IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx?

Abstract

Background: The role of Th2 cells (producing interleukin (IL-)4, IL-5 and IL-13) in allergic asthma is well-defined. A distinct proinflammatory T cell lineage has recently been identified, called Th17 cells, producing IL-17A, a cytokine that induces CXCL8 (IL-8) and recruits neutrophils. Neutrophilic infiltration in the airways is prominent in severe asthma exacerbations and may contribute to airway gland hypersecretion, bronchial hyper-reactivity and airway wall remodelling in asthma.

Aim: to study the production of IL-17 in asthmatic airways at the mRNA level, and to correlate this with IL-8 mRNA, neutrophilic inflammation and asthma severity.

Methods: We obtained airway cells by sputum induction from healthy individuals (n = 15) and from asthmatic patients (n = 39). Neutrophils were counted on cytospins and IL-17A and IL-8 mRNA expression was quantified by real-time RT-PCR (n = 11 controls and 33 asthmatics).

Results: Sputum IL-17A and IL-8 mRNA levels are significantly elevated in asthma patients compared to healthy controls. IL-17 mRNA levels are significantly correlated with CD3gamma mRNA levels in asthmatic patients and mRNA levels of IL-17A and IL-8 correlated with each other and with sputum neutrophil counts. High sputum IL-8 and IL-17A mRNA levels were also found in moderate-to-severe (persistent) asthmatics on inhaled steroid treatment.

Conclusion: The data suggest that Th17 cell infiltration in asthmatic airways links T cell activity with neutrophilic inflammation in asthma.

Figure 1

IL-17A and IL-8 mRNA levels in healthy controls and asthmatics. RNA was isolated from induced sputum of healthy controls (n = 11) and asthmatic patients (n = 33) and real time RT-PCR was performed with β-actin, IL-17A and IL-8 specific primers and with VIC-(β-actin) or FAM-(IL-17A, IL-8) labelled specific probes. Results were quantified by the use of a cDNA plasmid-standard and expressed as the ratio of cDNA copy numbers for IL-17A (A-C) or IL-8 (B-D) divided by the cDNA copy numbers for β-actin multiplied by 10⁴ (A-C) or 10¹ (B-D). Patients were divided in subgroups according to asthma severity following the reviewed GINA criteria [33] (A, B) and allergic state (allergic state was not known in one patient and two patients with only specific IgE for grass pollen were excluded for the comparison of allergic to non-allergic asthma) (C, D). Open symbols represent patients treated with corticosteroids, closed symbols represent patients without corticosteroid-treatment. Comparisons were performed using the Kruskall-Wallis and Mann-Whitney-U test. The median is indicated with a horizontal line. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and ns = not significant.

Figure 2

CD3γ mRNA levels in healthy controls and asthmatics, correlations between IL-17A and CD3γ and between IL-17A other cytokine/chemokine mRNA levels. CD3γ, IL-8, IL-17A and IL-5 mRNA levels in induced sputum of asthmatic patients were quantified as explained in figure 1 using IL-8, IL-17A and IL-5 specific primers and VIC-(β-actin) or FAM-(IL-8, IL-17A, IL-5) labeled specific probes. Asthma severity was determined as in figure 1A. Open symbols represent patients treated with corticosteroids, closed symbols represent patients without corticosteroid-treatment. Comparisons were performed using the Kruskall-Wallis test and Dunn's post test (C). Correlations were studied by Spearman non-parametric test (A-B, D). The median is indicated with a horizontal line. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and ns = not significant.

Figure 3

Sputum neutrophil counts in healthy controls and asthma patients and correlation of IL-17A and IL-8 mRNA levels with the neutrophil count in asthmatics. Cytospins of induced sputum of healthy controls (n = 11) and asthmatic patients (n = 32) were stained with May Grünwald Giemsa. Neutrophils are counted as percentage of the leukocytes. IL-17A and IL-8 mRNA levels in induced sputum of asthmatic patients (n = 27) were determined as explained in figure 1 and 2. Open symbols represent patients treated with corticosteroids, closed symbols represent patients without corticosteroid-treatment. The mean is indicated with a horizontal line. Differences are studied by student t-test (A) and correlation was studied by Spearman non-parametric test (B-C). * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.