Th2 response along with control proteins for assessing assay performance. Arrays were developed by sequential addition of known antigen amounts, detector antibodies and a fluorescent detection system followed by imaging and quantification. These arrays were used to determine the specificity, sensitivity and reproducibility of the assay and the performance compared with conventional ELISA. This multiplexed assay is able to measure human Th1/Th2 cytokines in sample volumes lower than 20 microl. The assay sensitivity for the eight cytokines range from 0.3 microg/l for IL-4 to 6.4 microg/l for IL-5 which are either comparable to or higher than those reported for conventional ELISA or bead-based multiplex ELISA methods. This assay can be automated to measure expression levels of multiple Th1/Th2 cytokines simultaneously from tens to hundreds of biological samples. This assay platform is more sensitive and has a larger dynamic range as compared to a conventional ELISA in addition to significantly reducing the time and cost of assay. This platform provides a versatile system to rapidly quantify a wide variety of proteins in a multiplex format.     

Quantitative analysis of human immunoregulatory cytokines by electrochemiluminescence method

Quantitative analysis of human immunoregulatory cytokines in physiological media and cell cultures plays an important role in fundamental and clinical research. Here we describe the quantification of interleukin (IL)-2, IL-4, IL-10 and interferon-gamma (IFN-gamma) in human serum and peripheral blood mononuclear cell (PBMC)-conditioned medium by electrochemiluminescence method (ECL). We demonstrate that this approach allows to detect cytokine concentration from 1 pg/ml. The high sensitivity in combination with accuracy and wide range of determined concentration indicates that ECL meets the standards of quantitative analysis of cytokines. Simplicity and short time of procedure, small assay volume and high reproducibility make ECL method competitive in practical use with conventional quantitative methods of cytokine detection.     

接种纯化人用狂犬病疫苗对Th1/Th2类细胞因子谱的影响

目的 研究人用狂犬病纯化疫苗(RV)对人体抗原特异性Th1/Th2细胞因子谱的影响.方法 对20例暴露者进行RV的全程接种,在开始接种RV的第0天、14天、45天时分别采血并分离人外周血单个核细胞(PBMC)与RV进行培养,采用ELISA法检测血清抗狂犬病病毒抗体,体外培养检测淋巴细胞转化增殖能力(MTT法),流式微球分析技术(CBA)法检测细胞培养上清液中Th1类细胞因子(IFN-γ、TNF、IL-2)及Th2类细胞因子(IL4、IL-5、IL-10)的水平.结果 暴露组全程注射RV后,19例于第45天、1例于第60天检测血清抗狂犬病病毒抗体阳性,阳性率100%.RV刺激后,暴露组第14天、第45天淋巴细胞增殖能力显著增高,第45天淋巴细胞增殖能力明显高于第0天(P<0.05);当RV刺激后,暴露组第14天、第45天产生IFN-γ、IL-2、IL-4、IL-5的含量高于未刺激组及暴露组第0天水平(P<0.05);TNF、IL-10的含量各组间比较差异无统计学意义(P>0.05).结论 RV在刺激机体产生体液免疫的同时,可以诱导机体产生特异性细胞免疫,Th1的免疫应答尤为显著,提示细胞免疫在预防和控制狂犬病病毒感染中发挥重要的协同作用.     

Expression and regulation of monocyte chemoattractant protein-1 by human eosinophils

Several recent studies have identified eosinophils as a cellular source of various cytokines, indicating that eosinophils play not only an effector role, but also a regulatory role within the allergic inflammatory cell network. In this study, we demonstrate that eosinophils can generate and secrete monocyte chemoattractant protein-1 (MCP-1), a prototype of C-C chemokines. Eosinophils generated immunoreactive MCP-1 in response to such diverse stimuli as C5a, formylmethionyl-leucyl-phenylalanine (FMLP) and ionomycin, but MCP-1 production was not induced by interleukin (IL)-1 or tumor necrosis factor-α. C5a- and FMLP-induced eosinophil MCP-1 production was absolutely dependent on pretreatment with cytochalasin B. Eosinophils elaborated significantly more MCP-1 than neutrophils. Immunoreactive MCP-1 was detected at 6 h of incubation with C5a or FMLP. Expression of MCP-1 mRNA reached a maximum within the first 3 h after stimulation and then declined rapidly to a very low and stable level by 18 h. Pretreatment with IL-5 markedly amplified C5a-induced MCP-1 production, and the enhancement occurred at the pretranslational level. Eosinophilactive chemokines such as eotaxin failed to induce MCP-1 generation, even when eosinophils were primed by IL-5. Since MCP-1 exerts a potent histamine-releasing effect on human basophils, our results indicate that eosinophils may regulate basophil mediator release with possible consequent contribution to the pathogenesis of allergic inflammation via a paracrine mechanism.     

Regulation of fetal allograft survival by hormone-controlled Th1- and Th2-type cytokines

There is clear evidence to suggest that the maternal immune system during pregnancy can enhance or inhibit the development of the fetoplacental unit. Recent data support the view that some cytokines produced by both T cells and non-T cells (IL-3, GM-CSF, TGF-β, IL-4, IL-10), favor fetal survival and growth. In contrast, other cytokines, such as IFN-γ, TNF-β and TNF-α, can rather compromise pregnancy. Accordingly, we show here that T-cell clones generated from the decidua of women with unexplained recurrent abortion produced significantly lower concentrations of IL-4 than clones derived from the decidua of voluntary abortions or the endometrium of nonpregnant women. Thus, despite the complexity of the cytokine network, it appears that cytokines favoring the maintenance of fetal survival mainly belong to the Th2 pathway, whereas the failure of pregnancy rather associates with the predominance of Th1-type cytokines and/or the absence of Th2-type cytokines. Interestingly, we also found that, at least in vitro, progesterone promotes the preferential development of Th2-like cells and induces transient IL-4 production by established Th1 cells, whereas relaxin, another corpus luteum-derived hormone, mainly promotes the development of Th1-like cells. These data provide an excellent basis for investigating the relationship between the endocrine and the immune system in the regulation of the maternal-fetal interaction.     

Human airway and peripheral blood eosinophils enhance Th1 and Th2 cytokine secretion

BACKGROUND: The effector function of eosinophils involves their release of toxic granule proteins, reactive oxygen species, cytokines, and lipid mediators. Murine studies have demonstrated that eosinophils can also enhance T cell function. Whether human eosinophils, in particular, airway eosinophils, have similar immunoregulatory activity has not been fully investigated. The aim of this study was to determine whether human blood and airway eosinophils can contribute to Th1 and Th2 cytokine generation from CD4+ T cells stimulated with superantigen. METHODS: Eosinophils were obtained from blood or bronchoalveolar lavage fluid 48 h after segmental allergen bronchoprovocation. Purified eosinophils were co-cultured with autologous CD4+ blood T cells in the presence of staphylococcal enterotoxin B (SEB). Cytokine levels in the supernatant fluid were determined by enzyme-linked immunosorbent assay (ELISA). Eosinophil expression of major histocompatibility complex (MHC) class II and co-stimulatory molecules was assessed by flow cytometry before culture, 24 h after granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation, and 24 h after co-culture with CD4+ T cells and SEB. RESULTS: Interleukin (IL)-5, IL-13, and interferon (IFN)-gamma generation increased when CD4+ T cells were co-cultured with either blood or airway eosinophils in the presence of SEB. The ability of eosinophils to enhance cytokine generation was independent of their source (blood vs airway), activation by GM-CSF, or detectable expression of human leukocyte antigen (HLA)-DR, CD80, or CD86. CONCLUSION: Our data demonstrate that SEB-induced generation of Th1 and Th2 cytokines is increased in the presence of human blood and airway eosinophils. Thus, eosinophils can have an immunoregulatory function in pathogen-associated allergic diseases such as atopic dermatitis, chronic sinusitis, and asthma exacerbations.     

Th1- and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts

Eosinophils (Eos) and fibroblasts are known to play a major role in the pathogenesis of bronchial asthma and fibrotic lung disease. Therefore, we investigated whether Th1 and Th2 cytokines stimulate the production of Eo-activating chemokines by lung fibroblasts. Analyses of the culture supernatant using multiple steps of high-performance liquid chromatography demonstrated that interleukin (IL)-4 preferentially stimulates lung fibroblasts to secrete a peak of eosinophil chemotactic activity (ECA) which, upon N-terminal analyses, showed similar sequence to eotaxin, whereas interferon (IFN)-gamma had negligible effect on the release of this chemokine. In contrast, tumor necrosis factor (TNF)-alpha stimulated lung fibroblasts to release two peaks of activity that were found to correspond to eotaxin and regulated on activation, normal T cells expressed and secreted (RANTES), respectively. Interestingly, IL-4 synergized with TNF-alpha to increase greatly the production of three biochemically distinct eotaxin forms. In contrast, IFN-gamma synergized with TNF-alpha to increase RANTES production. Neither IL-2, IL-5, IL-6 nor IL-10 had an effect on lung fibroblasts' capacity to express or release eotaxin and RANTES. Upon appropriate cytokine stimulation, lung fibroblasts were also found to express messenger RNA for monocyte chemotactic protein (MCP)-3 and MCP-4 but not eotaxin-2. However, no ECA like MCP-3 or MCP-4 was detected. These observations suggest that the release of Th1 or Th2 cytokines in the lung tissue polarizes lung fibroblasts to produce either RANTES or eotaxin as major Eo attractants.     

Th1, Th2, and Th17 cytokines in systemic lupus erythematosus

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the breakdown of immune tolerance leading to excessive inflammation and tissue damage. Imbalance in the levels of cytokines represents one of the multifactorial causes of SLE pathogenesis and it contributes to disease severity. Deregulated levels of T helper type 1 (Th1), type 2 (Th2), and type 17 (Th17) cytokines have been associated with autoimmune inflammation. Growing evidence has shown deregulated levels of Th1, Th2, and Th17 cytokines in SLE patients compared to healthy controls associated with disease activity and severity. In this review, we describe and discuss the levels of Th1, Th2, and Th17 cytokines in SLE patients, and clinical trials involving Th1, Th2, and Th17 cytokines in SLE patients. In particular, with the exception of IL-2, IL-4, and TGF-β1, the levels of Th1, Th2, and Th17 cytokines are increased in SLE patients associated with disease severity. Current phase II or III studies involve therapeutic antibodies targeting IFN-α and type I IFN receptor, while low-dose IL-2 therapy is assessed in phase II clinical trials.     

Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis.

In experimental Leishmania donovani infection in BALB/c mice, initial susceptibility gives way to T-cell-dependent acquired resistance and eventual control over visceral infection. Since various cytokines appear to underlie the host response to Leishmania infection, we examined infected liver tissue for gene expression of cytokines associated with Th1 (gamma interferon [IFN-gamma] and interleukin-2 [IL-2]) and Th2 cells (IL-4 and IL-10). By Northern (RNA) blot analysis, only IFN-gamma mRNA expression was detected in livers of infected euthymic mice. To determine whether activation of Th1 cells develops selectively in this model, qualitative PCR analysis was used. These results indicated that mRNAs for IFN-gamma, IL-2, IL-4, and IL-10 were all induced by L. donovani infection. The potentially negative Th2 cell-associated response did not appear to play a functional role, however, since resistance was acquired, anti-IL-4 monoclonal antibody treatment did not accelerate control over visceral infection, and serum immunoglobulin E levels remained low. As judged by PCR analysis, IL-4 and IL-10 mRNAs were also expressed under three other conditions without apparent effect: in naive euthymic mice treated with IL-2, which induces leishmanicidal activity; in rechallenged immune mice, which resist reinfection; and in nude mice, which fail to control L. donovani. These results suggest that, like other Leishmania species, L. donovani infection may trigger a potentially suppressive Th2 cell-associated cytokine response. However, in T-cell-intact mice able to control L. donovani, this response either is insufficient to influence outcome or more likely is overshadowed by the Th1 cell response.     

膜表达的HLA-G1对同种反应性PBMC分泌Th1/Th2型细胞因子的影响

目的研究人脐静脉内皮细胞系(ECV304)表达的HLA-G1对同种异体外周血单个核细胞(PBMC)Th1/Th2型细胞因子分泌的影响。方法采用脂质体介导的基因转染技术将pcDNA3-HLA-G1转入ECV304,以间接免疫荧光技术在蛋白质水平上检测HLA-G1分子在ECV304上的表达;以表达HLA-G1的ECV304作为刺激细胞,灭活后,与健康人PBMC共同培养,用ELISA检测上清液中Th1/Th2型细胞因子的浓度,观察HLA-G1对同种异体抗原激活的PBMC分泌Th1/Th2型细胞因子的影响。结果与转染pcDNA3空质粒的对照组相比,HLA-G1能使PBMC的IL-10分泌增加(P<0.05),而IL-2,IFN-γI、L-4分泌无明显影响。结论HLA-G1能引起由同种异体抗原激活的PBMC分泌IL-10增加,提示HLA-G1有可能使Th1/Th2型细胞因子向Th2型偏移。     

Expression of Th1 and Th2 type cytokines responding to HBsAg and HBxAg in chronic hepatitis B patients.

The cytokine pattern on viral antigen recognition is believed to exert a profound influence on the resolution of viral infections and viral clearance. This study was initiated to investigate whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analysed by RT-PCR. Upon HBsAg stimulation, expression of IFN-gamma, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of IFN-gamma was associated with high levels of serum AST/ALT. However, we could not prove that Th2 type cytokines had a protective effect on hepatocytes. Upon HBxAg stimulation, there was no recognizable association of cytokine patterns with AST/ALT levels. In conclusion, production of a Th1 cytokine, IFN-gamma, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B, while no counteracting effect of Th2 cytokines produced by those cells was observed.     

Infection of human dendritic cells by dengue virus activates and primes T cells towards Th0-like phenotype producing both Th1 and Th2 cytokines

Dengue viruses (DV) infection is an important public health issue all over the world. Although the pathogenesis remains unclear, the overwhelmingly triggered immune responses have been consistently observed. Recently, we and other researchers demonstrated that the natural hosts for DV are dendritic cells (DC), the primary sentinels of immune system. In light of the significance of T cells in dengue virus pathogenesis, here, we examine the possible consequences of DC-T cell interaction that is supposed to be happening in lymphoid tissues after infection. We showed that DV-infected DC induced the interacting T cells to proliferate, to produce interleukin-2 as well as to express activation markers on cell surface. Compared to mock-infected DC, the infection of DC by DV also induced T cells to produce interleukin-4, interleukin-10 and interferon-gamma, a cytokine pattern suggesting Th0 phenotype. Such an effect was either totally abolished or greatly reduced when DV were pre-inactivated with heat or ultraviolet before infection. In addition, we demonstrated that such a Th0 phenotype shift of T cells was affected neither by different dosages of viruses that infected DC nor by different durations of DC-T cell interaction. Our results provide a basic support for clinical observations and may be of help in understanding the pathogenesis of DV infection.