Peripheral blood CD4 but not CD8 t-lymphocytes in patients with exacerbation of asthma transcribe and translate messenger RNA encoding cytokines which prolong eosinophil survival in the context of a Th2-type pattern: effect of glucocorticoid therapy.

Abstract

T-lymphocyte (T-LC)-derived cytokines have been implicated in asthma pathogenesis. Activation of peripheral blood CD4 but not CD8 T-LC and a Th2-type pattern of elevated cytokine mRNA expression in BAL fluid T-LC have been observed in asthmatics, but the principal source (CD4 or CD8 T-LC) of these cytokines is unknown. Our objective was to measure expression of Th1- and Th2-type cytokine mRNA and spontaneous secretion of IL-3, IL-5, and GM-CSF by peripheral blood CD4 and CD8 T-LC from asthmatics before and after oral glucocorticoid therapy and non-asthmatic controls. We used in situ hybridization to detect mRNA expression in isolated CD4 and CD8 T-LC, and an in vitro eosinophil survival assay to detect secretion of IL-3, IL-5, and GM-CSF in T-LC culture supernatants. Comparing the asthmatics with the controls, elevated percentages of CD4 T-LC expressed mRNA encoding IL-5, IL-4, and GM-CSF (P < 0.02) but not IL-3, IL-2, or IFN-gamma. In CD8 T-LC, mRNA expression was generally low with no significant differences between the groups. In the asthmatics, the percentages of CD4 T-LC expressing IL-5 mRNA correlated with disease severity and the numbers of peripheral blood eosinophils (P < 0.01). Culture supernatants of asthmatic CD4 but not CD8 T-LC exhibited significantly higher (P = 0.0003) eosinophil survival-prolonging activity compared with controls, in which low activity was detected. Inhibition with anti-cytokine antibodies suggested that GM-CSF, and to a lesser extent IL-5 and IL-3, could account for this activity. After oral glucocorticoid therapy of the asthmatics, lung function improved and the percentages of CD4 T-LC expressing mRNA encoding IL-3, IL-5, and GM-CSF but not IL-2, IL-4, or IFN-gamma were reduced (P < 0.04). Secretion of eosinophil survival-prolonging activity by the CD4 T-LC was also reduced (P = 0.004). We conclude that peripheral blood CD4 but not CD8 T-LC from asthmatics express cytokine mRNA in a Th2-type pattern and show elevated secretion of cytokines prolonging eosinophil survival. Glucocorticoid therapy of asthmatics is associated with a reduction in the percentages of CD4 T-LC expressing IL-3, IL-5, and GM-CSF mRNA and secretion of the corresponding proteins.

Expression of Activation Markers and Cytokine mRNA by Peripheral Blood CD4 and CD8 T Cells in Atopic and Nonatopic Childhood Asthma: Effect of Inhaled Glucocorticoid Therapy

“…These studies support a role for activated "Th2-type" T cells and their eosinophil-active cytokine products in the pathogenesis of child asthma and have reinforced observations in adult asthmatics that the properties of peripheral blood T cells, at least in terms of expression of activation markers and cytokine mRNA, closely resemble those of T cells in the bronchial mucosa and lumen. [5][6][7][8][9]13 One of our principal aims in the present study was to compare functional aspects of both CD4 and CD8 T cells in atopic and nonatopic child asthmatics. The rationale for this was based partly on our previous studies 15,16 demonstrating that CD8, as well as CD4 T cells, show evidence of activation in child atopic asthma, and on recent studies in adults 27 showing that both CD8 and CD4 T cells within the asthmatic bronchial mucosa are a source of mRNA encoding both IL-5 (the eosinophil-specific cytokine most strongly implicated in the regulation of asthma severity) and IL-4 (1 of only 2 cytokines shown to induce IgE synthesis in B cells and therefore implicated in the pathogenesis of atopy).…”

“…7,16 Briefly, cytospins were rehydrated then permeabilized (20 minutes at 37°C) with Triton X-100 and proteinase K (1 mg/mL) in 0.1 M Tris containing 50 mM ethylene diamine tetraactic acid then incubated with 0.1 M triethanolamine and 0.5% acetic anhydride (20 minutes at 37°C) to inhibit nonspecific probe binding. Cells were prehybridized in 50% formamide in 2X standard saline citrate (15 minutes, 37°C), hybridized with 10 6 counts per minute of labeled probe in 10 L of hybridization buffer then washed to high stringency in decreasing concentrations of standard saline citrate at 42°C.…”

“…The autoradiographs were then developed in Kodak D-19 developer (Kodak, Toronto, Canada), fixed and counterstained with hematoxylin, then mounted with a cover slip and examined with a microscope fitted with a graduated eyepiece for positive signals as previously described. 7,16 Percentages of CD4 and CD8 T cells with overlying silver grains, showing specific riboprobe binding, were obtained from the counting of at least 200 cells per sample. An observer ignorant of the identity of the samples counted the slides.…”

“…Evidence accumulated over the past 2 decades suggests that selective eosinophil accumulation and activation in the asthmatic bronchial mucosa is mediated, at least in part, by the actions of eosinophil-active cytokines derived predominantly from activated CD4 T cells, of which interleukin-5 (IL-5) is uniquely eosinophilspecific. 4,[7][8][9] These cells have a "Th2-type" profile of cytokine production. 10 Interleukin-4 (IL-4) may also participate in selective eosinophil accumulation because it upregulates expression of vascular cell adhesion molecule on vascular endothelium, a ligand for very late antigen-4 expressed uniquely (among the granulocytes) on eosinophils.…”

“…Previous evidence suggests that they do this at least partly by reducing T-cell activation and concomitant cytokine production. 7,13,14 The relative lack of knowledge about the molecular immunopathology of asthma in children stems partly from the practical and ethical problems with obtaining access to the bronchial mucosa. Fortunately, the studies on the properties of peripheral blood T cells in adult asthma cited above suggest that the properties of these cells reflect those of cells in the bronchial mucosa, owing perhaps to a "spillover" or recirculation of these cells into the peripheral circulation.…”