Pulmonary CXCR2 regulates VCAM-1 and antigen-induced recruitment of mast cell progenitors

Abstract

Chemokine receptors regulate the trafficking of leukocytes by mediating chemotaxis and by their influence on the expression and/or affinity of leukocyte integrins. Using blocking mAb, we showed that antigen-induced recruitment of mast cell progenitors (MCp) to the lung requires interaction of a4 integrins on the MCp with endothelial vascular cell adhesion molecule 1 (VCAM-1). In seeking a chemokine component, we found that CXCR2-deficient but not CCR3- or CCR5-deficient sensitized and antigen-challenged mice have significantly fewer lung MCp 1 day after challenge and fewer tracheal intraepithelial MC 1 week after challenge, implying that recruited MCp provide the source for these mature MC. Unexpectedly, reconstitution of sensitized, sublethally irradiated +/+ and -/- mice with bone marrow cells of either genotype indicated that expression of CXCR2 by the migrating MCp was not required. Instead, receptor function by resident lung cells was required because normal BM did not reconstitute MCp recruitment in irradiated CXCR2(-/-) mice. The reduced MCp influx into the lung of CXCR2(-/-) mice was accompanied by reduced induction of VCAM-1 transcripts and reduced endothelial surface expression. Thus, these studies demonstrate a role for a chemokine receptor in regulating endothelial VCAM-1 expression, MCp migration, and the level of intraepithelial MC in the lung of aerosolized, antigen-challenged mice.

Fig. 1.

MCp recruitment to the lung is reduced in antigen-challenged CXCR2^−/− mice. (A) Total number of lung MCp per mouse from OVA-sensitized WT (black bars) and CXCR2^−/− (open bars) mice with and without aerosolized OVA challenges. Values are the mean ± SE from eight determinations in seven separate experiments. (B) Concentration of MCp (MCp per 10⁶ MNC) in the lungs of the same mice. (C) Number of lung MNC recovered per mouse in the same mice. The asterisk (*) indicates statistical significance (P < 0.05) as determined by a two-tailed Student's t test.

Fig. 2.

CXCR2^−/− BM reconstitutes MCp recruitment to the lung of sensitized, sublethally irradiated, antigen-challenged WT mice. (A) Total number of lung MCp per mouse from sensitized, antigen-challenged WT mice (black bar), from sensitized, sublethally irradiated, antigen-challenged WT mice without BM reconstitution (SI WT; open bar), from sensitized, sublethally irradiated, antigen-challenged, WT mice reconstituted with WT BM (SI WT +/+BM; gray bar), and from sensitized, sublethally irradiated, antigen-challenged WT mice reconstituted with CXCR2^−/− BM (SI WT −/−BM; striped bar). Values are the mean ± SE from four mice in two separate experiments. (B) Concentration of MCp (MCp per 10⁶ MNC) in the lungs of the same mice. (C) Number of lung MNC recovered per mouse in the same mice.

Fig. 3.

WT BM does not reconstitute MCp recruitment to the lung of sensitized, sublethally irradiated, antigen-challenged CXCR2^−/− mice. (A) Total number of lung MCp per mouse from sensitized, sublethally irradiated, antigen-challenged WT mice without BM reconstitution (black bar), from sensitized, sublethally irradiated, antigen-challenged WT mice reconstituted with WT BM (open bar) and from sensitized, sublethally irradiated, antigen-challenged CXCR2^−/− mice reconstituted with WT BM (gray bar). Values are the mean ± SE from six WT and seven CXCR2^−/− mice in three separate experiments, except for the no reconstitution control, which was done in two of the three experiments. (B) Concentration of MCp (MCp per 10⁶ MNC) in the lungs of the same mice. (C) Number of lung MNC recovered per mouse in the same mice. The asterisk (*) indicates statistical significance (P < 0.05) as determined by a two-tailed Student's t test.

Fig. 4.

VCAM-1 transcripts in lung and protein expression on lung endothelium are reduced in sensitized, antigen-challenged CXCR2^−/− mice. (A) Relative VCAM-1 mRNA expression (compared with GAPDH) in lung from sensitized, antigen-challenged WT (Left) and sensitized, antigen-challenged CXCR2^−/− (Right) mice 1 day after challenge (n = 7, from two separate experiments). The asterisk (*) indicates statistical significance (P < 0.05), as determined by a two-tailed Student's t test. (B–E) Representative pictures (of four mice of each genotype in two separate experiments) of VCAM-1 expression on lung endothelium in sensitized WT mice (B), sensitized CXCR2^−/− mice (C), sensitized, antigen-challenged WT mice (D), and sensitized, antigen-challenged CXCR2^−/− mice (E).

Fig. 5.

The number of tracheal IE MC is reduced in sensitized, antigen-challenged CXCR2^−/− mice. (A) The mean (±SE) number of IE MC per tracheal cross-section in sensitized, antigen-challenged WT and sensitized, antigen-challenged CXCR2^−/− mice 1 week after challenge. (B) The mean (±SE) number of submucosal (SM) MC per tracheal cross-section in sensitized, antigen-challenged WT and CXCR2^−/− mice 1 week after challenge. The asterisk (*) indicates statistical significance (P < 0.05, n = 9 WT and 10 CXCR2^−/−, from two separate experiments) as determined by a two-tailed Student's t test. (C) Representative images of tracheal epithelium demonstrating IE MC (arrows) in sensitized, antigen-challenged WT but not in the sensitized, antigen-challenged CXCR2^−/− mice. (D) Representative images of tracheal SM MC (arrows) in sensitized, antigen-challenged WT and sensitized, antigen-challenged CXCR2^−/− mice. (Scale bars: 10 μm.)