Takahiro Tsuburai

Cytokine Production Profile of CD4+ T Cells from Patients with Active Churg-Strauss Syndrome Tends Toward Th17

Background: Churg-Strauss syndrome (CSS) is a rare systemic necrotizing vasculitis that develops in some asthma patients. What types of asthma make patients prone to develop this condition remains unknown. We found that inhibition of regulatory T cells (Treg) differentiation, especially by Tr1 cells, due to a decreased ability of responder T cells to generate IL-2, is associated with the onset and pathogenesis of CSS. In contrast, recent evidence suggests that IL-17-producing Th17 cells play a crucial role in autoimmune inflammation. However, few studies have addressed the role of Th17 in the pathogenesis of CSS. Methods: Mononuclear leukocytes were obtained from healthy subjects, patients with bronchial asthma (BA), asthma-accompanying chronic eosinophilic pneumonia (BA+CEP) and CSS. The cells were stimulated for 4–5 h with PMA and ionomycin in the presence of brefeldin A to generate and accumulate cytokines. Intracellular cytokines were detected after fixation and permeabilization of these cells. Tr1 cells were evaluated as CD4+CD25+ T cells dominantly producing IL-10 and TGF-β, nTreg as CD4+CD25+ T cells expressing Foxp3, a master transcriptional factor, and Th17 were also evaluated as CD4+ T cells mainly producing IL-17. Patients with CSS were classified into an active group and an inactive group in accordance with the disease state after treatment. Results: The frequency of Th17 in peripheral blood was significantly higher in active CSS patients than in healthy subjects, BA, BA+CEP, and inactive CSS patients. In contrast, the Tr1 cell detection frequency was remarkably decreased in active CSS in comparison with BA, BA+CEP, and inactive CSS patients. Also, there was a significant relation between the condition of a given CSS patient and the detection frequencies of both CD4+ helper T cells when CSS cases were divided into inactive and active stages. The Th17 detection frequency was increased in the active stage of CSS, while the Tr1 frequency decreased sharply but then reversed in the inactive stage of CSS. Conclusion: Active CSS patients have elevated intracellular IL-17 and decreased IL-10 levels, which correlate with measures of disease activity, suggesting that Th17 and Tr1 may play important roles in the pathogenesis of CSS.

Differences in regulatory T cells between Churg-Strauss syndrome and chronic eosinophilic pneumonia with asthma

BACKGROUND Chronic eosinophilic pneumonia (CEP) with asthma precedes the onset of Churg-Strauss syndrome (CSS) in half of all patients with CSS. It is not known what determines whether patients with CEP after asthma will have CSS. OBJECTIVE We examined whether activation of regulatory T cells in patients with CEP inhibits CSS development and is otherwise involved in the mechanism of CSS disease. METHODS In patients with CSS (n = 38), CEP with asthma (n = 20), and general adult asthma (n = 108), we examined the number of CD4(+)CD25(+) T cells in peripheral blood, as well as levels of expression of the cytokines IL-2, IL-5, IL-10, and TGF-beta by CD4(+)CD25(+) T cells, CD4(+)CD25(-) T cells, or both. RESULTS At disease onset, patients with CSS, unlike patients with CEP, had significantly fewer CD4(+)CD25(+) T cells than patients with any step of asthma. CD4(+)CD25(+) T cells producing IL-10 were rarely detected in patients with CSS at disease onset or relapse, whereas the numbers of IL-10-producing T cells in patients with CEP were high at disease onset. There were fewer CD4(+)CD25(-) T cells producing IL-2 in patients with CSS before treatment than in patients with CEP at disease onset. The proportions of CD4(+)CD25(+) T cells producing IL-10 and CD4(+)CD25(-) T cells producing IL-2 in patients with CSS increased at remission. CONCLUSIONS Maintenance of the numbers of regulatory T cells in patients with CEP with asthma might inhibit CSS development through the action of cytokines, such as IL-10 and IL-2, produced by CD4(+)CD25(+) or CD4(+)CD25(-) T cells. This might be part of a mechanism that influences progression and prognosis in these diseases.

Reference Ranges for Exhaled Nitric Oxide Fraction in Healthy Japanese Adult Population

BACKGROUND The measurement of the exhaled nitric oxide fraction (FE(NO)) is proposed as a useful marker of airway inflammation. In healthy adults, there have been a few studies of the reference ranges for FE(NO) in Caucasians. A community study in other regions may reveal any possible ethnic differences in the FE(NO) levels. METHODS A total of 240 healthy adults aged between 18 to 74 years were recruited from four medical centers in Japan. Current smokers and subjects having a history of atopic disease were not included. FE(NO) was measured using an online electrochemical nitric oxide analyzer according to the current guidelines. The reference ranges for FE(NO) were estimated using two different statistical methods recommended by International Federation of Clinical Chemistry and Laboratory Medicine. RESULTS The mean FE(NO) was 16.9 ppb (parts per billion) with a 95% prediction interval (2.5 to 97.5 percentiles) of 6.5 to 35.0 ppb in healthy Japanese adults. Normality assumptions were met for the logarithm-transformed FE(NO). The geometric mean FE(NO) was 15.4 ppb with a mean ± two standard deviations of 6.5 to 36.8 ppb. Age, gender, height, and past smoking history were not associated with the FE(NO) levels. CONCLUSIONS The reference ranges for FE(NO) in healthy Japanese adults were similar to those of Caucasians. It seems reasonable that the upper limit of FE(NO) for healthy adults should be set at approximately 36.0 ppb irrespective of ethnic differences.

High-dose Intravenous Immunoglobulin Treatment Increases Regulatory T Cells in Patients with Eosinophilic Granulomatosis with Polyangiitis

Objective. We studied the effects of intravenous immunoglobulin (IVIG) treatment on clinical symptoms and regulatory T (Treg) cell frequency in patients with eosinophilic granulomatosis with polyangiitis (EGPA). Methods. Twenty-two EGPA patients with severe mononeuritis multiplex or cardiac dysfunction received IVIG therapy combined with conventional therapy (corticosteroid, immunosuppressants, or both). As a control, 24 EGPA patients without severe vasculitic symptoms were treated with conventional therapy. Before, during, and after treatment, we determined percentages of Treg cells and other relevant cells in patients’ peripheral blood. Results. The frequency of CD25+ among CD4+ T cells was lower at onset in the study group than in controls but increased significantly after IVIG treatment, relative to controls. The frequency of CD25+ among CD4+ T cells correlated with the frequency of FOXP3+ among CD4+ T cells and interleukin 10 produced by CD25+CD4+ T cells. Conclusion. The increase in Treg cells seen with the combination of IVIG and conventional therapy may promote remission in EGPA.

Pseudomonas aeruginosa-Induced Neutrophilic Lung Inflammation Is Attenuated by Adenovirus-Mediated Transfer of the Heme Oxygenase 1 cDNA in Mice

Heme oxygenase (HO) is well known as the rate-limiting enzyme in the oxidative degradation of heme to biliverdin, carbon monoxide (CO), and iron. Based on recent evidence that overexpression of HO-1 confers protection against various types of cell and tissue injury by regulating apoptotic cell death or cytokine expression profiles, the present study was performed to examine whether the transfer of exogenous HO-1 cDNA in the lung would provide therapeutic effect in a murine model of lung inflammation induced by Pseudomonas aeruginosa. HO-1 overexpression clearly attenuated neutrophil influx and decreased numbers of apoptotic bronchial epithelial cells. Interestingly, the overexpression of Bcl-2, a known antiapoptotic factor, was observed and thought to be the mechanism that inhibits bronchial epithelial cellular apoptosis. It is thus suggested that HO-1 overexpression is useful for treating P. aeruginosa-associated lung inflammation by attenuating neutrophil influx and apoptotic cell death.

Involvement of Regulatory T Cells in the Pathogenesis of Churg-Strauss Syndrome

Background: Churg-Strauss syndrome (CSS) is a rare systemic necrotizing vasculitis that develops in some asthma patients. However, it remains unknown what types of asthma patients can develop this condition. In addition, CSS patients experience eosinophilic pneumonia (EP) before the onset, but some asthma patients do not develop CSS following EP. Few studies have addressed the immunological differences in both cases. Additionally, it is well known that abnormality or failure of immunological tolerance by regulatory T cells (Treg) directly triggers the onset of autoimmune diseases. To elucidate the involvement of Treg in the difference in the pathogenesis of patients with and without CSS, the frequency of naturally occurring Treg (nTreg) and Treg which dominantly produce IL-10 and TGF-β (Tr1) was measured in peripheral blood. Methods: Mononuclear leukocytes were obtained from patients with asthma, asthma accompanying EP and CSS. The cells were stimulated with PMA and ionomycin in the presence of brefeldin A to generate and accumulate cytokines. Cytokines in the cells were detected after fixation and permeabilization of these cells. Tr1 cells were evaluated as CD4+CD25+ T cells dominantly producing IL-10 and TGF-β, and nTreg were also evaluated as CD4+CD25+ T cells expressing FOXP3, a master transcriptional factor. Results: There was a significantly greater number of nTreg in the peripheral blood of EP patients in comparison to both patients with asthma and whole CSS. However, no significant difference was observed regarding the number of nTreg when patients with CSS were divided into inactive and active stage. In contrast, the detection frequency of Tr1 remarkably decreased in active CSS, especially at the time of onset of CSS, in comparison with asthma, EP and inactive CSS, while there was no difference between EP and inactive CSS. Additionally, the ability of CD4+CD25– T cells (responder T cells) to generate IL-2 when stimulated with PMA and ionomycin sharply decreased in active CSS. Conclusion: In the patient who does not develop CSS even after repeat EP, the frequency of Tr1 and the ability of responder T cells to generate IL-2 do not decrease, but show a remarkable decrease in the EP patient who develops CSS. These findings strongly suggest that Tr1 is responsible for maintaining immunological tolerance in peripheral blood, while also inhibiting the onset of CSS from EP.

The Proportion of Regulatory T Cells in the Peripheral Blood Reflects the Relapse or Remission Status of Patients with Churg-Strauss Syndrome

Background: Churg-Strauss syndrome (CSS) is a rare, systemic, necrotizing vasculitis that develops in some asthma patients. We previously reported that maintenance of the proportion of type 1 regulatory T (Tr1) cells in patients with chronic eosinophilic pneumonia and asthma might inhibit the development of CSS through the action of cytokines, such as interleukin (IL)-10 and IL-2, produced by Tr1 and responder T cells. We also reported that IL-17-producing CD4+ helper T cells (Th17 cells) are involved in the pathogenesis of CSS because a higher proportion of Th17 cells was observed in CSS patients during relapses than during remissions. However, few studies have addressed the role of both Tr1 cells and Th17 cells in the status of CSS. Methods: We recruited 40 patients (25 in remission and 15 in relapse) for participation in this study. CSS was diagnosed on the basis of American College of Rheumatology criteria. Remission was defined as the absence of any clinical symptoms of active vasculitis. Tr1 cells were defined as CD4+CD25+ T cells that predominantly produce IL-10 when costimulated with phorbol myristate acetate (PMA) and ionomycin. Naturally occurring Treg (nTreg) cells were defined as CD4+CD25+ T cells that expressed Forkhead box P3 (FOXP3) and cytotoxic T-lymphocyte antigen 4 (CTLA-4). Th17 cells were identified as CD4+ T cells that mainly produced IL-17 and IL-22. Peripheral blood mononuclear cells (PBMCs) obtained from the subjects were costimulated with PMA and ionomycin, and intracellular cytokines were detected after fixing and permeabilizing the cells. Indoleamine 2,3-dioxygenase (IDO) expression was measured in PBMCs that had been treated with IFN-γ and then stimulated overnight with lipopolysaccharide (LPS) or lipopeptide Pam3CSK. Results: Lower expression of CTLA-4 was observed on the surface of CD4+CD25+ T cells obtained from patients with relapsed CSS versus patients in remission. Both FOXP3-expressing nTreg cells and IL-10-producing Tr1 cells were detected in a lower proportion in patients with a relapse compared to patients in remission, but the proportion of CD4+ T cells producing IL-17 was higher during relapse than during remission. In addition, the proportion of CD4+ T cells that produced IL-25, which promotes Th2 inflammation, was also higher in the relapsed patients. We observed a lower percentage of CD14+ monocytes expressing both TLR2 and TLR4 obtained from patients with a relapse of CSS versus patients in remission. Stimulation of CD14+ monocytes with LPS or Pam3CSK reduced IDO expression by the cells from patients with relapsed CSS. The level of IDO expression was positively correlated with the proportion of Tr1 cells in the peripheral blood and inversely correlated with the percentage of Th17 cells. Conclusion: CSS relapse may be linked to increased numbers of CD4+ T cells producing IL-25, which promotes Th2 inflammation, and to a decline in the Tr1 cell subpopulation resulting from lower IDO expression in monocytes. Thus, the proportions of Tr1 cells and Th17 cells reflect the status of CSS.

Comparison of cysteinyl leukotriene concentrations between exhaled breath condensate and bronchoalveolar lavage fluid

Background Collection of exhaled breath condensate (EBC) is a simple, non‐invasive method of obtaining samples from the airways and it can be repeated in short intervals without side effects; therefore, it provides an opportunity to monitor the changes in concentration of inflammatory mediators in the airways. However, EBC analysis still has several unresolved issues.

Decreases in the Numbers of Peripheral Blood Regulatory T Cells, and Increases in the Levels of Memory and Activated B Cells, in Patients with Active Eosinophilic Granulomatosis and Polyangiitis

PurposeEosinophilic granulomatosis with polyangiitis (EGPA), a rare disease characterized by the presence of allergic granulomatosis and necrotizing vasculitis, is often effectively treated with corticosteroids. However, relapse rates are high and, for unknown reasons, some EGPA patients suffer frequent relapses after entry into initial remission. Regulatory T (Treg) cells and B cells are implicated in the development and progression of EGPA. Here, we explored the influence of Treg cells and a co-stimulatory factor present on B cells on the development and course of EGPA.MethodsWe studied 45 EGPA patients (19 of whom experienced frequent relapses and 26 of whom seldom relapsed) and 67 (control) patients with general asthma. We determined the counts or percentages of whole-blood cells exhibiting the following characteristics: FOXP3+ cells among CD4+ Treg cells; CTLA-4+ cells among CD4+/CD25+ Treg cells; and CD27+, CD80+, CD86+, or CD95+ cells among CD19+ B cells. We also measured serum IgG concentrations.ResultsCompared with patients with asthma or seldom-relapsing EGPA, frequently relapsing EGPA patients with active disease exhibited decreased counts of Treg cells and increased percentages of B cells that scored as CD80+, CD27+, or CD95+. Patients with frequently relapsing EGPA had increased percentages of CD27+ and CD95+ B cells, and fewer CD19+ B cells, than did patients in the other two groups. Lower CD19+ B cell counts were associated with reduced Treg cell counts and a lower serum IgG concentration.ConclusionIn patients with frequently relapsing EGPA, decreases in Treg cell numbers and increased percentages of activated B cells may induce apoptosis of B cells.

Bronchial hyperresponsiveness to histamine correlates with airway remodelling in adults with asthma

BACKGROUND Chronic eosinophilic inflammation may promote bronchial hyperresponsiveness (BHR), which involves reversible reduction of airflow and airway remodelling such as a thickening of the reticular basement membrane (RBM) and hypertrophy and hyperplasia of airway smooth muscle (ASM). BHR to histamine (Hist) and acetylcholine (ACh) cannot differentiate airway inflammation and remodelling. OBJECTIVE To examine the correlations between eosinophilic inflammation or airway remodelling and BHR to Hist or ACh in adults with severe asthma. METHODS We examined eosinophils in the sputum of 50 adult patients with severe asthma before inhaled corticosteroid (ICS) treatment. Airway responses to ACh and Hist were measured on separate days after the first hospital visit and before bronchofiberscopy. Bronchial specimens were obtained by bronchofibrescopy for evaluation of RBM and ASM thickening after systemic corticosteroid treatment. RESULTS Eosinophil scores in the sputum before ICS treatment were correlated with BHR to ACh but not to Hist. Asthma duration was inversely correlated with % forced expiratory volume in 1 s, %V(50), %V(25), BHR to Hist, and ASM thickness, but not BHR to ACh or RBM. A multivariate logistic regression model showed that Long duration of asthma affected ASM thickness more than it affected %V(50). ASM thickness was inversely correlated with BHR to Hist but not to ACh. CONCLUSION In adult patients with severe asthma, BHR to ACh is related to the degree of eosinophilic airway inflammation, whereas BHR to Hist indicates airway remodeling, particularly ASM hypertrophy.