Protein microarray analysis in patients with asthma: elevation of the chemokine PARC/CCL18 in sputum

Abstract

Background: Microarray technology offers a new opportunity to gain insight into global gene and protein expression profiles in asthma. To identify novel factors produced in the asthmatic airway, we analyzed sputum samples by using a membrane-based human cytokine microarray technology in patients with bronchial asthma (BA).

Methods: Induced sputum was obtained from 28 BA subjects, 20 nonasthmatic atopic control (AC) subjects, and 38 nonasthmatic nonatopic normal control (NC) subjects. The microarray samples of subjects were randomly selected from nine BA subjects, three AC subjects, and six NC subjects. Sputum supernatants were analyzed using a custom human cytokine array (RayBio Custom Human Cytokine Array; RayBiotech; Norcross, GA) designed to analyze 79 specific cytokines simultaneously. The levels of growth-regulated oncogene (GRO)-alpha, eotaxin-2, and pulmonary and activation-regulated chemokine (PARC)/CCL18 were measured by sandwich enzyme-linked immunosorbent assays (ELISAs), and eosinophil-derived neurotoxin (EDN) was measured by radioimmunoassay.

Results: By microarray, the signal intensities for GRO-alpha, eotaxin-2, and PARC were significantly higher in BA subjects than in AC and NC subjects (p = 0.036, p = 0.042, and p = 0.033, respectively). By ELISA, the sputum PARC protein levels were significantly higher in BA subjects than in AC and NC subjects (p < 0.0001). Furthermore, PARC levels correlated significantly with sputum eosinophil percentages (r = 0.570, p < 0.0001) and the levels of EDN (r = 0.633, p < 0.0001), the regulated upon activation, normal T cell expressed and secreted cytokine (r = 0.440, p < 0.001), interleukin-4 (r = 0.415, p < 0.01), and interferon-gamma (r = 0.491, p < 0.001).

Conclusions: By a nonbiased screening approach, a chemokine, PARC, is elevated in sputum specimens from patients with asthma. PARC may play important roles in development of airway eosinophilic inflammation in asthma.

FIGURE 1

Custom human cytokine array (RayBio Custom Human Cytokine Arrey; RayBiotech). A total of 79 antibodies toward cytokines and chemokines were placed on the array. Representative membrane protein arrays incubated with sputum supernatant of a BA subject (top left, A), an AC subject (bottom left, B), and an NC subject (top right, C). Four spots in the upper left (row 1; columns A–D) and two spots in the lower right (row 8; columns J and K) of the membranes indicate the positive controls, and two spots in the upper left (row 1; columns E and F) and one in the lower right (row 8; column I) of the membranes indicate the negative controls. The names and locations of each cytokine/chemokine custom spots for this set of experiments are listed (bottom right, D).

FIGURE 2

PARC levels in sputum and serum from BA subjects, AC subjects, and NC subjects. The PARC levels in sputum specimens (top, A) and in serum specimens (bottom, B) from BA subjects, AC subjects, and NC subjects. Solid line shows medians; dashed line shows sensitivity level (top, A).

FIGURE 3

PARC levels, eosinophil inflammatory markers, and cytokines/chemokines in sputum specimens. Correlations are shown between PARC levels and eosinophils (top left, A), EDN (bottom left, B), RANTES (top middle, C), IL-5 (bottom middle, D), IL-4 (top right, E), and IFN-γ (bottom right, F) in sputum specimens.