Chemokine Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant

Abstract

Microbial infection is characterized by release of multiple proinflammatory chemokines that direct neutrophils to the insult site. How collective function of these chemokines orchestrates neutrophil recruitment is not known. Here, we characterized the role for heterodimer and show that the Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant in mice and can recruit more neutrophils than the individual chemokines. Chemokine-mediated neutrophil recruitment is determined by Cxcr2 receptor signaling, Cxcr2 endocytosis, and binding to glycosaminoglycans. We have now determined heterodimer's Cxcr2 activity using cellular assays and Cxcr2 density in blood and recruited neutrophils in heterodimer-treated mice. We have shown that the heterodimer binds glycosaminoglycans with higher affinity and more efficiently than Cxcl1 or Cxcl2. These data collectively indicate that optimal glycosaminoglycan interactions and dampened receptor activity acting in concert in a dynamic fashion promote heterodimer-mediated robust neutrophil recruitment. We propose that this could play a critical role in combating infection.

Keywords: GPCR; heterodimer; inflammation; leukocyte; proteoglycan.

Fig. 1.

Neutrophil recruitment of the Cxcl1–Cxcl2 heterodimer (HD). Peritoneal neutrophil (PMN) recruitment of Cxcl1–Cxcl2 heterodimer, Cxcl1, and Cxcl2 as a function of dose at 3 h postinjection (A) and as a function of time for the 10-µg dose (B). Neutrophil levels are expressed as mean ± SE from 4 or more mice from 1 to 5 independent experiments. The figure is a composite from a total of 16 sets of mice – PBS control (1 set); recruitment as a function of 3 doses for Cxcl1, Cxcl2, and the heterodimer (9 sets); and recruitment as a function of time at the 10-μg dose for Cxcl1, Cxcl2, and the heterodimer (6 sets). Of the 16 sets, 14 data sets are an average of 2 or more independent experiments, with each set containing 4 or more mice. The recruitment activity of Cxcl1 and Cxcl2 at 24 h for the 10-μg dose was from a single experiment containing a minimum of 4 mice. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Fig. 2.

Mobilization of reservoir neutrophils. Blood neutrophil (PMN) levels were measured at 3 h after chemokine administration using flow cytometry. Neutrophil levels are expressed as mean ± SE from 4 mice in the same experiment. Compared to the PBS control, neutrophils were elevated only for Cxcl1 and Cxcl2 at the 10-μg dose. *P < 0.05.

Fig. 3.

Cxcr2 activity of the Cxcl1–Cxcl2 heterodimer. (A) Calcium release activity was measured using mouse bone marrow neutrophils from fluorescence changes using Flexstation 3. (B) β-Arrestin recruitment activity was measured using mCxcr2 CHO.K1 cells from relative luminescence (RLU) that occurs upon β-arrestin–Cxcr2 interaction. Data were collected in triplicate and analyzed using GraphPad Prism software. The measured EC₅₀ and 95% confidence interval values are shown as a table in the supplementary material.

Fig. 4.

Neutrophil Cxcr2 levels. The blood (A) and peritoneal (B) and neutrophil Cxcr2 levels were measured at 3 h after chemokine administration using flow cytometry. Fluorescence-activated cell sorting (FACS) analysis was carried out by excluding the doublets (FSC-A/FSC-H), gating for Ly6G⁺/Cd11b⁺ cells (neutrophils) in the FSC-A/SSC-A subset, and then measuring Cxcr2 levels. Cxcr2 levels are expressed as means ± SE from 4 to 8 mice from 1 or 2 independent experiments, with each experiment containing a minimum of 4 mice. Significance of the blood neutrophil Cxcr2 levels is compared to the PBS control and of the peritoneal neutrophil Cxcr2 levels to the Cxcl1 0.1-μg data. Cxcr2 levels in the peritoneum for PBS and Cxcl2 at 10 μg are not reported due to an absence of neutrophils (#). *P < 0.05, **P < 0.01, ***P < 0.001.