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Comparative Study
. 2004 Dec;143(8):1033-41.
doi: 10.1038/sj.bjp.0705972. Epub 2004 Oct 11.

Beta 2-adrenergic receptor regulation of human neutrophil function is sexually dimorphic

Catherine de Coupade  1 , Robert W Gear, Paul F Dazin, Herve Y Sroussi, Paul G Green, Jon D Levine
Affiliations
Comparative Study

Beta 2-adrenergic receptor regulation of human neutrophil function is sexually dimorphic

Catherine de Coupade et al. Br J Pharmacol. 2004 Dec.

Abstract

While the mechanisms underlying the marked sexual dimorphism in inflammatory diseases are not well understood, the sexually dimorphic sympathoadrenal axis profoundly affects the inflammatory response. We tested whether adrenergic receptor-mediated activation of human neutrophil function is sexually dimorphic, since neutrophils provide the first line of defense in the inflammatory response. There was a marked sexual dimorphism in beta(2)-adrenergic receptor binding, using the specific beta(2)-adrenergic receptor ligand, [(3)H]-dihydroalprenolol, with almost three times more binding sites on neutrophils from females (20,878 +/- 2470) compared to males (7331 +/- 3179). There was also a marked sexual dimorphism in the effects of isoprenaline, a beta-adrenergic receptor agonist, which increased nondirected locomotion (chemokinesis) in neutrophils obtained from females, while having no effect on neutrophils from males. Isoprenaline stimulated the release of a chemotactic factor from neutrophils obtained from females, but not from males. This chemotactic factor acts on the G protein-coupled CXC chemokine receptor 2 (CXCR2) chemokine receptor, since an anti-CXCR2 antibody and the selective nonpeptide CXCR2 antagonist SB225002, inhibited chemotaxis produced by this factor. While interleukin- (IL-) 8 is a principal CXCR2 ligand, isoprenaline did not produce an increase in IL-8 release from neutrophils. IL-8-induced chemotaxis was inhibited in a sexually dimorphic manner by isoprenaline, which also stimulated release of a mediator from neutrophils that induced chemotaxis, that was inhibited by anti-CXCR2 antibodies. These findings indicate an important role for adrenergic receptors in the modulation of neutrophil trafficking, which could contribute to sex-differences in the inflammatory response.

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Figures

Figure 1
Figure 1
Human neutrophils express β2-adrenergic receptors. (a) RT–PCR analysis of β1 and β2-adrenergic receptor expressions in neutrophils from males and females. (b) β2-adrenergic receptor protein expression in the membrane fraction (left panel) and the cytosolic fraction (right panel) of male neutrophils. HEK-293 cells stably transfected with the human CCR5 gene (293/CCR5) were used as a negative control and HEK-293 cells stably transfected with the human β2-adrenergic receptor gene (293/β2-AR) as positive control. Data shown are representative of three independent experiments.
Figure 2
Figure 2
Checkerboard transmigration analysis of human neutrophils in response to serial dilutions of isoprenaline. (a) Chemotaxis analysis with isoprenaline added to the lower chamber; (b) chemokinesis analysis with isoprenaline added to both lower and upper chambers, and (c) fugetaxis with isoprenaline added to the upper chamber. (d) Inhibitory effect of ICI-118,551 on female chemokinesis and fugetaxis. Cells were preincubated with the β2-adrenergic receptor antagonist before the transmigration assay. Numbers shown represent mean of percentage of transmigrated cells±s.e.m. with n, number of donors. Asterisks indicates P<0.05 for a pairwise contrasts for each isoprenaline concentration compare to no isoprenaline.
Figure 3
Figure 3
β2-adrenergic receptor density on neutrophils is greater in females than males. (a) Saturation curves representing [3H]-DHA binding in neutrophils freshly isolated from males and females. (b) Specific binding values were obtained by subtracting nonspecific binding from total binding. Data were analyzed using a nonlinear regression plot. Data represent mean±s.e.m. with n, number of donors.
Figure 4
Figure 4
Determination of plasma adrenaline and noradrenaline concentrations in females and males donors. Data represent mean±s.e.m.; n, number of donors.
Figure 5
Figure 5
Inhibition of IL-8-induced neutrophil chemotaxis in the presence of isoprenaline. Neutrophil locomotion in females (a) and males (b) is expressed as percentage of migrated cells into the lower chamber. Dose–response to IL-8 is shown as a control curve. Neutrophil migration is shown in the presence of 10−10, 10−8 or 10−6 M isoprenaline for the three different concentrations of IL-8. Data are expressed as mean±s.e.m.; n, number of donors.
Figure 6
Figure 6
Neutrophil β2-adrenergic receptor activation stimulates the release of a mediator that enhances chemotaxis. (a) Freshly isolated neutrophils from females (left panel) and males (right panel) were incubated with different concentrations of isoprenaline for 3 h at 37°C. Cell-free supernatants were added to the lower chamber of transwells and tested for their chemoattractive properties with freshly neutrophils added to the upper chamber. Neutralizing antibody against human CXCR2 was incubated on ice for 15 min prior to isoprenaline pretreatment. Note that baseline migration levels of neutrophils were 35% (male) and 65% (females) less than the data shown in Figure 2 (checkerboard analysis). This difference could be due to the fact that in this experiment, neutrophils were used 5 h after blood harvesting, compared to 2 h for the checkerboard analysis. Values are mean±s.e.m.; n, number of donors. (b) Quantification of IL-8 released by isoprenaline-stimulated neutrophils. Level of IL-8 was determined by EIA from cell-free supernatants of isoprenaline-stimulated neutrophils from females and males. The values are mean±s.e.m.; n, number of donors. (c) Freshly isolated neutrophils from females were added to the upper chamber with or without the selective CXCR2 receptor antagonist, SB225002 (300 nM) and either cell-free supernatant from isoprenaline (10−8 M)-stimulated cells or the selective CXCR2 agonist GROα (10 nM) was added to the lower chamber. Asterisks indicate P<0.05 for a paired one-tailed t-test for the effect of SB225002.
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