Figure 5. Altered characteristics of CD11b⁺Gr1⁺ neutrophils isolated from Ifnb1^–/– mice.

(A) Percentage of CXCR4⁺ neutrophils in blood of tumor-bearing Ifnb1^–/– and C57BL/6 mice. (B) Percentage of CXCR4⁺ neutrophils isolated from tumors of Ifnb1^–/– and C57BL/6 mice. Tumors were removed; single-cell solutions were prepared, stained, and analyzed using the BD LSR II System. Data were analyzed with FACSDiva software. Experiments were done twice with at least 5 animals per group. Data represent mean ± SEM. *P ≤ 0.01. (C) IFN-β treatment downregulates Vegf, Mmp9, and Cxcr4 gene expression. Tumors were removed; single-cell solutions were prepared and stained, and CD11b⁺Gr1⁺ neutrophils were isolated. Monolayers of such cells were incubated with 5 U rmIFN-β; after 4 hours, RNA was isolated, cDNA prepared, and gene expression measured using real-time RT-PCR. Cells were derived from 5 pooled tumors. All experiments were repeated at least 2 times. (D) High expression of Cxcr4 correlates with higher expression of c-myc and Stat3, and rmIFN-β downregulates both c-myc and Stat3. CD11b⁺Gr-1⁺ neutrophils were sorted from tumors and placed in culture with 5 U rmIFN-β. After 4 hours, cDNA was prepared as described in C. Cells were sorted from 5 pooled tumors. All experiments were repeated at least 2 times. (E) Expression of intracellular pSTAT3 in blood and tumor-infiltrating neutrophils was compared between control and Ifnb1^–/– mice. Cell suspensions from blood and tumors were prepared, stained, and analyzed using the BD LSR II System. Data were analyzed with FACSDiva software. Experiments were done twice with at least 5 animals per group.