CXCR3 and CCR5 chemokines in induced sputum from patients with COPD

BACKGROUND: COPD is associated with increased numbers of CD4(+) and CD8(+) lymphocytes and macrophages in the small airways and lung parenchyma. The chemokines regulating T-cell recruitment into the lung are unknown but may involve CXCR3 and CCR5 chemoattractants. The aims of this study were to determine the concentrations of CXCR3 chemokines CXCL9, CXCL10, CXCL11, and the CCR5 chemokine CCL5 in induced sputum from patients with COPD, smokers, and nonsmokers, and to examine the relationship between chemokine expression, inflammatory cells, and airway obstruction. METHODS: Differential cell counts were performed and concentrations of CXCL9, CXCL10, CXCL11, and CCL5 were measured in induced sputum from nonsmokers (n = 18), smokers (n = 20), and COPD patients (n = 35) using an enzyme-linked immunosorbent assay. RESULTS: Concentrations of CXCL9, CXCL10, CXCL11, and CCL5 were significantly increased in the sputum of patients with COPD when compared with nonsmokers but not smokers without obstruction: CXCL9 (median, 14.3 pg/mL; interquartile range [IQR], 6.5 to 99.3; vs median, 1.4 pg/mL; IQR, 0 to 10.4 [p < 0.001]; vs 8.5 pg/mL; IQR, 0 to 16.0, respectively); CXCL10 (16.9 pg/mL; IQR, 6.2 to 148.8; vs 3.7 pg/mL; IQR, 0 to 18.8 [p < 0.05]; vs 11.3 pg/mL; IQR, 3.7 to 46.7); CXCL11 (58.1 pg/mL; IQR, 34.5 to 85.3; vs 33.5 pg/mL; IQR, 23.2 to 49.7 [p < 0.05]; vs 49.8 pg/mL; IQR, 32.6 to 105.6); and CCL5 (59.9 pg/mL; IQR, 57.1 to 67.8; vs 33.5 pg/mL; IQR, 31.6 to 36.9 [p < 0.001]). CCL5 in sputum from smokers was also significantly increased compared with that from nonsmokers (median, 63.0 pg/mL; IQR, 60.8 to70.2; p < 0.001). There was a negative correlation between FEV(1) percentage of predicted, FEV(1)/FVC ratio, and percentage of macrophages, and all the chemokines analyzed. Neutrophil numbers correlated positively with the concentrations of chemokines. CONCLUSIONS: CXCR3 chemokines and CCL5 are increased in sputum from COPD patients compared with nonsmokers, and may be important in COPD pathogenesis.     

Induced sputum CD8+ T-lymphocyte subpopulations in chronic obstructive pulmonary disease

BACKGROUND: Previous studies have shown that the inflammatory response to cigarette smoking differs between smokers who develop chronic obstructive pulmonary disease (COPD) and those who do not and that the CD8+ T-lymphocytes have been identified as a key player in this process. The aim of this study was to investigate further the role of CD8+ cells and their subtypes in sputum cells. METHODS: Sputum induction was performed in 36 COPD patients, 25 smokers without COPD and 10 non-smoking healthy controls. After stimulation of sputum lymphocytes with phorbol-myristate-acetate, we used double immunocytochemical methods to identify CD4+, CD8+ cells and CD8+ INFgamma or IL4 cells (Tc1,Tc2). RESULTS: COPD patients had an increased number of CD8+ cells in sputum as compared with smokers without COPD (P = 0.0001) and control subjects (P = 0.001). CD8+-IL4 cells were reduced both in COPD and in smokers without COPD compared to controls (P = 0.0001), while CD8+-IFNgamma cells were significantly reduced only in COPD (P = 0.001) as compared with controls. A significant (P = 0.02) relationship between the CD8+-IL4/CD8+-IFNgamma ratio and FEV1 (% pred) was found only in COPD patients. CONCLUSION: These findings suggest that an imbalance both in T-lymphocyte subpopulation (CD4/CD8) and in CD8+ cell subsets (Tc1/Tc2) characterizes the inflammatory responses of smokers with established COPD.     

CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease.

Previous studies have shown an increased number of inflammatory cells and, in particular, CD8+ve cells in the airways of smokers with chronic obstructive pulmonary disease (COPD). In this study we investigated whether a similar inflammatory process is also present in the lungs, and particularly in lung parenchyma and pulmonary arteries. We examined surgical specimens from three groups of subjects undergoing lung resection for localized pulmonary lesions: nonsmokers (n = 8), asymptomatic smokers with normal lung function (n = 6), and smokers with COPD (n = 10). Alveolar walls and pulmonary arteries were examined with immunohistochemical methods to identify neutrophils, eosinophils, mast cells, macrophages, and CD4+ve and CD8+ve cells. Smokers with COPD had an increased number of CD8+ve cells in both lung parenchyma (p < 0.05) and pulmonary arteries (p < 0.001) as compared with nonsmokers. CD8+ve cells were also increased in pulmonary arteries of smokers with COPD as compared with smokers with normal lung function (p < 0.01). Other inflammatory cells were no different among the three groups. The number of CD8+ve cells in both lung parenchyma and pulmonary arteries was significantly correlated with the degree of airflow limitation in smokers. These results show that an inflammatory process similar to that present in the conducting airways is also present in lung parenchyma and pulmonary arteries of smokers with COPD.     

Perforin down-regulation and adhesion molecules activation in pulmonary sarcoidosis: an induced sputum and BAL study

BACKGROUND: Sarcoidosis is thought to be a T-helper type 1 cytokine-mediated disorder. Sputum induction has been proposed as a useful noninvasive method mainly for the assessment of airway diseases. However, it is unknown whether the balance of T-cytotoxic (Tc1) type 1 and Tc2 cells is altered in sarcoidosis. STUDY OBJECTIVES: The primary aim of this study was to characterize the CD8+ T lymphocyte subpopulations in induced sputum from sarcoidosis patients, and to compare these subpopulations to those found in BAL fluid (BALF) from sarcoidosis patients. To further investigate the mechanism of the cytotoxic activity of CD8+ lymphocytes, we measured their perforin expression. Additionally, two adhesion molecules (CD62 and CD71), which are expressed on CD8+ T cells and may serve as novel immunologic markers, were detected. Settings: Department of Thoracic Medicine, University of Crete, and Department of Pneumonology, Democritus University of Thrace, Alexandroupolis, Greece. PATIENTS: We prospectively studied 22 patients with sarcoidosis (median age, 48 years; age range, 25 to 65 years) and 10 healthy subjects (5 female and 5 male; median age, 39 years; age range, 26 to 60 years). INTERVENTIONS: The stimulation of lymphocytes with phorbol 12-myristate 13-acetate was followed by the use of double immunocytochemical methods to identify CD8+ interferon (IFN)-gamma producing cells (ie, Tc1) and CD8+ interleukin-4 producing cells (ie, Tc2). MEASUREMENTS AND RESULTS: We found a significant decrease in the prestimulation percentage of IFN-gamma-positive CD8+ T cells in the BALF (p = 0.001) and induced sputum (p = 0.001) of sarcoidosis patients compared to the number in samples from healthy control subjects. However, no significant difference was documented between lymphocyte subsets poststimulation. Decreased levels of perforin expression were found in BALF (p = 0.001) and induced sputum (p < 0.001) of sarcoidosis patients compared to those in control subjects. The adhesion molecules were significantly increased in both the BALF and induced sputum of the sarcoid population compared to those in healthy control subjects. CONCLUSIONS: Our results suggest that inflammation could be effectively and noninvasively determined by using sputum induction in sarcoidosis patients. In addition, we have provided evidence suggesting the possibility that CD8+ lymphocytes might not play a major role in sarcoidosis.     

Increase in perforin-positive peripheral blood lymphocytes in extrinsic and intrinsic asthma

The cause of asthma, which has been linked to a chronic, T-cell-mediated bronchial inflammation, remains unclear. A number of other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with autoimmunity and there are data indicating that autoimmune phenomena might also be present in asthma. Expression of perforin, a cytotoxic molecule produced by lymphocytes, has been implicated in the pathogenesis of autoimmune diseases. We therefore tested the hypothesis that allergic and intrinsic asthma might be associated with an increase in lymphocytes producing perforin by comparing the expression of intracellular perforin in peripheral blood lymphocytes of patients with extrinsic asthma (n = 13), intrinsic asthma (n = 7), and healthy control subjects (n = 18). Lymphocytes were identified using flow cytometry and subdivided into CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) subpopulations after staining with appropriate monoclonal antibodies. The percentage of perforin-positive total lymphocytes was significantly elevated in patients with allergic as well as intrinsic asthma when compared with normal control subjects. Analysis of lymphocyte subpopulations also revealed a significant increase in the percentage of CD3(+), CD4(+), CD8(+), and CD56(+) cells expressing perforin in allergic asthma and a significant increase in the percentage of CD4(+) and CD56(+) cells in intrinsic asthma when compared with healthy control subjects. Perforin expression in CD4(+) cells in intrinsic asthma was also significantly elevated compared with allergic asthma. We conclude that allergic and intrinsic asthma is associated with increased expression of perforin in T-lymphocyte subsets.     

Increase in perforin-positive peripheral blood lymphocytes in extrinsic and intrinsic asthma

The cause of asthma which has been linked to a chronic, T-cell-mediated bronchial inflammation, remains unclear. A number of other T-lymphocyte-mediated, chronic inflammatory disorders have been associated with autoimmunity and there are data indicating that autoimmune phenomena might also be present in asthma. Expression of perforin, a cytotoxic molecule produced by lymphocytes, has been implicated in the pathogenesis of autoimmune disease. We therefore tested the hypothesis that allergic and intrinsic asthma might be associated with an increase in lymphocytes producing perforin by comparing the expression of intracellular perforin in peripheral blood lymphocytes of patients with extrinsic asthma (n = 13), intrinsic asthma (n = 7), and healthy (control subjects (n = 18). Lymphocytes were identified using flow cytometry and subdivided into CD3(+), CD4(+), CD8(+), CD16(+), and CD56(+) subpopulations after staining with appropriate monoclonal antibodies. The percentage of perforin-positive total lymphocytes as significantly elevated in patients with allergic as well as intrinsic asthma when compared with normal control subjects. Analysis of lymphocyte subpopulations also revealed a significant increase in the percentage of CD3(+), CD4(+), CD8(+), and CD56(+) cells expressing perforin in allergic asthma and a significant increase in the percentage of CD4(+), and CD56(+) cells in intrinsic asthma when compare with healthy control subjects. Perforin expression in CD4(+) cells in intrinsic asthma was also significantly elevated compared with allergic asthma. We conclude that allergic and intrinsic asthma is associated with increased expression of perforin in T-lymphocyte subsets.     

Enhanced cytotoxic function of natural killer and natural killer T‐like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway

Background and objective: Natural killer (NK) and natural killer T (NKT)‐like cells represent a small but important proportion of effector lymphocytes that we have previously shown to be major sources of pro‐inflammatory cytokines and granzymes. We hypothesized that these cells would be increased in the airway in chronic obstructive pulmonary disease (COPD), accompanied by reduced expression of the inhibitory receptor CD94 (Kp43) and increased expression of cytotoxic mediators granzyme B and perforin. Methods: We measured NK and NKT‐like cells and their expression of CD94 in the blood of COPD patients (n = 71; 30 current and 41 ex‐smokers), smokers (16) and healthy controls (25), and bronchoalveolar lavage fluid (BALF) from a cohort of subjects (19 controls, 12 smokers, 33 COPD). Activation was assessed by measuring CD69 in blood and the cytotoxic potential of NK cells by measuring granzymes A and B, and using a cytotoxicity assay in blood and BALF. Results: In blood in COPD, there were no significant changes in the proportion of NK or NKT‐like cells or expression of granzyme A or NK cytotoxic potential versus controls. There was, however, increased expression of granzyme B and decreased expression of CD94 by both cell types versus controls. The proportion of NK and NKT‐like cells were increased in BALF in COPD, associated with increased NK cytotoxicity, increased expression of granzyme B and decreased expression of the inhibitory receptor CD94 by both cell types. Conclusions: Treatment strategies that target NK and NKT‐like cells, their cytotoxicity and production of inflammatory mediators in the airway may improve COPD morbidity.     

Interleukin-10 level in sputum is reduced in bronchial asthma, COPD and in smokers.

Interleukin (IL)-10 is a potent regulatory cytokine that decreases inflammatory responses. This study investigated whether IL-10 levels in the airway are decreased in chronic airway inflammation associated with asthma or chronic obstructive pulmonary disease (COPD). Sputum was obtained from 12 healthy nonsmokers, 10 healthy smokers, 16 asthmatic patients and seven patients with COPD by means of the sputum-induction method. The IL-10 level was measured via enzyme-linked immunosorbent assay and immunocytochemical analysis. The IL-10 level in sputum was significantly lower in asthma and COPD patients and healthy smokers compared with that in healthy nonsmokers (nonsmokers, 68.0+/-11.3; smokers, 45.3+/-7.8; asthma, 26.7+/-4.0; COPD, 18.0+/-2.3 pg x mL(-1); p<0.05 for nonsmokers versus the other groups). The percentage of IL-10-positive cells in the sputum was also significantly lower in asthma and COPD and in smokers (nonsmokers, 13.2+/-1.7; smokers, 6.4+/-1.8; asthma, 5.4+/-3.5; COPD, 3.5+/-1.6%; p<0.05 for nonsmokers versus the other groups). The IL-10-positive cell appeared morphologically to be the macrophage. These data suggest that the reduced level of interleukin-10 within the airways plays a role in the pathogenesis of chronic airway inflammation in asthma and chronic obstructive pulmonary disease.     

Sputum substance P and neurokinin A are reduced during exacerbations of chronic obstructive pulmonary disease

Involvement of tachykinins in airway inflammation has been demonstrated in animal models, but evidence in humans is sparse. The aim of this study was to quantify the levels of substance P and neurokinin A in induced sputum of patients with chronic obstructive pulmonary disease (COPD) and to compare them with the levels in smokers with normal lung function and healthy nonsmokers. Content of tackykinins was measured in 12 sputum samples collected during stable condition and nine sputum samples collected during exacerbations from 13 COPD patients, in eight sputum samples from smokers with normal lung function and in nine from healthy nonsmokers. Patients with COPD exacerbations had a lower sputum content of substance P compared with the other 3 groups (p<0.05). No differences were found between patients with stable COPD, smokers with normal lung function, and nonsmokers. Sputum levels of neurokinin A were trending in the same direction of substance P, but the significant difference was reached for the paired sputum samples collected from the same COPD patients (n=8) during exacerbation and in stable condition. COPD exacerbations are associated with a reduced sputum content of substance P and neurokinin A. These tackykinins might be involved in COPD exacerbations.     

Increased expression of interleukin-18 and its receptor in peripheral blood of patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a complex systemic disorder characterized by both local pulmonary and systemic inflammation. Many studies suggested that activation of circulating inflammatory cells and increased circulating levels of inflammatory cytokines occur in COPD. Interleukin (IL)-18 is a unique proinflammatory cytokine that mediates its effects by binding to the IL-18 receptor (IL-18R). In the present study, the expression of IL-18 in serum and IL-18R on peripheral blood T lymphocytes was analyzed. Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum levels of IL-18 and interferon (IFN)-γ, and high sensitivity C-reactive protein (hsCRP) were measured by chemiluminiscent immunoassay. Expression of IL-18R was examined using a three-color flow cytometry method. In total, 120 subjects were recruited including 32 nonsmokers, 30 current smokers and 58 stable COPD patients. Serum levels of IL-18 and hsCRP were significantly higher in stable COPD patients than those in nonsmokers and current smokers. A significant negative correlation existed between pulmonary function and serum level of IL-18 rather than hsCRP in stable COPD patients. The proportions of IL-18Rα-expressing T lymphocytes and CD8(+) T lymphocytes were significantly higher in stable COPD patients than in nonsmokers and current smokers. The current study extended prior analyses by examining IL-18R expression in peripheral blood. The results suggested that IL-18/IL-18R system was active in peripheral blood of COPD patients.     

Tc2 response at the onset of COPD exacerbations

BACKGROUND: T lymphocytes and especially the subpopulations of CD8+ cells are believed to have a key role in COPD pathophysiology, but there are only few data regarding the role of these cells in COPD exacerbation. Aim: We aimed to study prospectively changes of CD8+ T-lymphocyte subpopulations in the sputum of COPD patients at the onset of mild exacerbations and at a stable condition in order to provide further insight in the pathophysiology of the disease. METHODS: Induced-sputum samples were collected from 24 COPD patients with median age of 52 years (interquartile range [IQR], 44 to 58 years) and FEV(1) percentage of predicted of 78.05% (IQR, 75.8 to 80.1%) at the onset of mild exacerbations not requiring hospitalization and when stable. Inflammatory cells and T-lymphocyte subpopulations (CD4+, CD8+, and cells producing interferon [IFN]-gamma or interleukin [IL]-4) were measured using flow cytometry and immunocytochemical methods. RESULTS: A significant increase in sputum CD8+ T lymphocytes (p < 0.0001) and significant decreases in CD4+ T lymphocytes as well as in CD4+/CD8+ (p = 0.0001) and CD8+IFN-gamma+/CD8+IL-4+ (p = 0.001), CD4+IFN-gamma+/CD4+IL-4+ (p = 0.0003) sputum cells ratios were found decreased at the onset of exacerbations compared to stable condition. The changes in T-lymphocyte subpopulations were not associated with smoking history, demographic characteristics, or disease severity. CONCLUSION: The findings of the present study suggest that CD8+ lymphocytes are increased and potentially polarized toward a Tc2 profile in the airways of COPD patients at the onset of COPD exacerbations with respect to stable condition. The clinical impact of the observed phenomenon requires further investigation.     

Blunted gamma delta T-lymphocyte response in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterised by an excessive inflammatory response to inhaled particles, mostly tobacco smoking. Although inflammation is present in all smokers, only a percentage of them develop COPD. T-lymphocytes are important effector and regulatory cells that participate actively in the inflammatory response of COPD. They comprise the T-cell receptor (TCR)-alpha beta (CD4+ and CD8+) and TCR-gamma delta T-lymphocytes. The latter represent a small percentage of the total T-cell population, but play a key role in tissue repair and mucosal homeostasis. To investigate TCR-alpha beta (CD4+ and CD8+) and TCR-gamma delta T-lymphocytes in COPD, the present authors determined, by flow cytometry, the distribution of both subpopulations in peripheral blood and bronchoalveolar lavage (BAL) samples obtained from patients with COPD, smokers with normal lung function and never-smokers. The present study found that: 1) the distribution of CD4+ and CD8+ lymphocytes in blood and BAL was similar in all three groups; 2) compared with nonsmokers, gamma delta T-lymphocytes were significantly increased in smokers with preserved lung function; and 3) this response was blunted in patients with COPD. These results highlight a novel, potentially relevant, pathogenic mechanism in chronic obstructive pulmonary disease.     

Abnormal peripheral blood T-lymphocyte subsets in a subgroup of patients with COPD

BACKGROUND: Smoking may induce changes in T-lymphocyte subsets in peripheral blood. Abnormalities of T-lymphocyte subsets in peripheral blood and in BAL fluid, and increased CD8+ T lymphocytes in the airways have been reported in patients with COPD. These findings suggest that T-lymphocyte abnormalities might be involved in the pathogenesis of airflow limitation in people who smoke. DESIGN: Cross-sectional study. SETTING: Outpatient pulmonary department of a university hospital. METHODS: To investigate this hypothesis, peripheral blood T-lymphocyte subsets were measured by flow cytometry using specific monoclonal antibodies in 20 healthy nonsmokers, 20 healthy smokers, and 20 smokers with stable COPD. No significant differences in the peripheral blood T-lymphocyte subsets were observed among the three groups. Because a previous study showed peripheral blood T-lymphocyte abnormalities in the subgroup of nonsmoking patients with COPD, we wanted to investigate what factors determine the subgroup of COPD with abnormal T-lymphocyte subsets. We tried to measure the relationship between T-lymphocyte subsets and physiologic indexes of pulmonary function tests in patients with COPD. The proportion of CD8+ T lymphocytes significantly correlated with diffusing capacity of the lung for carbon monoxide (DLCO) and DLCO per unit of alveolar volume (DLCO/VA), and CD4+/CD8+ ratio correlated with DLCO/VA. Therefore, we attempted to classify the patients with COPD into two subgroups on the basis of DLCO/VA: 10 COPD patients with low DLCO/VA (< 80% predicted) and 10 patients with normal DLCO/VA (> or = 80% predicted). RESULTS: The normal DLCO/VA subgroup had a significantly higher proportion of CD8+ T lymphocytes and a lower CD4+/CD8+ ratio than the healthy smokers or the low DLCO/VA subgroup. Moreover, FEV1/FVC significantly correlated with the CD4+/CD8+ ratio only in the normal DLCO/VA subgroup. CONCLUSION: These findings suggest that T-lymphocyte abnormalities might be involved in the pathogenesis of airflow limitation in a subgroup of patients with COPD, presumably with small airways disease, but not in all cases of COPD.     

8-Isoprostane as a marker of oxidative stress in nonsymptomatic cigarette smokers and COPD.

8-Isoprostane is a potential in vivo marker for oxidant burden, but its usefulness in induced sputum of smokers and chronic obstructive pulmonary disease (COPD) has not been investigated. The current study investigated 58 subjects comprising 11 never-smokers, 11 ex-smokers, 13 healthy current smokers and 23 COPD with stage 0-III disease (according to the Global Initiative for Chronic Obstructive Lung Disease criteria). 8-Isoprostane was determined from induced sputum by enzyme immunoassay. Sputum 8-isoprostane levels were similar in the never-smokers and ex-smokers, but were elevated in the healthy smokers compared with nonsmokers, and in those with stage I-III COPD. Sputum 8-isoprostane levels could not differentiate nonsymptomatic smokers from those with Stage 0 COPD. There was a correlation between sputum 8-isoprostane level and lung function parameters (forced expiratory volume in one second/forced vital capacity and sputum neutrophils. In conclusion, sputum 8-isoprostane levels correlate with the severity of chronic obstructive pulmonary disease. However, they do not appear to differentiate healthy smokers from those who are at risk of developing chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease stage 0).     

Long-term repeatability of induced sputum cells and inflammatory markers in stable, moderately severe COPD

STUDY OBJECTIVES: Neutrophilic inflammation is a major feature of COPD. Induced sputum is increasingly used to monitor inflammatory airway diseases. Although short-term repeatability of selected sputum markers has been extensively studied in several populations, data on the long-term repeatability of induced sputum markers in stable COPD are scant. DESIGN: Sputum supernatant of 12 patients with stable COPD was analyzed on three separate occasions with 4-weekly intervals. Sputum cells and inflammatory markers interleukin (IL)-8 and soluble intercellular adhesion molecule (sICAM)-1 were measured in supernatant using enzyme-linked immunosorbent assay. Repeatability of sputum markers was expressed by intraclass correlation coefficients (Ri). Measurements and results: Sputum induction was safe in all patients. None of the sputum parameters analyzed changed significantly throughout the study. The repeatability for cell differential counts in stable COPD was as follows: total cells, Ri = 0.07; neutrophils, Ri = 0.66; macrophages, Ri = 0.47; eosinophils, Ri = 0.49; and lymphocytes, Ri = 0.58. The repeatability of soluble markers was as follows: IL-8, Ri = 0.50; and sICAM, Ri = 0.58. Sputum neutrophils were negatively correlated with lung function on each separate occasion, whereas soluble markers were not correlated with sputum cells (p > 0.16, all correlations) or lung function (p > 0.24, all correlations). CONCLUSIONS: Clinically stable, moderate COPD is associated with equally stable sputum inflammatory markers. Repeatability of induced-sputum markers of neutrophilic inflammation in stable COPD is satisfactory, even over extended periods of time. These data support the usefulness of serial monitoring of induced-sputum inflammatory markers in COPD.     

Reduced perforin expression in systemic juvenile idiopathic arthritis is restored by autologous stem-cell transplantation

OBJECTIVES: Familial haemophagocytic lymphohistiocytosis (FHL) is a disorder characterized by deficient cytotoxic T-cell function and activated macrophages, owing to a defect in the perforin gene and absent perforin expression. Because symptoms of patients with systemic juvenile idiopathic arthritis (sJIA) are sometimes clinically very similar to those with FHL, we studied whether perforin expression in sJIA patients would be reduced also. METHODS: We determined the perforin expression levels on two subsets of CD8(+) cells (CD8(+)CD28(-)CD45RA(-) and CD8(+)CD28(-)CD45RA(+)) and natural killer (NK) cells from patients with sJIA under conventional treatment as well as before and after autologous stem-cell transplantation (ASCT). RESULTS: CD45RA(-) cytotoxic effector cells of sJIA patients (n=13) express significantly lower levels of perforin than polyarticular juvenile idiopathic arthritis (pJIA, n=9) patients [sJIA mean fluorescence intensity (MFI) 34.6; pJIA MFI 98.0] or control donors (MFI 124.6, n=5). A similar pattern was seen in the CD45RA(+) subset. Also NK cells from sJIA patients expressed significantly less intracellular perforin (sJIA MFI 398.4; controls MFI 972.4). In four patients with sJIA who were treated with ASCT, a clear increase in perforin expression was found at 12 months after ASCT in both cytotoxic effector cell subsets (CD45RA(-) subset before ASCT MFI 13.2; 12 months after ASCT MFI 172.3). CONCLUSION: We conclude that perforin expression can be severely reduced in sJIA. This finding may implicate defective cytotoxicity and haemophagocytosis and could thus explain why sJIA may be complicated by macrophage activation syndrome. ASCT leads to a reconstitution of the (T cell) immune system with a normal expression of perforin.     

The effects of cigarette smoking on T cell subsets. A population-based survey of healthy caucasians

To investigate the influence of cigarette smoking on mononuclear cell subsets, we determined T cell, B cell, monocyte, and HLA-DR+ subsets in a population-based, stratified, random sample of healthy Caucasians using monoclonal antibodies and flow cytometry. The study population consisted of 282 subjects 20 to 69 yr of age, including 108 smokers and 174 nonsmokers. Multivariate analysis techniques were used to assess the influence of cigarette smoking status after controlling for the effects of age and gender. Cigarette smoking was associated with a nonspecific increase in the leukocyte count involving all major cell types (smokers: 8.50 +/- 0.15 versus nonsmokers: 7.33 +/- 0.12 cells/mm3; p less than or equal to 0.0001). In addition, cigarette smokers had a selective increase in CD4+ cells (helper-inducer T cells) compared with nonsmokers (55.3 +/- 0.8 versus 52.2 +/- 0.6% of lymphoid cells; p = 0.002), resulting in a statistically significant increase in the CD4+/CD8+ (helper/suppressor) ratio (2.42 +/- 0.1 versus 2.13 +/- 0.16; p = 0.02). There was no significant difference between smokers and nonsmokers in the level of CD3+ cells (total T cells: 76.8 +/- 0.7 versus 76.1 +/- 0.5; p = 0.5), CD8+ cells (suppressor-cytotoxic T cells: 25.7 +/- 0.8 versus 27.0 +/- 0.5%; p = 0.1), CD19+ cells (B cells) (10.7 +/- 0.4 versus 10.0 +/- 0.3%; p = 0.2), CD14+ cells (monocytes) (18.0 +/- 0.6 versus 17.0 +/- 0.4%; p = 0.2), or HLA-DR+ cells (14.5 +/- 0.5 versus 14.0 +/- 0.4%; p = 0.4).(ABSTRACT TRUNCATED AT 250 WORDS)     

CD4+NKG2D+ T cells in Crohn's disease mediate inflammatory and cytotoxic responses through MICA interactions

BACKGROUND & AIMS: Crohn's disease (CD) is an inflammatory bowel disease characterized by uncontrolled immune responses to bacterial flora, with excessive activation of T lymphocytes. MICA is a stress-induced major histocompatibility complex-related molecule expressed on normal intestinal epithelial cells (IECs) and recognized by the NKG2D-activating receptor on CD8(+) T cells, gammadelta T cells, and natural killer cells. We examined the role of MICA-NKG2D interactions in the activation of T lymphocytes in CD. METHODS: MICA expression was analyzed by flow cytometry on IECs isolated from patients with active inflammatory bowel disease and controls. NKG2D expression and function were analyzed on lamina propria and peripheral blood lymphocytes. RESULTS: MICA expression was significantly increased on IECs in CD, with higher expression in macroscopically involved areas. A subset of CD4(+) T cells expressing NKG2D was increased in the lamina propria from patients with CD compared with controls and patients with ulcerative colitis. CD4(+)NKG2D(+) T cells with a Th1 cytokine profile and expressing perforin were increased in the periphery and in the mucosa in CD. CD4(+)NKG2D(+) T-cell clones were functionally active through MICA-NKG2D interactions, producing interferon-gamma and killing targets expressing MICA. IECs from patients with CD had the ability to expand this subset in vitro. CD4(+)NKG2D(+) lamina propria lymphocytes from patients with CD highly expressed interleukin-15R alpha, and interleukin-15 increased NKG2D and DAP10 expression in CD4(+)NKG2D(+) T-cell clones. CONCLUSIONS: These findings highlight the role of MICA-NKG2D in the activation of a unique subset of CD4(+) T cells with inflammatory and cytotoxic properties in CD.     

Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members.

Mutations in the perforin gene have been described in some patients with hemophagocytic lymphohistiocytosis (HLH), but the role of perforin defects in the pathogenesis of HLH remains unclear. Four-color flow cytometric analysis was used to establish normal patterns of perforin expression for control subjects of all ages, and patterns of perforin staining in cytotoxic lymphocytes (natural killer [NK] cells, CD8(+) T cells, CD56(+) T cells) from patients with HLH and their family members were studied. Eleven unrelated HLH patients and 19 family members were analyzed prospectively. Four of the 7 patients with primary HLH showed lack of intracellular perforin in all cytotoxic cell types. All 4 patients showed mutations in the perforin gene. Their parents, obligate carriers of perforin mutations, had abnormal perforin-staining patterns. Analysis of cytotoxic cells from the other 3 patients with primary HLH and remaining family members had normal percentages of perforin-positive cytotoxic cells. On the other hand, the 4 patients with Epstein-Barr virus-associated HLH typically had depressed numbers of NK cells but markedly increased proportions of CD8(+) T cells with perforin expression. Four-color flow cytometry provides diagnostic information that, in conjunction with evidence of reduced NK function, may speed the identification of life-threatening HLH in some families and direct further genetic studies of the syndrome.