The monomer-dimer equilibrium and glycosaminoglycan interactions of chemokine CXCL8 regulate tissue-specific neutrophil recruitment

Abstract

Chemokines exert their function by binding the GPCR class of receptors on leukocytes and cell surface GAGs in target tissues. Most chemokines reversibly exist as monomers and dimers, but very little is known regarding the molecular mechanisms by which the monomer-dimer equilibrium modulates in vivo function. For the chemokine CXCL8, we recently showed in a mouse lung model that monomers and dimers are active and that the monomer-dimer equilibrium of the WT plays a crucial role in regulating neutrophil recruitment. In this study, we show that monomers and dimers are also active in the mouse peritoneum but that the role of monomer-dimer equilibrium is distinctly different between these tissues and that mutations in GAG-binding residues render CXCL8 less active in the peritoneum but more active in the lung. We propose that tissue-specific differences in chemokine gradient formation, resulting from tissue-specific differences in GAG interactions, are responsible for the observed differences in neutrophil recruitment. Our observation of differential roles played by the CXCL8 monomer-dimer equilibrium and GAG interactions in different tissues is novel and reveals an additional level of complexity of how chemokine dimerization regulates in vivo recruitment.

Figure 1.. Schematic of CXCL8 monomer and dimer.

Structures of the CXCL8-trapped monomer (A) and WT dimer (B) are shown. The two monomers within the dimer structure are shown in different colors, and the structures reveal that the structure of the monomer is similar to the monomer structure in the dimer.

Figure 2.. Neutrophil recruitment activity of CXCL8 variants.

Total neutrophils recruited into the peritoneum are plotted for each of the variants at different doses. Data are presented as mean ± sem and are from 10 to 19 animals from a total of two to four independent experiments with four to six animals/group for each experiment. One-way ANOVA was used to determine statistical significance. The recruitment of the WT compared with the monomer and dimer is significant at all doses (P<0.01), except at 1 μg between WT and monomer. The recruitment of WT and monomer compared with the control was significant at all doses (P<0.01) and of the dimer compared with control was significant only at the 10 μg dosage. Inset: The R6K mutation renders CXCL8 completely inactive, and its recruitment at 10 μg dose is similar to that of control PBS, indicating that recruitment as a result of spurious contamination or bacterial byproducts can be ruled out.

Figure 3.. Recruitment of neutrophils using MPO assay.

MPO is expressed predominantly only in neutrophils, and the measured MPO activity correlates with the neutrophil levels shown in Fig. 2. Each data set represents an average of two to three experiments using four to six animals/group. One-way ANOVA was used to determine statistical significance. P is at least <0.01 between monomer and dimer, monomer and WT, and dimer and WT at both doses.

Figure 4.. Neutrophil recruitment profiles in the peritoneum versus lung.

Neutrophil recruitment mediated by CXCL8 WT, monomer, and dimer in the peritoneum is compared with the previously reported recruitment in the lung [9]. The recruitment profile of the WT is distinctly different between the peritoneum and lung, indicating differential roles for the monomer-dimer equilibrium in regulating neutrophil recruitment.

Figure 5.. Neutrophil recruitment activity of CXCL8 GAG mutants in the peritoneum.

Recruitment activity of the GAG mutants was measured at three different doses: 0.25 μg (A), 1 μg (B), and 10 μg (C). Data are presented as mean ± sem and represent four to six animals/group. Two-way ANOVA with post-test was used to determine statistical significance. Reduced recruitment of all of the mutants was significant (P<0.01), except for K67A at the 0.25-μg dose.

Figure 6.. Neutrophil recruitment activity of CXCL8 GAG mutants in the lung.

Recruitment activity of the GAG K64A and R60A mutants was measured at two different doses: 1 μg (A) and 10 μg (B). Data are presented as mean ± sem and represent four to six animals/group. Two-way ANOVA with post-test was used to determine statistical significance. Both mutants showed higher activity at the 1-μg (P<0.01) and 10-μg (P<0.05) doses.