IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis

Background  Atopic dermatitis (AD) is a chronic disease with a Th2-type-cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half-life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)-4/IL-13 antagonist (IL-4DM), which blocks both IL-4 and IL-13 signal transductions.
Objective  To examine the effects of IL-4DM in vivo in an AD model induced by the repeated exhibition of oxazolone (OX).
Methods  Plasmid DNA was injected intraperitoneally to cause an experimental AD-like dermatitis. The effect was evaluated by ear thickness, histological findings, and mast cells counts in the inflamed skin. The plasma IgE and histamine levels were measured. Cytokine production in skin and splenocytes were also analysed.
Results  Mice treated with control plasmid developed marked dermatitis with mast cells and eosinophil infiltration, and had increased plasma IgE and histamine levels with a Th2 type splenocyte cytokine profile. Treatment with mouse IL-4 DNA augmented the ear swelling and thickness with an increased dermal eosinophil count, plasma histamine level, and production of splenocyte IL-4. However, IL-4DM treatment successfully controlled the dermatitis, decreased the mast cell and eosinophil count, and suppressed plasma IgE and histamine levels. Splenocytes produced an increased level of IFN-γ.
Conclusion  These data showed that the simultaneous suppression of IL-4/IL-13 signals successfully controlled Th2-type chronic dermatitis. IL-4DM DNA treatment is a potent therapy for AD and related diseases.     

Human keratinocytes constitutively express IL-4 receptor molecules and respond to IL-4 with an increase in B7/BB1 expression

Abstract In certain pathological conditions, such as atopic dermatitis, interleukin-4 (IL-4) can be detected in the skin. As the rôle of this cytokine in inflammatory skin lesions is not completely clear, we investigated its biological effects on skin keratinocytes. It was found that freshly isolated as well as cultured keratinocytes obtained from normal individuals express mRNA for the IL-4 receptor (IL-4R) and produce IL-4R protein, as determined by How cytometry. Moreover, IL-4 induced a proliferative response in keratinocyles after 1 day of culture and enhanced B7/BB1 expression in these cells. B7-2 (CD86) mRNA and protein were neither detected on untreated nor IL-4 treated keratinocytes. In contrast to interferon-γ (IFN-γ), IL-4 did not induce ICAM-1 (CD54) or HLA-DR-expression. Keratinocytes which had been treated with IL-4 showed an enhanced ability to stimulate allogeneic T-cell proliferation in the presence of staphylococcus enterotoxin B (SEB),(p<0.01). Neutralizing anti- B7/BBI monoclonal antibodies did not block this effect. These results indicate that other molecules than B7/BB-I, HLA-DR or ICAM-1 on IL-4-activated keratinocytes may be involved in T-cell stimulation. In conclusion our results suggest, that locally produced IL-4, besides modulating keratinocyte membrane molecules, may enable keratinoeytes to interact with skin infiltrating lymphocytes.     

Differential role of MAPK signaling in human dendritic cell maturation and Th1/Th2 engagement

Dendritic cells (DCs) are potent antigen-presenting cells that can stimulate resting T cells in the primary immune response. During the maturation process, immature DCs lose their ability to internalize antigens and they acquire the capacity to present antigens to naive T cells. Many observations have suggested that distinct DC subsets might differentially regulate Th responses. However, recent reports suggest that specific subsets of either murine or human DCs cultured in vitro with different stimuli respond with great plasticity in terms of both gene expression and cytokine secretion. Thus, the microenvironment of DCs may determine the nature of mature DCs and the subsequent immune response. The mechanism by which the character of DCs is determined is unknown. The in vitro maturation process of human monocyte-derived DCs (MoDC) can be initiated by various stimuli. Many stimuli induce phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 MAPK in DCs during maturation. Such kinase-specific inhibitors help to reveal the functions of MAPKs in the maturation of human MoDCs. Recent studies suggest that three MAPK signaling pathways differentially regulate all aspects of phenotypic maturation, cytokine production, and functional maturation of MoDCs. Thus, distinct maturation of DCs may be induced by modulating the balance of phosphorylation of the three MAPKs. In this review, we summarize the role of MAPK signaling pathways in the maturation of human MoDCs.     

IL-10 secretion of allergen-specific skin-derived T cells correlates positively with that of the Th2 cytokines IL-4 and IL-5*

Abstract In atopic individuals, allergen-specific CD4+ T lymphocytes often belong to the T-helper 2 (Th2) subset as they secrete the marker cylokincs interleukin-4 (IL-4) and IL-5 but not intcrfcron-y (INF-y). IL-10 is a cytokine the production of which, in the mouse system has been described to be restricted to the Th2 subset, but in the human was found to be produced by both ThI and Th2 T cell clones (TCC). We have recently shown that house dust mite antigen (Dermatophagoides pteron-yssinus)-specific TCC isolated from skin of patients with atopic dermatitis have a more polarized Th2 cytokine production profile than TCC obtained from the peripheral blood of these patients. In this study, we report that skin-derived TCC secrete more IL-10, IL-4 and IL-5, than TCC isolated from the blood of the same individual (p < 0.05). The difference was more significant with specific TCC than with non-specific TCC. Furthermore, there was a positive correlation between the production of IL-10 and that of IL-4 and IL-5, respectively. In addition, the amount of IL-4 and IL-5 secreted by specific TCC from the skin correlated positively. These results were confirmed by the detection of mRNA by PCR. Finally, our data confirm that in human blood-derived TCC IL-10 secretion is not related to a particular cytokine production profile. We suggest that the skin of AD provides an unique environment for the development of aTh2-likc secretion pattern not only with respect to IL-4 and IL-5 but also regarding IL-10.     

IL-4 and IL-13 alter plasmacytoid dendritic cell responsiveness to CpG DNA and herpes simplex virus-1

Human plasmacytoid dendritic cells (pDCs) are found in skin lesions in a wide variety of diseases. The role of the microenvironment in these lesions on the function of human pDCs remains elusive. We sought to determine the effect of T(h)2 cytokines on the ability of human pDCs to respond to CpG oligodeoxynucleotides (ODNs) and herpes simplex virus in vitro. In this study, we found that the T(h)2 cytokines, IL-4 and IL-13, modulate Toll-like receptor 9 (TLR-9)- and herpes simplex virus-induced pDC phenotype and enhance the ability of these cells to induce allogeneic T-cell responses. Moreover, T(h)2 cytokines impaired TLR-9-induced secretion of inflammatory cytokines and chemokines. Taken together, these results demonstrate that T(h)2 cytokines are involved in the modulation of pDC function and responsiveness to bacterial- and viral-derived stimuli.     

复发性生殖器疱疹患者外周血IL-12与Th1/Th2细胞因子的检测

目的检测复发性生殖器疱疹(RGH)患者不同病期外周血CD4+T细胞内IL-12,IFN-,γIL-4的水平,探讨IL-12,Th1与Th2亚群在疾病中的可能作用。方法应用流式细胞仪对20例发作期、15例恢复期RGH患者和15名健康人外周血CD4+T细胞IL-12,IFN-γ和IL-4进行检测。结果发作期患者外周血IFN-γ+-CD4+T细胞百分率显著低于正常对照组(P<0.05),IL-4+-CD4+T细胞百分率明显高于正常对照组(P<0.01),Th1/Th2比值显著低于正常对照组(P<0.01),同时IL-12+-CD4+T细胞百分率显著降低(P<0.01)。恢复期患者外周血IL-12+-CD4+T细胞百分率仍显著低于正常(P<0.05)。结论RGH患者存在Th1/Th2比例失衡和IL-12水平低下,而后者可能是导致Th1/Th2比例失衡和病情反复发作的重要原因。     

Evaluation of mite allergen-induced Th1 and Th2 cytokine secretion of peripheral blood mononuclear cells from atopic dermatitis patients: association between IL-13 and mite-specific IgE levels

There have been several reports about Th1/Th2 imbalances in atopic dermatitis (AD), but there have been few precise investigations about the differences between Th1 and Th2 cytokine secretion patterns of peripheral blood mononuclear cells (PBMCs) in such patients. We cultured PBMCs, taken from AD patients and healthy subjects, with dust mite extract (DME) and measured subsequent immunoreactive interferon (IFN)-gamma (Th1 cytokine), interleukin (IL)-4, IL-5, and IL-13 (Th2 cytokines) levels in the supernatants by ELISA assays. There is a difference between IL-4 and IL-13 secretion patterns by DME-stimulated PBMCs in AD subjects; immunoreactive IL-4 levels were detectable maximally within 24-h cultures, while IL-13 levels increased time-dependently within 7-day cultures. IL-13 levels were significantly elevated in AD subjects compared to healthy subjects, while IFN-gamma levels did not significantly differ between the two groups. IL-13 levels were significantly higher in AD patients who had high levels (>100 U/ml) of Dermatophagoides pteronyssinus-specific IgE (Dp-IgE) than in those AD patients who had low levels (<10 U/ml) of Dp-IgE. Tacrolimus (FK-506), at a concentration of 10(-8) M, significantly inhibited DME-induced IL-13 production from PBMCs. These findings suggest that IL-13 produced by Th2 cells are involved in IgE overproduction in AD subjects.     

CO_2激光联合5-ALA光动力治疗尖锐湿疣及对Th1/Th2细胞因子水平的影响

目的采用CO_2激光与氨基乙酸乙酯光动力疗法(ALA-PDT)联合治疗尖锐湿疣(CA),了解其疗效、复发情况、不良反应等,比较外周血中Th1/Th2细胞因子水平。方法选取2017年12月至2019年6月兰州市第一人民医院皮肤性病门诊诊治的216例尖锐湿疣患者作为研究对象。将这216例患者随机分为联合治疗组和激光治疗组,每组各108例。联合治疗组患者应用CO_2激光联合5-ALA光动力治疗,激光组患者仅使用CO_2激光治疗。比较两组患者的总有效率、复发率和不良反应发生率,并在治疗后6个月测定外周血细胞因子Th1/Th2。结果两组患者的年龄和性别差异无统计学意义。联合治疗组患者与激光组患者相比,联合治疗组患者的痊愈率、显效率、有效率、总有效率均高于激光组患者,差异具有统计学意义(P0.05);联合治疗组患者的复发率明显低于激光组患者,差异具有统计学意义(P0.05);联合治疗组患者的不良反应发生率与激光组患者比较,差异无统计学意义(P0.05);联合治疗组患者IL-2和IFN-γ水平显著高于激光组患者,差异具有统计学意义(P0.05);而联合治疗组患者IL-4、10水平明显低于激光组患者,差异具有统计学意义(P0.05)。结论 CO_2激光联合5-ALA光动力疗法治疗尖锐湿疣不增加不良反应发生率,是安全有效的,值得临床推广。     

Therapeutic effects of streptococcal preparation OK-432 on atopic dermatitis-like lesions in NC/Nga mice: possible shift from a Th2- to Th1-predominance

BACKGROUND: The inducement of Th1 cell-mediated immune response, possibly brought about through bacterial stimulation, may serve to control atopic disorders such as atopic dermatitis (AD). The streptococcal preparation, OK-432, has been shown a potent Th1 inducer through the action of IL-12. NC/Nga mice under ordinary conditions have been found to contract dermatitis similar to human AD. OBJECTIVE: Examination was made of the therapeutic effects of OK-432 local intra- and/or subcutaneous injections on AD-like lesions in NC/Nga mice. METHODS: Immunohistochemical staining with IL-4/IL-12p40 and CD80/86 and phosphorylated STAT4/p-STAT6 and RT-PCR for IL-4/IL-12p40 and STAT6/STAT4 mRNA was conducted for the evaluation of OK-432 treatment of spontaneous AD-like lesions in NC/Nga mice. RESULTS: At 5 weeks following injection of OK-432, for treating head and back lesions in NC/Nga mice, 10 of 12 OK-432 treated NC mice were found to have clinically improved quite considerably. On the head and back skin of OK-432-treated mice, IL-12p40/CD80 positive cellular infiltration was conspicuous, in contrast to non-treated mice. IL-4/CD86 positive cellular infiltrates in OK-432-treated mice had decreased significantly more than in non-treated mice and IL-4 mRNA expression was virtually absent in OK-432-treated mice. P-STAT4 positive cells could be seen abundantly present in OK-432-treated mice, and p-STAT6 positive cells were much fewer than in non-treated mice. CONCLUSIONS: OK-432-treatment appears to induce Th1 cellular response and to down-regulate that of the Th2 pathway in AD-like lesions of NC/Nga mice. The present results demonstrate bacterial components from such Streptococcus to likely constitute an effective new therapeutic approach in the treatment of AD.     

Flow cytometric analysis of IL-4, IL-13 and IFN-gamma expression in peripheral blood mononuclear cells and detection of circulating IL-13 in patients with atopic dermatitis provide evidence for the involvement of type 2 cytokines in the disease

Recent studies have shown increased T-helper (Th) 2 cytokine expression and decreased IFN-gamma expression in peripheral blood from patients with atopic dermatitis (AD). In the present study, we examined the cytokine expression in peripheral blood mononuclear cells in patients with AD and tested how the cytokine profile correlated with the patients' age, severity and laboratory findings. Peripheral blood mononuclear cells obtained from 20 patients with AD were stimulated with phorbol 12-myristate 13-acetate and ionomycin, and were examined for the frequencies of CD4+ cells expressing IL-4, IL-13 and IFN-gamma at the single cell level using intracellular cytokine staining and flow cytometry. There was a significant positive correlation between IL-4 and IL-13 expression in CD4+ cells. Expression levels of both IL-4 and IL-13 in CD4+ cells were significantly correlated with peripheral blood eosinophilia. These findings suggest that CD4+ cells producing IL-4 and IL-13 play important roles in the pathogenesis of AD. IFN-gamma expression did not correlate with either IL-4 or IL-13 expression, or with blood eosinophilia. We also measured serum levels of IL-13 in 144 patients with AD using enzyme-linked immunosorbent assay, and found 16 patients with detectable levels of serum IL-13. IL-13+ patients had significantly higher serum IgE levels than IL-13- patients, suggesting a direct association between serum levels of IL-13 and IgE. It was also shown that the IL-13+ group was significantly younger in age than the IL-13- group, although the implication of this finding is not clear at present. The sum of these findings suggested that the predominance of Th2 cells and the consequent overexpression of IL-4 and IL-13 in peripheral blood may be deeply involved in the pathogenetic process of AD.     

A rapid flow cytometric assay to detect CD4+ and CD8+ T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy

BACKGROUND: The immune response in atopic dermatitis (AD) is thought to be driven by T-helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)-4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)-gamma have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL-5, IL-13 and tumour necrosis factor (TNF)-alpha are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD. OBJECTIVES: To use a rapid (4-h) flow cytometric assay to study a wide range of Th1 and Th2 cytokine patterns in peripheral blood lymphocytes from patients with AD, comparing them with non-atopic healthy controls. To correlate cytokine patterns with the degree of eosinophilia observed and in the case of one patient with severe disease, to observe the effect of cyclosporin therapy on peripheral blood cytokine patterns. METHODS: Peripheral blood from eight patients with AD and 23 healthy controls was examined for the frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4, IL-5, IL-13, IFN-gamma and TNF-alpha using flow cytometry. RESULTS: Significantly higher frequencies of CD4+/IL-4+ (P < 0.005) and CD4+/IL-13+ (P < 0.0001) and lower frequencies of CD4+/IFN-gamma+ (P < 0.002) and CD8+/TNF-alpha+ (P < 0.05) T lymphocytes were found in patients with AD compared with controls. There were significant positive correlations with the increased percentages of CD4+/IL-4+ and CD4+/IL-13+ T lymphocytes and the degree of eosinophilia observed (P < 0.05, P < 0.001) and a negative correlation between the percentage of CD4+/IFN-gamma+ T lymphocytes and eosinophilia (P < 0.05). In one patient examined before and 8 days after cyclosporin therapy, 50% or greater reductions were observed in percentages of peripheral blood CD8+/IL-5+, CD8+/IL-13+, CD4+/IL-4+ and CD4+/IL-5+ T lymphocytes following cyclosporin therapy. A smaller reduction of 15% after cyclosporin therapy was found in percentages of CD4+/IL-13+ T cells. CONCLUSIONS: These data strongly support a Th2 predominance in the peripheral blood of AD. The results suggest that administration of cyclosporin therapy in patients with AD may help to restore the Th2 cytokine imbalance seen in these patients.     

重组人干扰素凝胶联合光动力治疗尖锐湿疣疗效及对血清Th1/Th2细胞因子水平影响研究

目的:观察重组人干扰素凝胶联合光动力治疗尖锐湿疣疗效及对血清Th1/Th2细胞因子水平的影响。方法:选择2015年3月至2016年3月我院收治的符合尖锐湿疣诊断标准患者110例入选本研究,随机分为两组,每组55例。对照组给予光动力治疗,观察组采取重组人干扰素凝胶联合光动力治疗,比较两组疗效、复发情况及治疗前后血清Th1/Th2细胞因子水平变化。结果:与对照组比较,观察组治疗后总有效率显著升高(P0.05);与对照组比较,观察组复发发生率及复发后测定病毒体数量均显著降低(P0.05);与对照组比较,观察组血清Th1/Th2水平显著升高(P0.05)。两组不良反应发生率比较差异无统计学意义(P0.05)。结论:重组人干扰素凝胶联合光动力治疗尖锐湿疣疗效显著,且有助于维持血清Th1/Th2细胞因子平衡,并不增加不良反应发生率,临床应用安全、有效。