The Chemokine, CCL3, and Its Receptor, CCR1, Mediate Thoracic Radiation–Induced Pulmonary Fibrosis

The Chemokine, CCL3, and Its Receptor, CCR1, Mediate Thoracic Radiation–Induced Pulmonary Fibrosis Xuebin Yang1, William Walton1, Donald N. Cook2, Xiaoyang Hua3, Stephen Tilley3, Christopher A. Haskell4, Richard Horuk5, A. William Blackstock6, and Suzanne L. Kirby1* 1Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; 2Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; 3Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; 4Bayer Healthcare Pharmaceuticals, Davis, California; 5Department of Pharmacology, University of California at Davis, Davis, California; and 6Department of Radiation Oncology, Wake Forest University, Winston-Salem, North Carolina Patients receiving thoracic radiation often develop pulmonary injury and fibrosis. Currently, there are no effective measures to prevent or CLINICAL RELEVANCE treat these conditions. We tested whether blockade of the chemokine, CC chemokine ligand (CCL) 3, and its receptors, CC chemokine Our research demonstrates the requirement of the chemo- receptor (CCR) 1 and CCR5, can prevent radiation-induced lung kine, CC chemokine ligand 3, and its receptor, CC chemokine inflammation and fibrosis. C57BL/6J mice received thoracic radia- receptor (CCR) 1, in radiation-induced lung inflammation tion, and the interaction of CCL3 with CCR1 or CCR5 was blocked and fibrosis. A small molecule inhibitor of CCR1 prevented using genetic techniques, or by pharmacologic intervention. Lung fibrosis in mice, and might therefore have a similar benefit in inflammation was assessed by histochemical staining of lung tissue humans. and by flow cytometry. Fibrosis was measured by hydroxyproline assays and collagen staining, and lung function was studied by invasive procedures. Irradiated mice lacking CCL3 or its receptor, irradiation, and is characterized by alveolar cell depletion and CCR1, did not develop the lung inflammation, fibrosis, and decline in inflammatory cell accumulation. The fibroproliferative phase oc- lung function seen in irradiated wild-type mice. Pharmacologic curs approximately 6 months after irradiation, and includes fibro- treatment of wild-type mice with a small molecule inhibitor of blast proliferation, collagen accumulation, and alveolar septal CCR1 also prevented lung inflammation and fibrosis. By contrast, thickening (2). Although the pulmonary inflammation seen dur- mice lacking CCR5 were not protected from radiation-induced injury and fibrosis. The selective interaction of CCL3 with its receptor, ing the early phase is thought to trigger the later fibroprolifer- CCR1, is critical for radiation-induced lung inflammation and fibro- ative phase, the specific leukocyte subsets and molecules that sis, and these conditions can be largely prevented by a small link these two phases have not been identified (3, 4). molecule inhibitor of CCR1. Chemokines are low–molecular weight, secreted proteins that promote directional chemotaxis of leukocyte migration through Keywords: chemokine; fibrosis; radiation; lung; inflammation 7-TM–spanning, G protein–coupled receptors (5). Chemokines are divided into four subgroups based on the organization of the Thoracic radiation is commonly used to treat cancers of the lung, first pair of conserved cysteine motifs (6). Blocking leukocyte esophagus, thymus, breast, and lymph nodes. Whereas radiation recruitment to inflamed tissue should, therefore, be beneficial in treatments can reduce or eliminate tumor burden, many patients some inflammatory diseases (6–8). Certainly, chemokines are im- also develop lung inflammation and fibrosis, which is associated portant mediators in the pathogenesis of lung injury in several with considerable morbidity and mortality. The histopathologic settings (9–11). For example, binding of the chemokine, CXC, to changes to the irradiated lung are well described, and include its receptor, CXCR2, drives neutrophil recruitment in hypoxia- oxidative damage to fibroblasts and the vascular endothelium, induced lung injuries (11). We and others have also found that followed by vascular congestion, inflammation, mesenchymal cell a different chemokine, CC chemokine ligand (CCL) 3, is required proliferation, extracellular matrix deposition, and extensive pa- for leukocyte recruitment in several models of lung inflammation, renchymal remodeling (1). As with other forms of fibrosis, the including infections with influenza virus (12) or Aspergillus cellular and molecular events that give rise to these conditions are fumigatus (13), and in graft-versus-host disease (14). Here, we poorly understood, and there are currently no effective treatments show that both CCL3 and its receptor, CC chemokine receptor for them. Although corticosteroids are frequently administered to (CCR) 1, are required for radiation-induced lung inflammation these patients, this approach often fails to improve the prognosis and fibrosis, and that these conditions can be largely prevented for many patients, and is associated with numerous side effects. with a small molecule inhibitor of CCR1. Radiation-induced lung injury can be divided into two phases: an early inflammatory phase, and a late fibroproliferative phase. The inflammatory phase generally occurs within 1–3 months after MATERIALS AND METHODS Animals (Received in original form June 25, 2010 and in final form September 10, 2010) All animal studies were approved by the institutional animal care and This work was supported by National Institutes of Health grants 5R0 1CA098641- use committee at University of North Carolina at Chapel Hill (Chapel 02 and 5-U19-AI067798. Hill, NC). All mice were on a fully backcrossed C57Bl/6 background with age-matched controls. Ccl32/2 mice were described previously Correspondence and requests for reprints should be addressed to Suzanne L. 2/2 Kirby, M.D., Ph.D., Department of Pathology, University of North Carolina at (12). Ccr1 mice were purchased from Jackson Laboratories (Bar 2/2 Chapel Hill, Chapel Hill, NC 27599. E-mail: skirby7001@gmail.com Harbor, ME), and Ccr5 mice were obtained from Dr. William Kuziel (Protein Design Laboratories, Incline Village, NV). This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Thoracic Irradiation Am J Respir Cell Mol Biol Vol 45. pp 127–135, 2011 Originally Published in Press as DOI: 10.1165/rcmb.2010-0265OC on September 24, 2010 Anesthetized mice at the age of 10–12 weeks were immobilized, with Internet address: www.atsjournals.org a lead shield excluding all but the thoracic cavity. Animals were then 128 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 45 2011 irradiated with a single dose of 14.5 Gy from a cesium source (J. L. noma cells, with a single dose of 5 Gy and supernatants ware Shepherd and Associates, San Fernando, CA) at a dose rate of 1.65 Gy/min. collected 12 hours later for CCL3 protein quantitation. Al- LD50 for C57BL/6J mice is 14.5 Gy. At this dose, survival is sufficient to though not detectable (,1.5 pg/ml) before irradiation, CCL3 permit adequate numbers of animals for long-term analyses. production increased significantly in both the MRC5 (5.22 6 Histopathology and Morphometric Analyses 1.23 pg/ml) and SKLu-1 (8.12 6 2.49 pg/ml) cell lines after irradiation. Thus, CCL3 is increased by radiation in both normal Lung sections were stained with hematoxylin and eosin for routine and malignant lung-derived cell lines. histology, and with Masson’s trichrome for collagen analysis. A total of We next investigated if pulmonary levels of CCL3 are also 10 representative 53 images were analyzed in a blinded fashion for septal thickening using ImageJ software (National Institutes of Health, induced in vivo by thoracic irradiation. C57Bl/6J mice were Bethesda, MD) software, as previously described (15). irradiated and harvested at various time points thereafter for analysis of lung CCL3 protein. By 1 week after irradiation, CCL3 Quantification of Lung Hydroxyproline levels dropped to below levels seen in untreated mice (possibly The right lung was acid digested and hydroxyproline content was an- due to the death of chemokine-producing cells). By 2 weeks after alyzed as previously (16) (see the online supplement for details). irradiation, CCL3 had returned to baseline levels, and continued to rise further, reaching a peak at 4 weeks, and a second peak at Measurements of Lung Mechanics 20 weeks after irradiation (Figure 1A). The mRNA expression of Anesthetized mice were mechanically ventilated on a computer- CCL3 showed a similar pattern in lungs of irradiated mice with controlled small-animal ventilator (Scireq, Montreal, PQ, Canada) at peak expression at 4 and 20 weeks after irradiation (Figure 1B). 350 breaths/min, 6 ml/kg tidal volume, and positive end-expiratory To investigate the relevance of CCL3 expression to radiation- pressure of 3–4 cm H2O. Lung mechanics measurements were per- induced lung injury, we compared radiation-induced responses in formed as previously described (17). WT and CCL3-deficient mice. As expected, about half of the WT mice died by 32 weeks after irradiation (Figure 2A). Flow Cytometric Analyses However, only 16% of the Ccl32/2 mice died after thoracic Excised lungs were minced and digested in collagenase A for 1 hour. Red blood cells were lysed with ACK buffer, and resultant nucleated cells separated on a 20% Percoll gradient were stained with PE- or FITC-labeled monoclonal antibodies to Gr1, FoxP3, CD3,

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