Eotaxin-2/CCL24 and eotaxin-3/CCL26 exert differential profibrogenic effects on human lung fibroblasts Martin Kohan, MSc*; Ilaria Puxeddu, MD, PhD†; Reuven Reich, PhD†; Francesca Levi-Schaffer, PhD†; and Neville Berkman, MBBCh, FRCP* Background: Eotaxin-2/CCL24 and eotaxin-3/CCL26 play an important role in eosinophil chemotaxis and activation in asthma. We previously demonstrated that eotaxin/CCL11 is profibrogenic for human lung fibroblasts. The effect of eotaxin-2/ CCL24 and eotaxin-3/CCL26 on lung fibroblasts has not yet been investigated. Objective: To evaluate whether eotaxin-2/CCL24 and eotaxin-3/CCL26 modulate fibrotic properties of lung fibroblasts. Methods: Fibroblast proliferation was evaluated by means of 3-hydroxythymidine incorporation. Collagen production was assessed by means of 3-hydroxyproline incorporation and biochemical staining. Chemotaxis was determined using Boyden chambers. Expression of -smooth muscle actin was evaluated by means of immunostaining. Transforming growth factor 1 release was assessed using enzyme-linked immunosorbent assay. Parametric analysis of variance, followed by the Tukey-Kramer multiple comparisons test, was used to calculate statistical significance. Results: Eotaxin-2/CCL24 but not eotaxin-3/CCL26 stimulated human lung fibroblast proliferation and collagen synthesis. In contrast, eotaxin-3/CCL26 but not eotaxin-2/CCL24 promoted fibroblast migration. Neither eotaxin-2/CCL24 nor eotaxin-3/ CCL26 induced the expression of -smooth muscle actin or transforming growth factor 1 from lung fibroblasts. Conclusions: Eotaxin-2/CCL24 and eotaxin-3/CCL26 have differential profibrogenic effects on human lung fibroblasts. These CC chemokines may, therefore, contribute to airway remodeling in asthma. Ann Allergy Asthma Immunol. 2010;104:66 –72. INTRODUCTION ptk;4Asthma is a complex chronic disease characterized by airway obstruction, airway hyperresponsiveness, inflamma- tion, and remodeling. 1,2 Airway remodeling is defined as the qualitative and quantitative changes observed in structural components of the airways, including hyperplasia of mucous- secreting cells, increased subepithelial deposition of extracel- lular matrix, increased smooth muscle, and angiogenesis. 3 The molecular mechanisms that underlie the remodeling changes in chronic asthma are not yet fully understood. Fibroblast activation and differentiation to a myofibroblas- tic phenotype is a feature of airway remodeling 1–3 and has been reported in asthmatic patients. 4 Fibroblasts/myofibro- blasts are a major source of collagen production, and after activation, they account for the increased extracellular matrix deposition and subsequent subepithelial fibrosis seen in chronic asthma. 5 Eotaxin-1/CCL11, 6 eotaxin-2/CCL24, 7 and eotaxin-3/ CCL26 8 are members of the CC family of chemokines. Several studies provide evidence of a role for CC chemo- kines in human asthma, where they play an important role in airway inflammation, particularly via induction of eo- sinophil chemotaxis and activation. 9,10 Increased levels of eotaxin/CCL11 and its receptor, CC chemokine receptor 3 (CCR-3), were observed in bronchial biopsy samples from asthmatic patients 11 and in lungs and sputum after allergen challenge. 12 Eotaxin-2/CCL24 expression is also increased in endobronchial biopsy samples and bronchial epithelium of asthmatic patients. 13,14 Eotaxin-3/CCL26 is upregulated in bronchial epithelium and airways of asthmatic individ- uals 24 hours after allergen challenge. 13,14 We have previously shown that human lung fibroblasts express CCR-3 and that eotaxin/CCL11 has selective pro- fibrogenic properties on human lung fibroblasts. 15 Al- though eotaxin-2/CCL24 and eotaxin-3/CCL26 are ex- pressed by human lung and dermal fibroblasts, 16,17 there are no data, to our knowledge, that explore whether these chemokines can modulate fibroblast behavior. In this study, we demonstrate that eotaxin-2/CCL24 and eotaxin- 3/CCL26 have differential profibrogenic activity on lung fibroblasts. Affiliations: * Lung Cellular and Molecular Biology Laboratory, Insti- tute of Pulmonology, Hadassah-Hebrew University Medical Center, Jerusa- lem, Israel; † Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel. Disclosures: Authors have nothing to disclose. Funding Sources: This study was supported by grant 5878 –1 from the Chief Scientist Office of the Ministry of Health, Israel, and by the Israel Lung Association, Tel Aviv. Received for publication June 11, 2009; Received in revised form July 20, 2009; Accepted for publication July 28, 2009. © 2010 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.anai.2009.11.003 66 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY