Sex differences in prostaglandin biosynthesis in neutrophils during acute inflammation

Abstract

The severity and course of inflammatory processes differ between women and men, but the biochemical mechanisms underlying these sex differences are elusive. Prostaglandins (PG) and leukotrienes (LT) are lipid mediators linked to inflammation. We demonstrated superior LT biosynthesis in human neutrophils and monocytes, and in mouse macrophages from females, and we confirmed these sex differences in vivo where female mice produced more LTs during zymosan-induced peritonitis versus males. Here, we report sex differences in PG production in neutrophils during acute inflammation. In the late phase (4-8 hrs) of mouse zymosan-induced peritonitis and rat carrageenan-induced pleurisy, PG levels in males were higher versus females, seemingly due to higher PG production in infiltrated neutrophils. Accordingly, human neutrophils from males produced more PGE₂ than cells from females. Increased PG biosynthesis in males was accompanied by elevated cyclooxygenase (COX)-2 expression connected to increased nuclear factor-kappa B activation, and was abolished when LT synthesis was pharmacologically blocked, suggesting that elevated PG production in males might be caused by increased COX-2 expression and by shunting phenomena due to suppressed LT formation. Conclusively, our data reveal that the biosynthesis of pro-inflammatory PGs and LTs is conversely regulated by sex with consequences for the inflammatory response.

Figure 1

PGE₂, 6-keto-PGF_1α and LTC₄ peritoneal levels in male and female mice after zymosan-induced peritonitis. Mice were treated with zymosan (i.p.), sacrificed at the indicated time points and (A) PGE₂ and (C) LTC₄ were measured in peritoneal exudates from male and female mice, and (B) 6-keto-PGF_1α was measured in peritoneal exudates 4 hrs after zymosan injection. Time zero (T0) corresponded to untreated mice. Values represent means ± S.E.M; n = 10 mice/T0; n = 10 mice/2 hrs; n = 10 mice/4 hrs; n = 6 mice/8 hrs. ^###p < 0.001 and ^#p < 0.05, male vs female mice; two-way ANOVA plus Bonferroni (A and C) and two-tailed Student’s t test (B).

Figure 2

Sex differences in eicosanoid biosynthesis in carrageenan-induced pleurisy in rats. Rats were treated with carrageenan (intrathoracic injection), sacrificed at the given time points, and (A) LTB₄ and (B) PGE₂ in the thoracic exudates were measured by EIA and RIA, respectively. (C) 6-keto-PGF_1α levels in thoracic exudates were measured 4 hrs after carrageenan injection by EIA. Time zero (T0) corresponds to untreated rats. Values represent means ± S.E.M; n = 6 rats/T0; n = 13 rats/2 hrs; n = 6 rats/4 hrs; n = 6 rats/8 hrs; ^#p < 0.05; ^###p < 0.001, male vs female; two-way ANOVA plus Bonferroni (A and B) and two-tailed Student’s t test (C).

Figure 3

Cyclooxygenase expression and NF-κB activation in thoracic exudates differ between male and female cells. Rats were treated with carrageenan (intra thoracic injection) and sacrificed after 4 hrs. (A) Numbers of infiltrated cells (10⁶/rat) were measured in a Burker chamber using light microscopy. (B) Analysis of COX-1/2, (E) phospho-NF-κB p65 and (G) phospho-p38 MAPK by Western blot in total cell lysates of thoracic exudates from male and female carrageenan-treated rats. Densitometric analysis of (C) COX-1, (D) COX-2, (F) phospho-NF-κB p65 and (H) phospho-p38 MAPK protein normalized to β-actin expression. Values represents means ± S.E.M., n = 4 rats. ^#p < 0.05; ^##p < 0.01, male vs female; two-tailed Student’s t test.

Figure 4

Inhibition of 5-LO product formation by MK886 abolishes the sex differences in PG formation in the thoracic exudates of carrageenan-treated rats. Rats were pre-treated with MK886 (1.5 mg/kg, i.p.) or vehicle (DMSO 2%, i.p.) 30 min prior to carrageenan intrathoracic injection and sacrificed after 4 hrs. (A) PGE₂, (B) 6-keto-PGF_1α, and (C) LTB₄ levels in the exudates. (D) Number of infiltrated cells. Values represent means ± S.E.M; n = 6 rats; ^#p < 0.05, male vs female; ^°p < 0.05, female control rats vs female rats treated with MK886; two-tailed Student’s t test.

Figure 5

Capacity of human neutrophils for PGE₂ production is sex-dependent. PGE₂ production in human neutrophils from male and female donors after LPS or A23187 stimulation. Neutrophils from male and female donors were incubated with (A) LPS (1 µg/ml) for 0, 0.5, 3 and 20 hrs, or (B) with 0.5 µM A23187 for 0, 5, 15, 30, 60, 120, and 240 min. The reactions were stopped on ice, and PGE₂ levels were measured by ELISA. (C) After selected time points (0, 5, 15, 30, 60, 120 min) upon stimulation with A23187 (0.5 µM), free ³H-AA content in the supernatant of ³H-AA-pre-labelled neutrophils was evaluated by scintillation counting. Values represents means ± S.E.M of n = 3–6 experiments each in duplicate. ^#p < 0.05, ^##p < 0.01, male vs female cells; two-way ANOVA plus Bonferroni.

Figure 6

COX-2 expression is higher in neutrophils from male versus female donors; effects of 5-LO pathway inhibition on PGE₂ production. (A–C) Western blot analysis of total cell lysates of neutrophils from male and female donors. (A) COX-1 and -2 protein expression. Densitometric analysis of (B) COX-1 and (C) COX-2 protein normalized to β-actin expression. Western blots are representatives of 11 independent experiments. Values represents means ± S.E.M of densitometric analyses of COX-1/-2 protein bands, normalized to β-actin. ^#p < 0.05, male vs female cells. (D) PGE₂ production in human neutrophils from male and female donors after pre-treatment with MK886 (30 nM, 15 min) upon A23187 stimulation (0.5 µM, 4 hrs). Values represents means ± S.E.M of n = 5 experiments, each in duplicate. ^#p < 0.05, male vs female; ^°°p < 0.01, female cells with MK886 vs female control cells; two-tailed Student’s t test.