Chemokine (C-C Motif) Receptor-Like 2 is not essential for lung injury, lung inflammation, or airway hyperresponsiveness induced by acute exposure to ozone

Abstract

Inhalation of ozone (O₃), a gaseous air pollutant, causes lung injury, lung inflammation, and airway hyperresponsiveness. Macrophages, mast cells, and neutrophils contribute to one or more of these sequelae induced by O₃ Furthermore, each of these aforementioned cells express chemokine (C-C motif) receptor-like 2 (Ccrl2), an atypical chemokine receptor that facilitates leukocyte chemotaxis. Given that Ccrl2 is expressed by cells essential to the development of O₃-induced lung pathology and that chemerin, a Ccrl2 ligand, is increased in bronchoalveolar lavage fluid (BALF) by O₃, we hypothesized that Ccrl2 contributes to the development of lung injury, lung inflammation, and airway hyperresponsiveness induced by O₃ To that end, we measured indices of lung injury (BALF protein, BALF epithelial cells, and bronchiolar epithelial injury), lung inflammation (BALF cytokines and BALF leukocytes), and airway responsiveness to acetyl-β-methylcholine chloride (respiratory system resistance) in wild-type and mice genetically deficient in Ccrl2 (Ccrl2-deficient mice) 4 and/or 24 hours following cessation of acute exposure to either filtered room air (air) or O₃ In air-exposed mice, BALF chemerin was greater in Ccrl2-deficient as compared to wild-type mice. O₃ increased BALF chemerin in mice of both genotypes, yet following O₃ exposure, BALF chemerin was greater in Ccrl2-deficient as compared to wild-type mice. O₃ increased indices of lung injury, lung inflammation, and airway responsiveness. Nevertheless, no indices were different between genotypes following O₃ exposure. In conclusion, we demonstrate that Ccrl2 modulates chemerin levels in the epithelial lining fluid of the lungs but does not contribute to the development of O₃-induced lung pathology.

Keywords: CXCR2; Cmklr1; Gpr1; chemerin; methacholine; osteopontin.

Figure 1

Relative abundance of chemokine (C‐C motif) receptor‐like 2 (Ccrl2) messenger ribonucleic acid (mRNA) in the left lung lobe of wild‐type C57BL/6 mice 4 and 24 hours following cessation of a 3‐hour exposure to ozone (O₃; 2 parts/million). The abundance of Ccrl2 mRNA in O₃‐exposed mice was expressed relative to Ccrl2 mRNA in the left lung lobe of wild‐type C57BL/6 mice 4 and 24 hours following cessation of a 3‐hour exposure to filtered room air (air). All data were normalized to the abundance of hypoxanthine guanine phosphoribosyl transferase mRNA, a reference gene, in the left lung lobe. Each value is expressed as the mean ± the standard error of the mean. n = 8–10 mice in each group.

Figure 2

The concentration of chemerin in (A) bronchoalveolar lavage fluid (BALF) and (B) serum from wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 4 and 24 hours following cessation of a 3‐hour exposure to either filtered room air (air) or ozone (O₃; 2 parts/million). Each value is expressed as the mean ± the standard error of the mean. n = 8–10 mice in each group. *P < 0.05 compared to genotype‐matched mice exposed to air. ^# P < 0.05 compared to wild‐type mice with an identical exposure.

Figure 3

(A) The concentration of protein and (B) the number of epithelial cells in bronchoalveolar lavage fluid from wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 4 and 24 hours following cessation of a 3‐hour exposure to either filtered room air (air) or ozone (O₃; 2 parts/million). Each value is expressed as the mean ± the standard error of the mean. n = 8–10 mice in each group. *P < 0.05 compared to genotype‐matched mice exposed to air.

Figure 4

(A−D) Representative light photomicrographs of hematoxylin‐ and eosin‐stained lung sections and (E) bronchiolar epithelial injury scores from wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 24  hours following cessation of a 3‐hour exposure to either filtered room air (air) or ozone (O₃; 2 parts/million). A and B are lung sections from air‐exposed wild‐type and Ccrl2‐deficient mice, respectively. C and D are lung sections from O₃‐exposed wild‐type and Ccrl2‐deficient mice, respectively. The black arrows in A and B are directed at bronchiolar epithelial cells that appear normal and are attached to the basement membrane, whereas the blue arrows in C and D are directed at detached bronchiolar epithelial cells. In D, the detached, degenerate epithelial cells are associated with flattening and erosion of the underlying mucosa. In A−D, the images have been magnified with a 40 ×  objective lens while each of the scale bars in A−D represent 50 μm. In E, each value is expressed as the mean ± the standard error of the mean. n = 8 mice in each group. *P < 0.05 compared to genotype‐matched mice exposed to air.

Figure 5

The concentration of (A) adiponectin, (B) eotaxin, (C) hyaluronan, (D) interleukin (IL)‐6, (E) keratinocyte chemoattractant (KC), (F) macrophage inflammatory protein (MIP)‐2, (G) MIP‐3α, and (H) osteopontin (OPN) in bronchoalveolar lavage from wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 4 and 24 hours following cessation of a 3‐hour exposure to either filtered room air (air) or ozone (O₃; 2 parts/million). Each value is expressed as the mean ± the standard error of the mean. n = 8–10 mice in each group. *P < 0.05 compared to genotype‐matched mice exposed to air.

Figure 6

The number of (A) macrophages and (B) neutrophils in bronchoalveolar lavage fluid from wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 4 and 24 hours following cessation of a 3‐h exposure to either filtered room air (air) or ozone (O₃; 2 parts/million). Each value is expressed as the mean ± the standard error of the mean. n = 8–10 mice in each group. *P < 0.05 compared to genotype‐matched mice exposed to air.

Figure 7

(A) Quasistatic respiratory system pressure–volume (PV) curves, (B) area (hysteresis) of PV curves normalized for A, an estimate of inspiratory capacity, (C) A, (D) K, the parameter from the Salazar–Knowles equation reflecting the curvature of the upper portion of the expiratory limb of the PV curve, and (E) C_stat, static compliance of the respiratory system. PV curves were initiated from functional residual capacity, which is defined as lung volume at 3 cm H₂O positive end‐expiratory pressure, and generated by subsequent volume displacement. The PV curves and associated parameters were obtained from wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 24 hours following cessation of a 3‐hour exposure to filtered room air. In B − E, each value is expressed as the mean ± the standard error of the mean. n = 10–12 mice in each group.

Figure 8

Responses to aerosolized acetyl‐β‐methylcholine chloride (methacholine) for respiratory system resistance (R_RS) in wild‐type C57BL/6 mice and mice genetically deficient in chemokine (C‐C motif) receptor‐like 2 (Ccrl2‐deficient mice) 24 hours following cessation of a 3‐hour exposure to either filtered room air (air) or ozone (O₃; 2 parts/million). Each value is expressed as the mean ± the standard error of the mean. n = 10–13 mice in each group. *P < 0.05 compared to genotyped‐matched mice exposed to air. ^# P < 0.05 compared to wild‐type mice with an identical exposure.