Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Leukocytes in expressed breast milk of asthmatic mothers

ObjectiveInfants are born immunologically immature. However, breastfeeding mothers retain an immunological link to their infants. While it is generally accepted that infants are at an immunological advantage when compared with formula-fed infants, the benefit of long-term exclusive breastfeeding by atopic mothers remains controversial. Inconsistency in the conferral of benefit may be due to differences in the immunological constituents passed to the recipient infant. The aim of this investigation was to examine the profile of human milk cells and cytokines from asthmatic compared to non-asthmatic mothers.MethodsTwenty-five exclusively breastfeeding mothers with a clinical diagnosis of asthma were postpartum age matched in a double-control 2:1 design with 50 non-asthmatic controls. Each mother provided a single milk sample which was assayed for cell differential by flow cytometry, for ex vivo cytokine production in culture and for aqueous phase cytokines.ResultsMilks from asthmatic mothers differed from non-asthmatics in that they contained a higher proportion of polymorphonuclear (PMN) cells and lower proportion of lymphocytes, predominantly CD3⁺/CD4⁺ T helper cells, reflected by a decrease in the chemokine CCL5 in the milk aqueous phase. More PMN and lymphocytes from asthmatic mothers expressed the adhesion molecule CD11b and lymphocytes the IgE receptor CD23, than those from non-asthmatic mothers.ConclusionsChanges to human milk leucocyte prevalence, activation state and cytokines due to maternal asthma may result in changes to immunological priming in the infant. Consequently, the protective effect of long-term breastfeeding may be altered in these mother-infant pairs.     

Different IL‐5 and IFN‐γ Production from Peripheral Blood T‐Cell Subsets in Atopic and Nonatopic Asthmatic Children

Defective Th1 and enhanced Th2‐type cytokine responses have been implicated in the development of atopic disease. However, the immunopathology of nonatopic asthma, especially in children, remains unclear, and there have been few studies to compare the cytokine profile in peripheral blood T‐cell subsets between atopic and nonatopic asthmatic children. To document whether atopic asthmatic children have a cytokine imbalance and to compare the cytokine profile between atopic and nonatopic asthmatic children, we investigated the interleukin (IL)‐5‐producing and interferon (IFN)‐γ‐producing T‐cell subsets from peripheral blood mononuclear cells (PBMC). The percentages of IFN‐γ‐producing CD4⁺ and CD8⁺ T cells from atopic asthmatic children were decreased, but those in nonatopic asthmatic children were not decreased. In both groups of asthmatic children, the percentages of IFN‐γ‐producing CD4⁺ T cells were inversely correlated with the peripheral blood eosinophils and had a significant correlation with airway responsiveness (PC₂₀). Thus, we found that the mechanism underlying allergic inflammation of nonatopic asthma is not simple a Th1/Th2 cytokine imbalance. Considering the inverse relationship between IFN‐γ‐producing CD4⁺ T cells and eosinophilia or airway hyperresponsiveness, IFN‐γ from CD4⁺ T cells may play an important role in allergic inflammation and airway hyperresponsiveness in asthmatic children.     

Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Th1/2 cytokine and IL-10 production

Acute exacerbations are the major cause of asthma morbidity, mortality, and health-care costs and are difficult to treat and prevent. The majority of asthma exacerbations are associated with rhinovirus (RV) infection, but evidence supporting a causal relationship is weak and mechanisms are poorly understood. We hypothesized that in asthmatic, but not normal, subjects RV infection would induce clinical, physiologic, and pathologic lower airway responses typical of an asthma exacerbation and that these changes would be related to virus replication and impaired T helper 1 (Th1)/IL-10 or augmented Th2 immune responses. We investigated physiologic, virologic, and immunopathologic responses to experimental RV infection in blood, induced sputum, and bronchial lavage in 10 asthmatic and 15 normal volunteers. RV infection induced significantly greater lower respiratory symptoms and lung function impairment and increases in bronchial hyperreactivity and eosinophilic lower airway inflammation in asthmatic compared with normal subjects. In asthmatic, but not normal, subjects virus load was significantly related to lower respiratory symptoms, bronchial hyperreactivity, and reductions in blood total and CD8⁺ lymphocytes; lung function impairment was significantly related to neutrophilic and eosinophilic lower airway inflammation. The same virologic and clinical outcomes were strongly related to deficient IFN-γ and IL-10 responses and to augmented IL-4, IL-5, and IL-13 responses. This study demonstrates increased RV-induced clinical illness severity in asthmatic compared with normal subjects, provides evidence of strong relationships between virus load, lower airway virus-induced inflammation and asthma exacerbation severity, and indicates augmented Th2 or impaired Th1 or IL-10 immunity are likely important mechanisms.     

A role for mitogen kinase kinase 3 in pulmonary inflammation validated from a proteomic approach

Proteomics is a powerful tool to ascertain which proteins are differentially expressed in the context of disease. We have used this approach on inflammatory cells obtained from patients with asthma to ascertain whether novel drugs targets could be illuminated and to investigate the role of any such target in a range of in vitro and in vivo models of inflammation.A proteomic study was undertaken using peripheral blood mononuclear cells from mild asthmatic subjects compared with healthy subjects. The analysis revealed an increased expression of the intracellular kinase, mitogen activated protein kinase (MKK3), and the function of this protein was investigated further in preclinical models of inflammation using MKK3 knockout mice.We describe a 3.65 fold increase in the expression of MKK3 in CD8⁺ T lymphocytes obtained from subjects with asthma compared with healthy subjects using a proteomic approach which we have confirmed in CD8⁺, but not in CD4⁺ T lymphocytes or human bronchial epithelial cells from asthmatic patients using a Western blot technique. In wild type mice, bacterial lipopolysaccharide (LPS) caused a significant increase in MKK3 expression and significantly reduced airway neutrophilia in MKK3^−/− mice (median, 25, 75% percentile; wild/LPS; 5.3 (0.7–9.9) × 10⁵ cells/mL vs MKK3^−/−/LPS; 0 (0–1.9) × 10⁵ cells/mL, P < 0.05). In contrast, eosinophilia in sensitized wild type mice challenged with allergen (0.5 (0.16–0.65) × 10⁵ cells/mL) was significantly increased in MKK3^−/− mice (2.2 (0.9–3.5) × 10⁵ cells/mL, P < 0.05).Our results suggest that asthma is associated with MKK3 over-expression in CD8⁺ cells. We have also demonstrated that MKK3 may be critical for airway neutrophilia, but not eosinophilia, suggesting that this may be a target worthy of further consideration in the context of diseases associated with neutrophil activation such as severe asthma and COPD.     

Recent advances in the role of NKT cells in allergic diseases and asthma

Asthma is the result of chronic airway inflammation that is dominated by the presence of eosinophils and CD4⁺ T lymphocytes. CD4⁺ T cells include several subsets and play a critical role in orchestrating the inflammation, predominantly by secreting interleukin-4 and interleukin-13. Recently, research identified a new subset of T cells, natural killer T (NKT) cells, which also express the CD4 marker. In contrast to conventional CD4⁺ T cells, NKT cells do not respond to peptide antigens, but rather to glycolipids. In animal models of asthma, direct activation of NKT cells by glycolipids results in the secretion of extensive amounts of cytokines and triggers the development of airway hyperreactivity. Moreover, in patients with chronic asthma, NKT cells can be found in bronchoalveolar lavage fluids in significant amounts. These data strongly suggest that NKT cells play an important role in asthma pathogenesis.     

Modèle expérimental de dermatite atopique

Skin lesions in the allergic form of atopic dermatitis (AD) are induced by allergen-specific T cells which infiltrate the skin at the site of allergen exposure. The pathophysiology of atopic dermatitis is not entirely defined. Although Th2-type CD4⁺ T cells appear to be crucial in AD pathophysiology, little is known about the contribution of CD8⁺ T cells in the development of the allergic skin inflammation. In the present study, we have developed a mouse model of allergen-induced AD and we have analyzed the respective roles of CD8⁺ and CD4⁺ T cells in the development of AD skin lesions. In sensitized mice, CD8⁺ T cells are rapidly and transiently recruited to the allergen-exposed site and initiate the inflammatory process, leading to skin infiltration with eosinophils and Th1/Th2 producing cells. CD8⁺ T cell-depleted mice show no inflammation, demonstrating that these cells are mandatory for the development of AD. In contrast, CD4⁺ T cell-depleted mice develop a severe form of eczema. Furthermore, adoptive transfer of CD8⁺ T cells from sensitized mice into naive recipient mice leads to skin inflammation soon after allergen exposure. These data indicate that allergen-primed CD8⁺ T cells are required for the development of AD-like lesions in mice. Ongoing studies may allow us to confirm these findings in humans.     

Effect of Acute Stress on Immune Cell Counts and the Expression of Tight Junction Proteins in the Duodenal Mucosa of Rats

Background/AimsDuodenal immune alterations have been reported in a subset of patients with functional dyspepsia (FD). The aim of this study was to investigate the effect of acute stress on immune cell counts and the expression of tight junction proteins in the duodenal mucosa.MethodsTwenty-one male rats were divided into the following three experimental groups: 1) the nonstressed, control group, 2) the 2-hour-stressed group, and 3) the 4-hour-stressed group. Eosinophils, mast cells and CD4⁺ and CD8⁺ T lymphocytes in the duodenal mucosa were counted. The protein and mRNA expressions of occludin and zonula occludens-1 (ZO-1) were examined.ResultsEosinophils, mast cells and CD8⁺ T lymphocyte counts did not differ between the stressed and control groups. The number of CD4⁺ T lymphocytes and the protein and mRNA expressions of occludin and ZO-1 were significantly lower in the 4-hour-stressed group compared with the control group. The plasma adrenocorticotrophic hormone and cortisol levels of the 4-hour-stressed group were significantly higher than those of the control group.ConclusionsAcute stress reduces the number of CD4⁺ T lymphocytes and the expression of tight junction proteins in the duodenal mucosa, which might be associated with the duodenal immune alterations found in a subset of FD patients.     

支气管哮喘患者痰白细胞介素16的测定及其意义

目的探讨白细胞介素１６（ＩＬ１６）在支气管哮喘发病机制中的作用。方法收集１２例过敏性哮喘患者、８例非过敏性哮喘患者、１０例过敏性体质患者和１０名正常对照者的痰液,以酶联免疫吸附测定法测定痰中ＩＬ１６的含量,并以免疫染色技术检测ＣＤ＋４细胞和活化嗜酸细胞（ＥＧ２细胞）数。结果哮喘患者无论是过敏性还是非过敏性,痰液中ＩＬ１６水平与过敏体质但无哮喘的患者和正常组比较,差异有显著性（Ｐ＜０．０１）。急性发作期哮喘患者经治疗后ＩＬ１６水平能达缓解期水平。此外,痰液中ＣＤ＋４细胞和ＩＬ１６水平比较呈显著正相关（ｒ＝０．７６,Ｐ＜０．０１）,痰液中ＥＧ２细胞数与ＩＬ１６水平呈显著正相关（ｒ＝０．６１,Ｐ＜０．０１）。结论ＩＬ１６参与了哮喘的发病过程,其机制可能是通过募集ＣＤ＋４Ｔ细胞从而在ＣＤ＋４Ｔ细胞所介导的哮喘气道嗜酸细胞炎症反应中发挥作用。     

Magnesium Affects the Cytokine Secretion of CD4+ T Lymphocytes in Acute Asthma

Introduction. Magnesium (Mg) administration has been shown to promote bronchodilation and to improve lung function in asthma. It also plays an additional role in modulating the immune responses. This study was initiated to explore if Mg supplementation could affect the secretion of cytokines in acute asthmatic CD4⁺ T cells. Methods. Total serum Mg concentrations of the acute asthmatic patients and healthy controls were determined. CD4⁺ T cells were isolated from the blood of the acute asthmatic patients. They were cultured in various concentrations of Mg-supplemented (0.8, 5, 10, 15, and 20 mmol/l) medium. Cytokine (IL-5, IL-13, and IFN-γ) levels were determined by Enzyme-Linked Immunosorbnent Assay (ELISA). Results. Serum Mg concentration was lower in the acute asthmatic patients than that in the healthy controls (p < .05). The secretion of IL-5 and IL-13 was decreased, while the acute asthmatic CD4⁺ T cells were cultured in 10 and 15 mmol/l Mg-supplemented medium, respectively, as compared to the 0.8 mmol/l Mg group (p < .05). The secretion of IFN-γ increased in the 10 mmol/l Mg group (p < .05). Conclusion. Mg supplementation was able to modulate the immune responses of acute asthmatic CD4⁺ T cells and decrease the secretion of type 2 CD4⁺ T lymphocytes cytokines.     

Asthma death, CD8+ T cells, and viruses

Despite advances in the understanding of the pathophysiology of asthma and the availability of effective treatment, the World Health Organization estimates that asthma accounts for 1 in every 250 deaths worldwide. Viruses are associated with half of all asthma exacerbations. The immune response to viral infection may enhance preexisting airway inflammation via the release of chemokines and cytokines and local recruitment of inflammatory cells. Murine models have provided evidence for a deleterious role for CD8+ T cells in the context of respiratory viral infection. Passive transfer of respiratory syncytial virus-specific cytotoxic T lymphocytes (CTLs) to infected mice results in virus clearance, which is associated with acute and sometimes fatal pulmonary disease. Compared with control subjects, CD8+ cells appear to be preferentially sequestered in the airways of individuals with asthma during acute exacerbations. In addition, an expanded CD8+ T cell population, dominated by activated cytotoxic CD8+ lymphocytes, has been documented in biopsies from patients dying as a result of acute asthma in association with a viral infection. Undoubtedly, CD8+ CTLs are a crucial in cell-mediated immunity in response to respiratory viruses. However, it would appear that an aberrant CD8+ T cell response in the context of a viral infection may place individuals with asthma at risk for fatal asthma.     

Immunological milieu in the peritoneal cavity at laparotomy for gastric cancer

AIM: To investigate the immunological repertoire in the peritoneal cavity of gastric cancer patients.METHODS: The peritoneal cavity is a compartment in which immunological host-tumor interactions can occur. However, the role of lymphocytes in the peritoneal cavity of gastric cancer patients is unclear. We observed 64 patients who underwent gastrectomy for gastric cancer and 11 patients who underwent laparoscopic cholecystectomy for gallstones and acted as controls. Lymphocytes isolated from both peripheral blood and peritoneal lavage were analyzed for surface markers of lymphocytes and their cytokine production by flow cytometry. CD4⁺CD25^high T cells isolated from the patient’s peripheral blood were co-cultivated for 4 d with the intra-peritoneal lymphocytes, and a cytokine assay was performed.RESULTS: At gastrectomy, CCR7^- CD45RA^- CD8⁺ effector memory T cells were observed in the peritoneal cavity. The frequency of CD4⁺ CD25 ^high T cells in both the peripheral blood and peritoneal cavity was elevated in patients at advanced stage [control vs stage IV in the peripheral blood: 6.89 (3.39-10.4) vs 15.34 (11.37-19.31), P < 0.05, control vs stage IV in the peritoneal cavity: 8.65 (5.28-12.0) vs 19.56 (14.81-24.32), P < 0.05]. On the other hand, the suppression was restored with CD4⁺ CD25^high T cells from their own peripheral blood. This study is the first to analyze lymphocyte and cytokine production in the peritoneal cavity in patients with gastric cancer. Immune regulation at advanced stage is reversible at the point of gastrectomy.CONCLUSION: The immunological milieu in the peritoneal cavity of patients with advanced gastric cancer elicited a Th2 response even at gastrectomy, but this response was reversible.     

Circulating activated innate lymphoid cells and mucosal-associated invariant T cells are associated with airflow limitation in patients with asthma

BackgroundA variety of innate subsets of lymphoid cells such as natural killer (NK) cells, several populations of innate lymphoid cells (ILCs), and mucosal-associated invariant T (MAIT) cells as innate-like T lymphocytes are involved in asthma and may have important effector functions in asthmatic immune responses. In the present study, we investigated whether NK cells, ILCs, and MAIT cells in the peripheral blood of patients with asthma would be associated with clinical asthma parameters.MethodsWe recruited 75 adult patients with mild to severe asthma. The peripheral blood mononuclear cells in peripheral venous blood samples from the patients were purified and stained with different combinations of appropriate antibodies. The cells were analyzed by flow cytometry.ResultsThe percentage of activated (i.e., CD69⁺) NK cells in the total NK cell population was negatively correlated with FEV₁% which is calculated by the forced expiratory volume in 1 s (FEV₁)/the forced vital capacity (FVC). The percentages of CD69⁺ ILC1s and ILC2s were negatively correlated with FEV₁% and %FEV₁. The percentage of CD69⁺ ILC3s was positively correlated with BMI, and the percentage of CD69⁺ MAIT cells was negatively correlated with FEV₁%. Moreover, the percentage of CD69⁺ NK cells, ILC1s, ILC2s, ILC3s, and MAIT cells were positively correlated with each other.ConclusionsFor the first time, our data showed that activated NK cells, ILC1s, ILC2s, ILC3s, and MAIT cells were positively correlated with each other and may be associated with airflow limitation in patients with asthma.     

支气管哮喘儿童急性发作期诱导痰液T细胞亚群及自然杀伤T细胞的变化

目的 分析支气管哮喘(简称哮喘)儿童急性发作期及缓解期诱导痰液T细胞亚群及自然杀伤T细胞(NKT细胞)的变化,探讨T细胞及NKT细胞在儿童哮喘气道炎症中的作用.方法 选取18例哮喘急性发作、21例哮喘缓解期及12名正常儿童的痰液,采用流式细胞术比较各组儿童诱导痰液CD3、CD4、CD8、NKT(CD3~+ CD56~+)百分比及CD4/CD8比值.结果 哮喘急性发作组CD4细胞百分比[(43.75±13.5)%]明显高于缓解期组[(37.04±7.11)%]和正常对照组[(33.57±7.54)%](P＜0.05),CD8细胞百分比[(21.10±6.10)%]明显低于缓解期组[(28.67±5.32)%]和正常对照组[(28.31±9.46)%](P<0.05),CD4/CD8比值(2.14±0.94)高于缓解期组(1.33±0.35)和正常对照组(1.31±0.42)(P＜0.05),CD4、CD8细胞百分比及CD4/CD8缓解期组和正常对照组差异无统计学意义(P>0.05),急性发作组CD4/CD8比值与嗜酸粒细胞百分比呈正相关(r=0.559,P＜0.05).缓解期组CD4/CD8比值与嗜酸粒细胞无相关性(r=0.398,P>0.05).急性发作组CD3+ CD56+细胞百分比[(3.33±1.69)%]略高于缓解组[(3.09±1.23)%]及对照组[(2.94±0.87)%],但差异无统计学意义(P>0.05).CD3- CD56+细胞百分比在三组之间无统计学差异(P>0.05).结论 儿童哮喘急性发作期,气道内CD4细胞占优势,CD4/CD8细胞比例失衡,CD4细胞可能介导儿童哮喘气道炎症过程.     

CD8+ T Cells of Tc2 Phenotype Mediate a GVL Effect and Prevent Marrow Rejection

Donor T cells mediate both beneficial and detrimental immune reactions in the setting of allogeneic BMT (alloBMT). T cells mediate the GVL effect and prevent marrow rejection, but also induce GVHD. In an attempt to favorably influence the balance of these allogeneic responses, we have evaluated the effect of donor CD4⁺, Thl/Th2 and CD8⁺, Tcl/Tc2 functional T cell subsets in murine marrow transplantation models. Our studies have identified the CD8⁺Tc2 population (which is a cytolytic effector secreting the type II cytokines IL-4, IL-5, and IL-10) as a subset capable of mediating the GVL effect and preventing marrow rejection with reduced GVHD. We have also shown that the Tc2 subset can be generated in humans. These studies indicate that administration of donor CD8⁺ T cells of Tc2 phenotype represents a strategy for improving alloBMT outcome.     

Les lymphocytes TH17 : différenciation, phénotype, fonctions, et implications en pathologie et thérapeutique humaine

Differentiation of naive CD4⁺ T helper (T_H) cells is a major step of the adaptative immune response. When activated by pathogens in a specific cytokine environment, CD4⁺ T cells differentiate into different subsets of T_H cells with specific effector functions. T_H1 lymphocytes orchestrate cellular immune response by producing interferon-γ and stimulating cytotoxic cells whereas T_H2 cells orchestrate humoral immune response by producing interleukin-4 (IL-4), IL-5 and IL-10, leading to immunoglobulin production. Conversely, regulatory T cells (Treg) are capable of inhibiting immune response. Recently discovered, T_H17 cells are characterized by their ability to produce IL-17 and play an important role in anti-infectious and inflammatory immune responses. This review focuses on present knowledge about T_H17 cells: their induction, phenotype, functions, implications in host defense and human disease, and their potential to represent possible therapeutic targets.     

Peripheral killer cells do not differentiate between asthma patients with or without fixed airway obstruction

Objective: The three main types of killer cells – CD8⁺ T cells, NK cells and NKT cells – have been linked to asthma and chronic obstructive pulmonary disease (COPD). However, their role in a small subset of asthma patients displaying fixed airway obstruction (FAO), similar to that seen in COPD, has not been explored. The objective of the present study was to investigate killer cell numbers, phenotype and function in peripheral blood from asthma patients with FAO, asthma patients without FAO, and healthy individuals. Methods: Peripheral CD8⁺ T cells (CD8⁺CD3⁺CD56^?), NK cells (CD56⁺CD3^?) and NKT-like cells (CD56⁺CD3⁺) of 14 asthma patients with FAO (post-bronchodilator FEV/FVC <0.7, despite clinician-optimised treatment), 7 asthma patients without FAO (post-bronchodilator FEV/FVC ≥ 0.7), and 9 healthy individuals were studied. Results: No significant differences were seen between the number, receptor expression, MAPK signalling molecule expression, cytotoxic mediator expression, and functional cytotoxicity of peripheral killer cells from asthma patients with FAO, asthma patients without FAO and healthy individuals. Conclusions: Peripheral killer cell numbers or functions do not differentiate between asthma patients with or without fixed airway obstruction.