Figure 1.
Neutrophil recruitment at 6 hours post-inoculation of various doses of CXCL8 variants.
(A) Neutrophil recruitment by CXCL8 monomer, dimer, and wild type (WT) variants for 0.1, 1.0, and 10.0 µg doses. Bronchoalveolar lavage fluid (BALF) samples from mice treated with CXCL8 variants were processed as described in Methods. Each data set represents an average of 2–3 experiments using 4–6 animals/group. P<0.05 between monomer and dimer, monomer and WT and dimer and WT at all doses. (B–C) Neutrophil levels for 0.1 µg and 1 µg doses are shown on an expanded scale to highlight the differences in neutrophil recruitment among the different CXCL8 variants. (D) Profile of neutrophil recruitment by monomer at various doses. (E) Estimation of neutrophil levels using fluorescence-activated cell sorting (FACS). Representative data of neutrophil recruitment in lungs for the 10 µg dose are shown. BALF cells were stained with neutrophil specific Gr-1 antibody conjugated with a fluorescent dye phycoerythrin (PE). (F) Neutrophil recruitment by the inactive R6K CXCL8 mutant is negligible, and similar to the control. The inset shows levels of R6K and control recruitment on an expanded scale for better clarity. Statistical analyses were carried out using ANOVA (Graph Pad prism 4); p<0.05 between monomer and dimer, monomer and WT, and dimer and WT at all doses, and more significant if so indicated (***p<0.001).
Figure 2.
Temporal variation in neutrophil recruitment.
(A) Neutrophil recruitment by CXCL8 monomer, dimer, and WT variants for the 10 µg dose at different time points. For clarity, recruitment by the individual variants is shown in panels B, C, and D. BAL samples were processed as described in Methods. Each data set represents an average of two experiments using 4–6 animals/group.
Figure 3.
Trafficking of CXCL8 in mouse lung.
Levels of CXCL8 WT (grey), monomer (black), and dimer (white) were measured in BAL (A) and serum (B) using ELISA, at different time points post-inoculation in the mouse lung. Monomeric CXCL8 shows higher serum levels (evident at the 4 h time point) than WT or trapped dimer.
Figure 4.
Expression of endogenous mouse chemokines and cytokines.
Cell-free supernatants of BAL from mice treated with CXCL8 variants were analyzed for mouse cytokines and chemokines at different time points. Representative data of GCSF, KC and IL-6 levels from mice treated with 10 µg of trapped dimer (which shows robust recruitment) and R6K mutant (inactive, no recruitment) are shown for 2 and 6 h time points. No correlation is seen between KC levels and neutrophil recruitment profiles.
Figure 5.
Tissue damage due to CXCL8-mediated neutrophil influx.
Representative lung sections of mice treated with (A) 50 µl phosphate buffer saline (PBS), (B) 10 µg CXCL8 dimer in 50 µl PBS after 6 h, and (C) 107 PFU of human metapneumovirus in 50 µl PBS after 24 h. Lungs were fixed with 10% formaldehyde in PBS, and histological sections were stained with haematoxylin and eosin (20× magnification; scale bar = 200 µm). Markers of tissue damage such as airway obliteration (arrow heads), peribronchiolar (short arrow), and perivascular (long arrow) inflammatory cell accumulation are obvious in the virus-infected lung tissue, and are completely absent in CXCL8 dimer treated lung tissue. (D) Total protein in cell free BALF from mice treated with 10 µg of CXCL8 WT, trapped monomer at various time intervals. Statistical analyses show no significant differences in protein levels among different CXCL8 variants at all time points.
Figure 6.
(A) Monocyte and neutrophil levels in BALF of mice treated with 10 µg of dimer over a period of 72 h. Dynamics of alveolar monocytes and neutrophil levels indicate resolution of the inflammation. (B) Phagocytosis of neutrophils by monocytes (arrow) from mice treated with 10 µg dose of trapped dimer for 24 h. (C) Alveolar monocytes in various stages of mitosis are seen in BALF of mice treated with 10 µg dose of trapped dimer for 48 h. The observed cell division could explain the increase in monocytes numbers observed. Scale bar = 20 µm.
Figure 7.
Chemotactic activity of CXCL8 variants.
(A) Chemotaxis of dHL60-CXCR2 cells, in response to gradients of CXCL8 variants, was measured in the microfluidic gradient chambers. Chemotaxis for all variants was measured as a function of both varying steepness and concentration. The data are shown as the mean chemotactic index (C.I.), which is defined as the displacement of cells that move along the Y-axis (direction of gradient) divided by the total migration distance. Statistical analyses were performed with two-way ANOVA with Bonferroni posttests, *p<0.05; ***p<0.001. (B) Movement tracks of ten randomly picked cells in response to different concentration gradients of CXCL8 variants. Gradients of monomer, dimer or WT CXCL8 were delivered into the device by a constant flow (the direction shown by the arrow). The cell movements (Supplemental Videos S1 and S2) were recorded every 20 sec for 30 min and data were analyzed with Metamorph software.
Figure 8.
A schematic showing processes involved in neutrophil recruitment in lung tissue.
CXCL8 produced at the site of insult migrates to the bloodstream where they form a concentration gradient, made up of monomers and dimers in solution, and monomers and dimers bound to GAGs. Free and GAG-bound monomers and dimers exist in equilibrium, and each species can bind and activate cognate receptors on neutrophils. Local concentrations and gradients dictate the spatial and temporal predominance of each species, which in turn modulates overall recruitment.