Qiu Meng

Thymic Stromal Lymphopoietin Expression Is Increased in Asthmatic Airways and Correlates with Expression of Th2-Attracting Chemokines and Disease Severity

Thymic stromal lymphopoietin (TSLP) is said to increase expression of chemokines attracting Th2 T cells. We hypothesized that asthma is characterized by elevated bronchial mucosal expression of TSLP and Th2-attracting, but not Th1-attracting, chemokines as compared with controls, with selective accumulation of cells bearing receptors for these chemokines. We used in situ hybridization and immunohistochemistry to examine the expression and cellular provenance of TSLP, Th2-attracting (thymus and activation-regulated chemokine (TARC)/CCL17, macrophage-derived chemokine (MDC)/CCL22, I-309/CCL1) and Th1-attracting (IFN-γ-inducible protein 10 (IP-10)/CXCL10, IFN-inducible T cell α-chemoattractant (I-TAC)/CXCL11) chemokines and expression of their receptors CCR4, CCR8, and CXCR3 in bronchial biopsies from 20 asthmatics and 15 normal controls. The numbers of cells within the bronchial epithelium and submucosa expressing mRNA for TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in asthmatics as compared with controls (p ≤ 0.018). TSLP and TARC/CCL17 expression correlated with airway obstruction. Although the total numbers of cells expressing CCR4, CCR8, and CXCR3 did not significantly differ in the asthmatics and controls, there was evidence of selective infiltration of CD4+/CCR4+ T cells in the asthmatic biopsies which correlated with TARC and MDC expression and airway obstruction. Epithelial cells, endothelial cells, neutrophils, macrophages, and mast cells were significant sources of TSLP and chemokines. Our data implicate TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10 in asthma pathogenesis. These may act partly through selective development and retention, or recruitment of Th2 cells bearing their receptors.

Expression and Cellular Provenance of Thymic Stromal Lymphopoietin and Chemokines in Patients with Severe Asthma and Chronic Obstructive Pulmonary Disease

Asthma and chronic obstructive pulmonary disease (COPD) are associated with Th2 and Th1 differentiated T cells. The cytokine thymic stromal lymphopoietin (TSLP) promotes differentiation of Th2 T cells and secretion of chemokines which preferentially attract them. We hypothesized that there is distinct airways expression of TSLP and chemokines which preferentially attract Th1- and Th2-type T cells, and influx of T cells bearing their receptors in asthma and COPD. In situ hybridization, immunohistochemistry, and ELISA were used to examine the expression and cellular provenance of TSLP, Th2-attracting (TARC/CCL17, MDC/CCL22, I-309/CCL1), and Th1-attracting (IP-10/CXCL10, I-TAC/CXCL11) chemokines in the bronchial mucosa and bronchoalveolar lavage fluid of subjects with moderate/severe asthma, COPD, and controls. Cells expressing mRNA encoding TSLP, TARC/CCL17, MDC/CCL22, and IP-10/CXCL10, but not I-TAC/CXCL11 and I-309/CCL1, were significantly increased in severe asthma and COPD as compared with non-smoker controls (p < 0.02). This pattern was reflected in bronchoalveolar lavage fluid protein concentrations. Expression of the same chemokines was also increased in ex- and current smokers. The cellular sources of TSLP and chemokines were strikingly similar in severe asthma and COPD. The numbers of total bronchial mucosal T cells expressing the chemokine receptors CCR4, CCR8, and CXCR3 did not significantly differ in asthma, COPD, and controls. Both asthma and COPD are associated with elevated bronchial mucosal expression of TSLP and the same Th1- and Th2-attracting chemokines. Increased expression of these chemokines is not, however, associated with selective accumulation of T cells bearing their receptors.

Basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin☆☆☆★

BACKGROUND Previous studies used indirect methods to identify basophils in the bronchi in asthma, and the numbers were not compared with eosinophils and mast cells. Furthermore, differences in basophil numbers between atopic and nonatopic asthma at baseline and between late-phase skin and asthmatic reactions have not been previously documented. OBJECTIVE The basophil granule-specific mAb BB1 was used to identify basophils in (1) bronchial biopsy specimens from atopic asthmatic subjects and nonatopic asthmatic subjects and control subjects, (2) biopsy specimens from atopic asthmatic subjects before and after inhalational allergen challenge, and (3) late-phase skin reactions. Basophil numbers were compared with EG2(+) eosinophils and tryptase(+) mast cells. METHODS Cells were enumerated in bronchial and skin biopsy specimens by means of immunohistochemistry with the alkaline phosphatase-antialkaline phosphatase method. RESULTS There were elevated numbers of basophils in baseline biopsy specimens in atopic asthmatic subjects compared with atopic control subjects or normal control subjects, although eosinophils and mast cells were 10-fold higher. There was an intermediate number of basophils in nonatopic asthmatic subjects. Basophils increased after allergen inhalation, but again basophils were less than 10% of eosinophils. In contrast, basophils in cutaneous late-phase reactions were approximately 40% of infiltrating eosinophils. The peak of basophil accumulation was at 24 hours, whereas maximal eosinophil infiltration occurred at 6 hours. One third of cutaneous basophils had morphologic appearances suggestive of degranulation. CONCLUSION Numerous basophils infiltrated cutaneous late-phase reactions in atopic subjects. However, this cell was not prominent in bronchial biopsy specimens of asthmatic subjects, either at baseline or after allergen challenge.

Kinetics of cell infiltration and cytokine messenger RNA expression after intradermal challenge with allergen and tuberculin in the same atopic individuals

BACKGROUND Previous studies, in which one time point was used, have shown that cells infiltrating skin biopsy specimens taken during allergen-induced late-phase responses (LPR) had a TH2-like (interleukin-4 [IL]-4 and IL-5 mRNA+) cytokine profile, whereas in delayed-type hypersensitivity (DTH) there was a predominant TH1-type pattern. OBJECTIVE The study was designed to examine the kinetics of accumulation of inflammatory cells and cells expressing mRNA for TH2- or TH1-type cytokines in LPR and DTH elicited simultaneously in the same subjects. METHODS Immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique) and in situ hybridization were used to analyze skin biopsy specimens taken during allergen-induced LPR. RESULTS In LPR elevated numbers of CD3+ and CD4+ cells, eosinophils, neutrophils, and IL-4 and IL-5 mRNA+ cells were detected as early as 1 hour after allergen challenge, with a peak at 6 hours, which was maintained for up to 96 hours. A small but significant delayed increase in macrophages, CD8+ and CD25+ cells, and IL-2 and interferon-gamma mRNA+ cells was also observed, but only at the 48-hour and 96-hour time points. In contrast, in DTH the numbers of CD3+, CD4+, and mRNA+ cells for IL-2 and interferon-gamma were not elevated until 24 hours after challenge and peaked at 48 hours after injection. At 48 hours there was an additional small but significant increase in IL-4 and IL-5 mRNA+ cells. For both LPR and DTH the kinetics of the increases in inflammatory cells and cytokine mRNA-expressing cells paralleled the clinical response. CONCLUSIONS In LPR accumulation of T cells and granulocytes, together with cells expressing mRNA encoding for TH2-type cytokines, is relatively rapid (i.e., within 1 to 6 hours), whereas in DTH the T cell/macrophage infiltration and appearance of cells expressing TH1-type cytokines are not apparent until 24 to 48 hours. In LPR there is a TH1-type (or possibly TH0) component at 48 to 96 hours, and in DTH there is an additional TH2/TH0 response.

T-helper cell type 2 (Th2) memory T cell-potentiating cytokine IL-25 has the potential to promote angiogenesis in asthma

IL-25 (IL-17E) is a T-helper cell type 2 (Th2) cytokine best described as a potentiator of Th2 memory responses. Reports of expression of its receptor, IL-25R, on airways structural cells suggest a wider role for IL-25 in remodeling. We hypothesized that IL-25 stimulates local angiogenesis in the asthmatic bronchial mucosa. Immunoreactive IL-25+, IL-25R+, and CD31+ (endothelial) cells in sections of bronchial biopsies from asthmatics and controls were detected by immunohistochemistry. The effect of IL-25 on angiogenesis was examined using an in vitro assay. Real-time PCR was used to detect expression of IL-25R and VEGF mRNA in cultured human vascular endothelial cells (HUVEC), and a cell proliferation kit (WST-8) was used to measure the effect of IL-25 on HUVEC proliferation. Immunostaining showed that IL-25+, IL-25R+, and CD31+/IL-25R+ cells were significantly elevated in the bronchial mucosa of asthmatics compared with controls (P < 0.003). In asthmatics, the numbers of IL-25+ cells correlated inversely with the forced expiratory volume in 1 s (r = −0.639; P = 0.01). In vitro, HUVEC constitutively expressed IL-25R, which was up-regulated further by TNF-α. IL-25 and TNF-α also increased expression of VEGF and VEGF receptors. IL-25 increased HUVEC proliferation and the number, length, and area of microvessel structures in a concentration-dependent manner in vitro. VEGF blockade, the PI3K-specific inhibitor LY294002, and the MAPK/ERK1/2 (MEK1/2)-specific inhibitor U0126 all markedly attenuated IL-25–induced angiogenesis, and the inhibitors also reduced IL-25–induced proliferation and VEGF expression. Our findings suggest that IL-25 is elevated in asthma and contributes to angiogenesis, at least partly by increasing endothelial cell VEGF/VEGF receptor expression through PI3K/Akt and Erk/MAPK pathways.

Il-4- and IL-5-positive T lymphocytes, eosinophils, and mast cells in allergen-induced late-phase cutaneous reactions in atopic subjects

It has previously been shown that cells mRNA+ for T(H2)-type cytokines (IL-4 and IL-5) infiltrate the site of allergen-induced cutaneous late-phase reactions (LPR) in atopic subjects. In this study we have used the same experimental model to identify the cell source of both IL-4 and IL-5 mRNA and protein product. Allergen-induced LPRs were provoked in the skin of atopic individuals and the sites microscopically examined at 6, 24, and 48 hours. Using single in situ hybridization and immunohistochemistry, we first showed that the numbers of IL-4 and IL-5 mRNA and protein product positive cells peaked at 24 hours. This coincided with the magnitude of the LPR. By double in situ hybridization/immunohistochemistry, we then established (in 24-hour biopsy specimens) that the percentage of CD3+ T lymphocytes, EG2+ eosinophils, and tryptase-positive mast cells that were either IL-4 or IL-5 mRNA+ was 19%, 24%, and 5% and 19%, 20%, and 5%, respectively. Conversely, the percentage of EG2+ and tryptase-positive cells that were IL-4 or IL-5 protein product positive were 62% and 53% and 72% and 29%, respectively. IL-4 and IL-5 protein did not colocalize to CD3+ cells. CD68+ macrophages were negative in both in situ hybridization and immunohistochemistry. With eosinophils we obtained direct evidence of time-dependent stimulus-induced IL-4 and IL-5 mRNA transcription by semiquantitative reverse transcription-polymerase chain reaction of cells incubated with either IgG- or sIgA-coated particles in vitro. Taken together, these experiments suggest that eosinophils, mast cells, and T cells all contribute in variable degrees to the expression of IL-4 and IL-5 in human cutaneous LPR. The failure to colocalize IL-4/IL-5 protein (as opposed to mRNA) to CD3+ cells is attributed to the inability of T lymphocytes to store and concentrate sufficient intracellular amounts of these cytokines to produce positive immunostaining.

Interleukin-10 levels increase in cutaneous biopsies of patients undergoing wasp venom immunotherapy

We have studied the influence of wasp venom immunotherapy (VIT) on cellular recruitment and cytokine mRNA expression during allergen‐induced cutaneous late‐phase responses (LPR). Nine subjectswith a history of wasp sting anaphylaxis, and specific IgE in their sera underwent wasp VIT. Skin biopsies were taken 24 h after intradermal diluent and allergen before and after 3 months VIT. Pre‐immunotherapy, there were significant allergen‐induced increases in EG2+ eosinophils, elastase+ neutrophils, CD68+ macrophages and IL–10 protein+ cells, and increased expression of mRNA for IL–4, IL–13, IFN–γ, IL–12, IL–10, TGF‐β, RANTES and eotaxin. When these allergen‐induced changes in cytokine mRNA and cellular profiles were compared with those obtained after 3 months VIT there was a significant reduction in IL–4 mRNA (p=0.012) and increase in IL–10 protein+ cells (p=0.004) with a trend to an increase in IL–10 mRNA (p=0.054). There were also significant reductions in eosinophils (p<0.004) and the size of the cutaneous LPR (p<0.01) but no change in mRNA to IFN–γ, IL–13 or IL–12. Therefore, VIT is associated with a significant increase in cells positive for IL–10 protein but not IL–12 or IFN–γ. These results suggest that induction of IL–10 may be important in VIT and occur independently of the switch to a Th1 phenotype. IL–10 generation may down‐regulate IL–4 expression and eosinophil recruitment.

Increased mature and immature CCR3 messenger RNA+ eosinophils in bone marrow from patients with atopic asthma compared with atopic and nonatopic control subjects

BACKGROUND Eosinophil infiltration of the bronchial mucosa is characteristic of asthma. Eosinophils differentiate from CD34(+) progenitors. Animal models suggest cooperation between IL-5 and eotaxin to allow rapid mobilization of a pool of bone marrow eosinophils followed by recruitment to the airway mucosa. OBJECTIVE The purpose of this study was to enumerate CD34(+) cell numbers in blood and bone marrow from atopic asthmatics and control subjects and to test the hypothesis that there is an increased bone marrow pool of CCR3(+) eosinophils in patients with atopic asthma, as compared with control subjects. METHODS Bone marrow aspirates and peripheral blood were obtained from volunteers with asthma and control volunteers. CD34(+) cell numbers were evaluated by flow cytometry, and eosinophil colony-forming activity was evaluated by methylcellulose cultures. Mature eosinophils, eosinophil myelocytes, metamyelocytes, and band forms (immature eosinophils) were enumerated by morphologic findings and immunocytochemistry for eosinophil cationic protein. CCR3 and eotaxin mRNA expression was examined by in situ hybridization, and protein expression was examined by immunocytochemistry. CCR3(+) cells were further identified with Chromotrope 2R staining. RESULTS CD34(+) cell numbers in bone marrow were increased in atopic subjects. Numbers of eosinophil colony-forming units in blood and bone marrow did not differ between groups. Percentages of both mature and immature eosinophils were increased in bone marrow from patients with atopic asthma, but not atopic patients with no asthma or normal control subjects. CCR3 was expressed by immature and mature bone marrow eosinophils. Eotaxin was expressed by bone marrow cells from all 3 groups, but there was no increase in subjects with asthma. CONCLUSION These findings suggest that in humans there is an increased bone marrow pool of CCR3(+) mature and immature eosinophils available for rapid mobilization in subjects with asthma but not in atopic subjects with no asthma.

Early production of thymic stromal lymphopoietin precedes infiltration of dendritic cells expressing its receptor in allergen- induced late phase cutaneous responses in atopic subjects

Background:  Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)‐7‐like cytokine that triggers dendritic cell‐mediated T helper (Th)2 inflammatory responses through a receptor consisting of a heterodimer of the IL‐7 receptor alpha (IL‐7Rα) chain and the TSLP receptor (TSLPR), which resembles the cytokine receptor common gamma chain. Dendritic cells activated by TSLP prime development of CD4+ T cells into Th2 cells contributing to the pathogenesis of allergic inflammation. We hypothesized that allergen exposure induces expression of TSLP and results in recruitment of TSLPR bearing cells in the cutaneous allergen‐induced late‐phase reaction (LPR) in atopic subjects.

Increases in eotaxin-positive cells in induced sputum from atopic asthmatic subjects after inhalational allergen challenge

Background: Eosinophils are believed to be critical proinflammatory cells in airway mucosal damage in asthma. Eotaxin is a C‐C chemokine with selective activity for eosinophils and basophils. Previous studies have shown increased expression of eotaxin in the airways of asthmatics at baseline. We aimed to investigate eotaxin expression during the late‐phase reaction to allergen inhalation in atopic asthmatics.