Sex-Based Disparities in Leukocyte Migration and Activation in Response to Inhalation Lung Injury: Role of SDF-1/CXCR4 Signaling

Abstract

The aim of the study was to determine whether sex-related differences exist in immune response to inhalation lung injury. C57BL/6 mice were exposed to Cl₂ gas (500 ppm for 15, 20, or 30 min). Results showed that male mice have higher rates of mortality and lung injury than females. The binding of the chemokine ligand C-X-C motif chemokine 12 (CXCL12), also called stromal-derived-factor-1 (SDF-1), to the C-X-C chemokine receptor type 4 (CXCR4) on lung cells promotes the migration of leukocytes from circulation to lungs. Therefore, the hypothesis was that elevated SDF-1/CXCR4 signaling mediates exaggerated immune response in males. Plasma, blood leukocytes, and lung cells were collected from mice post-Cl₂ exposure. Plasma levels of SDF-1 and peripheral levels of CXCR4 in lung cells were higher in male vs. female mice post-Cl₂ exposure. Myeloperoxidase (MPO) and elastase activity was significantly increased in leukocytes of male mice exposed to Cl₂. Lung cells were then ex vivo treated with SDF-1 (100 ng/mL) in the presence or absence of the CXCR4 inhibitor, AMD3100 (100 nM). SDF-1 significantly increased migration, MPO, and elastase activity in cells obtained from male vs. female mice post-Cl₂ exposure. AMD3100 attenuated these effects, suggesting that differential SDF-1/CXCR4 signaling may be responsible for sex-based disparities in the immune response to inhalation lung injury.

Keywords: chlorine; gender; leukocyte; lung injury.

Figure 1

Cl₂ exposure increases mortality in male vs. female mice. Kaplan–Meyer curves show that adult male C57BL/6 mice exposed to Cl₂ gas (500 ppm) for 30 min (A) or 20 min (B) have 0% survival within 1-day. In comparison, adult female C57BL/6 mice have 40% and 50% survival rates 7-days post-exposure to Cl₂ gas (500 ppm) for 30 (A) or 20 min (B), respectively. Upon exposure to Cl₂ gas (500 ppm) for 15 min, both male and female mice have a more than 70 percent survival rate over 14-days (C). ** p < 0.01, *** p < 0.001 vs. male mice; log-rank (Mantel–Cox) test.

Figure 2

Cl₂ increases BALF protein and total cell count levels primarily in male mice. Male C57BL/6 mice exposed to Cl₂ gas (500 ppm, 15 min) have significantly elevated parameters of lung injury such as BALF protein levels (A) and cell count (B) compared to their female counterparts (n = 5–13). Individual values and means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. groups at the end of individual lines; two-way ANOVA followed by Tukey’s post-hoc testing.

Figure 3

Cl₂ increases SDF-1 and CXCR4 levels primarily in male mice. Exposure of adult C57BL/6 mice to Cl₂ gas (500 ppm, 15 min) increases BALF SDF-1 levels in male mice on days 1, 7, and 14, but only on day 14 in female mice (A). Plasma SDF-1 levels are elevated in male but not female mice at 1- and 7- days post-exposure (B). FACS analysis (C) of lung cells isolated from male mice shows significantly higher peripheral levels of CXCR4 compared to their female counterparts (D) (n = 5–7). Individual values and means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. groups at the end of individual lines; two-way ANOVA followed by Tukey’s post-hoc testing.

Figure 4

Cl₂ increases migration of lung leukocytes in response to SDF-1 in male mice. Under basal conditions, migration of blood (A) and lung (B) leukocytes is higher in male vs. female mice 14 days post-Cl₂ (500 ppm, 15 min) exposure. Then SDF-1 (100 ng/mL) was added to media in the lower chamber, while some lung cells were treated with AMD3100 (100 nM) overnight and migration of lung leukocytes was measured next morning. SDF-1 does not alter lung leukocyte migration in air-exposed animals (C). However, SDF-1 increases migration (three-fold) in leukocytes obtained from male mice 1-day post-Cl₂ exposure (D), which is attenuated with AMD3100 treatment. SDF-1 also increases leukocyte migration by two-fold in cells obtained from male mice 14 days post-Cl₂ exposure (E). However, there is no significant increase in migration of leukocytes obtained from female mice (n = 5). Individual values and means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. groups at the end of individual lines; two-way ANOVA followed by Tukey’s post-hoc testing.

Figure 5

Lung leukocyte myeloperoxidase (MPO) activity increases more in response to SDF-1 in male vs. female mice post-Cl₂ exposure. Leukocyte MPO activity was assessed in isolated blood leukocytes and lung cells from air-exposed and Cl₂ (500 ppm, 15 min)-exposed mice, 1- and 14- days post-exposure. Blood and (A) lung leukocyte (B) MPO activity is elevated in male mice but not in female mice 1-day post-Cl₂ exposure. Lung cells were then treated with SDF-1 (100 ng/mL) in the presence or absence of AMD3100 (100 nM) and leukocyte MPO activity assessed 1-day post-treatment. SDF-1 increases MPO activity in air-exposed male and female mice (C). SDF-1 increases MPO activity in lung cells obtained from male mice 1- (D) and 14- (E) days post-Cl₂ exposure, which is attenuated by AMD3100. There is no change in MPO activity in cells obtained from female mice post-Cl₂ exposure (n = 5). Individual values and means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. groups at the end of individual lines; two-way ANOVA followed by Tukey’s post-hoc testing.

Figure 6

Cl₂ exposure increases elastase activity of lung leukocytes in response to SDF-1 in male mice. Leukocyte elastase activity was assessed in isolated blood leukocytes and lung cells from air-exposed mice and from mice exposed to Cl₂ (500 ppm, 15 min), 1- and 14- days post-exposure. Elastase activity is not changed in blood leukocytes post-Cl₂ exposure (A). However, lung leukocyte elastase activity in male mice is significantly elevated 1- day post-Cl₂ exposure. Lung cells were then treated with SDF-1 (100 ng/mL) in the presence or absence of AMD3100 (100 nM) and leukocyte elastase activity assessed 1-day post-treatment (B). SDF-1 does not increase elastase activity in lung cells of air-exposed mice (C) but increases the enzymatic activity in cells isolated from male mice 1- (D) and 14- (E) days post-Cl₂ exposure. AMD3100 attenuates this increase. SDF-1 does not significantly alter elastase activity in cells isolated from female mice post-Cl₂ exposure (n = 5). Individual values and means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. groups at the end of individual lines; two-way ANOVA followed by Tukey’s post-hoc testing.