Abstract
The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury.
Highlights
Peritoneal cells were harvested 6 h post-injection and total cells were counted along with differential counting to measure neutrophil levels
We showed that basic residues located in two distinct domains on opposite faces of the protein orchestrate GAG interactions, and some of the GAG-binding residues are located in the receptor-binding domain indicating that GAG-bound chemokine cannot activate the receptor[18]
We observed that the recruitment profiles vary, and in particular, the monomer and dimer were minimally active at low doses, and the WT was more active than the monomer and dimer at all doses
Summary
Peritoneal cells were harvested 6 h post-injection and total cells were counted along with differential counting to measure neutrophil levels. We recently showed that both monomers and dimers are agonists for the CXCR2 receptor and that the dimer binds GAG with higher affinity[17,18]. On the basis of our current and previous studies on the related chemokine CXCL8, we propose that GAG interactions and receptor activity of the monomer and dimer collectively determine in vivo gradients in a chemokine-specific manner to direct neutrophils to the target site.
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