UVB-induced sensory changes in human and rat skin 40 hours after UVB irradiation. (A and B) Three MEDs and UVB (1000 m J/cm²) to human and rat skin, respectively, caused a significant increase in blood flow to the irradiated area when compared to control skin sites. (C to F) In the same area, UVB irradiation caused a significant reduction in thresholds to mechanical (C and D) and thermal (E and F) stimulation in human and rat skin, respectively (paired t test or Wilcoxon signed-rank test (depending on data distribution). n = 10 (A, C, and E). n = 8 (B, D, and F).**P < 0.01; ***P < 0.001. All data are presented as the means ± SEM. AU, arbitrary unit.

Correlation of UVB-induced chemokine and cytokine expression in the human and rat. Human and rat gene expression after UVB irradiation was plotted against each other. The Pearson’s correlation coefficient, r = 0.435, showed that there is a positive relationship between the two data sets. This relationship is also significant with P < 0.001. Potential outliers (▲, see Results). n = 73.

The chemokine CXCL5 causes mechanical pain-related hypersensitivity and induces an infiltration of neutrophils and macrophages. (A) When compared to control rats, intraplantar injection of CXCL5 (3 μg) produced a significant reduction in paw withdrawal thresholds to mechanical stimulation at 0.5 and 6 hours after treatment. No significant changes were measured in mechanical thresholds at doses of 0.1 and 1 μg. (B) In the same animals, no change was observed in withdrawal latencies to thermal stimulation at any dose [**P < 0.001; *P < 0.05, two-way repeated-measures analysis of variance (ANOVA); vehicle, 0.1 μg, 1 μg (n = 11), and 3 μg (n = 6)].(C and D) Plantar skin preparations stained with H&E showed a marked inflammatory cell infiltrate within the dermis 6 hours after intraplantar injection of CXCL5 (3 μg). A representative section at low magnification is shown in (C) (scale bar, 200 μm). In (D), a high-magnification section is shown (scale bar, 25 μm) and infiltrating cells, both monocytes (red arrows) and PMNs (blue arrows), are indicated. (E and F) A low level of macrophages (as shown by IBA1 staining) is seen in the dermis of control animals (E), which markedly increases at 6 hours after CXCL5 (3 μg) injections (F) (scale bar, 200 μm). (I) The increase in IBA1 expression after CXCL5 injections was confirmed with qPCR compared to mRNA expression in vehicle-treated skin. (G and H) Immunopositive CD3 cells (a marker of lymphocytes) were detected at low levels in control skin (G) and did not change after CXCL5 treatment (H). (J) There was no difference in the mRNA levels of CD3 in treated versus control animals. (K and L) The mRNA of the neutrophil marker G-CSFR (K) and the cognate receptor for CXCL5, CXCR2 (L), were significantly up-regulated in the chemokine-treated skin compared to control [*P < 0.05; **P < 0.01, Mann-Whitney rank sum test or t test (depending on data distribution); n = 3 to 5]. All data are presented as means ± SEM.

Time course of CXCL5 expression and pain-related hypersensitivity in the rodent UVB model. (A and B) UVB irradiation caused a significant reduction in mechanical (A) and thermal thresholds (B) at 24 and 48 hours after treatment. Mechanical thresholds were also reduced 8 hours after UVB treatment. (C) CXCL5 mRNA expression measured at 0, 4, 8, 24, and 48 hours and 10 days after UVB (1000 m J/cm²). Compared to 0 hours, CXCL5 expression significantly increased at 48 hours (y axis has a logarithmic scale). No difference was measured at other time points (Kruskal-Wallis one-way ANOVA on ranks, n = 4 to 5).(D) Expression of CXCL5 protein was measured with a specific ELISA. Control skin samples contained nondetectable levels of CXCL5. However, 48 hours after UVB irradiation, CXCL5 was significantly increased (n = 4 per group, Mann-Whitney rank sum test, P = 0.029). (E to H) UVB irradiation increased the expression of IBA1 (E) and G-CSFR (G), but not CXCR2 (H) or CD3 (F) [*P < 0.05; **P < 0.01; ***P < 0.001, Mann-Whitney rank sum test or t test (depending on data distribution); n = 5 to 7]. All data are presented as means ± SEM.

CXCL5 contribution to UVB-induced pain-related hypersensitivity. (A and B) Animals received either a functional blocking CXCL5 antibody (Ab, 10 μg) or a control nonimmune IgG (10 μg) immediately after UVB irradiation, and again at 10 and 20 hours [shown by arrows in (A) and (B)]. Antibody neutralization of CXCL5 significantly attenuated the reduction in mechanical thresholds 30 hours after UVB (1000 mJ/cm²) (A). However, neutralization of this chemokine did not have an effect on thermal thresholds after UVB irradiation (B) (two-way repeated-measures ANOVA, n = 11). (C and D) At 8 hours after UVB irradiation, there was no change in infiltrating cell markers between treated and control animals. IBA1 mRNA expression was similar between CXCL5 antibody and the control IgG groups (C). Also, G-CSFR mRNA expression showed no change between groups (D). (E to H) At 30 hours after UVB irradiation, a time point at which the CXCL5-neutralizing antibody is effective in decreasing hypersensitivity behaviors, there was a significant reduction in IBA1 (E), G-CSFR (G), and CXCR2 mRNA expression (H) in animals treated with CXCL5-neutralizing antibody. No difference was found in CD3 mRNA expression (F) (*P < 0.05, t test; n = 4 to 6) (I to L) IBA1 staining in irradiated skin was reduced in animals treated with the CXCL5 antibody (K) versus vehicle (I). Low levels of CD3 staining were similarly detected in both groups (J and L) (scale bar, 200 μm). All data are presented as means ± SEM.

Exposure of macrophages to CXCL5 produced an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). (A to D) Macrophages in which application of ATP (25 μM) produced a change in [Ca²⁺]_i [as seen in (D)] were included in the analysis (thus excluding dead or nonfunctional cells). CXCL5 at a dose of 100 nM increased [Ca²⁺]_i in a number of cells as indicated by the red arrows (C). (E) The percentage of cells responding to 100 nM CXCL5 was significantly larger than after vehicle treatment (E) (Kruskal-Wallis one-way ANOVA on ranks, n = 3 to 6 independent experiments with at least 100 cells per concentration, data are presented as mean% ± SEM). (F and G) Representative response traces of a single cell for macrophages (F) and DRG neurons (G).

CXCL5 attracts cultured peritoneal macrophages. (A) When compared to control, 1 and 10 nM CXCL5 significantly increased the migration of macrophages (Kruskal-Wallis one-way ANOVA on ranks, n = 3 to 5).(B and C) Counterstained filters show cells in a control well (B) and a well containing CXCL5 (10 ng/ml) (C). (D) To test whether CXCL5 induces migration through a gradient (chemotaxis) or solely random nondirectional migration (chemokinesis), we used the checkerboard analysis. In a Boyden chamber, cells were suspended in increasing concentrations of CXCL5 and allowed to migrate toward increasing concentrations of CXCL5 in the lower wells. The gray boxes show the results obtained when the same concentration was used in both wells (no gradient). When there is an increasing gradient, migration is further enhanced, indicating that CXCL5 induces directional chemoattraction on macrophages (Kruskal-Wallis one-way ANOVA on ranks, n = 3 to 5). Significant migration across a gradient versus migration when the concentration is the same. *P < 0.05 compared to 0 versus 0 nM; ⁺P < 0.05 compared to 1 versus 1 nM; ^#P < 0.05 compared to 10 versus 10 nM. All data are presented as the means ± SEM.