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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: J Immunol. 2012 Aug 29;189(7):3741–3750. doi: 10.4049/jimmunol.1201162

Figure 3. Decreased expression of CCR2 and CXCR4 on Irf5−/− monocytes.

Figure 3

Blood and bone marrow cells were collected from naïve and pristane-injected Irf5+/+ (wt) and Irf5−/− littermates. Surface staining was performed with anti-CD11b, -Ly6G, -Ly6C and -CCR2 antibodies, and intracellular staining with anti-CXCR4 antibodies; receptor expression was analyzed by flow cytometry. (A) Representative histogram of CCR2 expression on naïve blood Ly6C(hi) monocytes from littermates and (B) quantification of the mean fluorescence intensity (MFI) on Ly6C(hi) and Ly6C(lo) monocytes; n = 10–11 mice per genotype. Gray area represents isotype control staining in (A and D). (C) Quantification of the MFI of CXCR4 on bone marrow monocytes from naïve mice. (D) Same as in (A) except CXCR4 expression was analyzed on bone marrow monocytes 4 weeks post-pristane injection. (E) Quantification of the MFI of CXCR4 and (F) percentage of CXCR4 expressing cells in Ly6C(hi) bone marrow monocytes from pristane-injected mice. n = 3 mice per genotype. (G) Irf5−/− monocytes are defective in their response to CCR2 and CXCR4 ligands. Enriched naïve bone marrow monocytes from Irf5+/+ and Irf5−/− littermates were seeded inside transwells and medium alone or supplemented with 50 ng/ml CCL2, CXCL12, RANTES or 300 ng/ml IP10 added to the other side as chemoattractants. Cells that migrated to bottom wells were quantified. n = 6 mice per genotype. Data are representative of two independent experiments. * p < 0.05; ** p < 0.01 by Mann-Whitney U test. (H) IRF5 regulates chemokine receptor promoter reporter activity. HEK 293T cells were transiently co-transfected with pGL3b-CCR2 or CXCR4 promoter reporters with pCAGEN-mIRF5 or empty vector and pRL. Luciferase activity was measured and normalized to Renilla activity. Data is presented as relative induction of luciferase activity by IRF5 over control empty vector. Data are representative of three independent experiments performed in duplicate. * p < 0.05; ** p < 0.01; *** p< 0.001 by unpaired Student t test.