Upregulation of IL-1Ra in the Tumor and Spleen of CCR5−/− Mice
To investigate the difference in the cytokine levels between the tumor and spleen tissues of the CCR5+/+ and CCR5−/− mice, we conducted a cytokine array assay using a Mouse Proteome Array (Figure 4A). Among 40 tested cytokines, the levels of IL-1Ra, MIG, MIP-1α increased about 686, 9 and 25-fold respectively, in CCR5−/− mice compared to CCR5+/+ mice. Using this technique, no other cytokines were found to be different between the CCR5−/− mice and the CCR5+/+ mice (Figure 4B). Similar altered cytokine levels were found in the spleens of CCR5−/− mice (Figure 4C). Since the IL-1Ra level was the most significantly elevated, the expression of IL-1Ra in tumor and spleen tissues was analyzed by immunofluorescence and western blotting. The expression of IL-1Ra was upregulated in tumor and spleen tissues of the CCR5−/− mice (Figure 4D-G).
10.1371/journal.pone.0033747.g004 Figure 4  Upregulation of IL-1Ra in the tumor and spleen of CCR5−/− mice.
A, Figure 4A indicates mouse cytokine array panel coordinates. Nitrocellulose membranes contain 40 different anti-cytokine antibodies printed in duplicate. B, Mouse cytokine array panel indicate the cytokine expression difference in tumor tissues of CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra. Representative blot from three independent experiments is shown. Positive controls show the manufacturer’s internal positive control samples on the membrane. C, Protein immune-arrays were performed using Mouse cytokine array in spleen tissues. There were differences in cytokines between CCR5+/+ mice and CCR5−/− mice, especially IL-1Ra. Representative blot from three independent experiments is shown. Positive controls show the manufacturer’s internal positive control samples on the membrane. D and E, Immunolfluorescence analysis was used to determine the expression levels of IL-1Ra in tumor and spleen tissues. The reactive cell number was determined as the number of DAPI-stained, IL-1Ra antibody-positive cells that were counted. Values are the mean ± S.D. of four experimental animals. * indicates statistically significant differences from CCR5+/+ mice. Scale bar indicates 50 µm. F and G, Expression of IL-1Ra was analyzed by western blotting in tumor and spleen tissues. Each band is representative of three independent experimental results. Data are means ± S.D. of three experimental animals. * indicates significant difference from CCR5+/+ mice (p<0.05).