Figure 1
Figure 1. MIF and MIF receptor expression in human and murine CLL. (A) Quantitative RT-PCR from purified B cells of healthy donors (n = 10) and of CLL patients (n = 30). Mean ± SEM of MIF mRNA expression in CLL patients was 8.9 ± 2.8 compared with 2.3 ± 1.3 in healthy donors relative to peptidylprolylisomerase A mRNA levels (P = .015, Mann-Whitney U test). (B) Representative immunoblot for MIF and β-actin protein in lysates of B cells from healthy donors (n = 5; > 95% CD19+ cells) and leukemic B cells from CLL patients (n = 10; > 95% CD19+ cells). Overexpression of MIF protein in human CLL cells (P = .0007). (C) MIF plasma levels in 49 patients with CLL and 50 age-matched healthy donors. Significantly elevated MIF plasma levels were found in CLL patients (7353 ± 1618 pg/mL for Binet A, n = 26; and 10 360 ± 2508 pg/mL for Binet B + C, n = 23) compared with healthy controls (105 ± 17 pg/mL; P < .0001). The difference of MIF levels in early stage (Binet A) versus advanced stage (Binet B + C) CLL patients was not significant. (D) MIF protein levels by ELISA in cell culture supernatants of purified CLL cells and CD19+ peripheral blood B cells (n = 5 each) cultured for 0-48 hours. CLL cells release more MIF than healthy B cells (P < .01 by a 2-way ANOVA). (E) Representative immunoblot for MIF, TCL1, and β-actin in lysates of splenocytes from leukemic Eμ-TCL1+ mice (n = 6) and nonleukemic wild-type control animals (n = 3). Overexpression of MIF protein in murine CLL cells (P = .0017). (F) Surface expression of MIF receptors CXCR2, CD44, and CD74 on TCL1+/wt nonleukemic (IgM+CD5−; n = 5) and leukemic (IgM+CD5+; n = 5) murine B cells. Increased receptor expression on leukemic versus nonleukemic B cells seen for CXCR2 and CD44. No difference in CD74 receptor expression in leukemic and nonleukemic B cells. MFI indicates mean fluorescence intensity. (G) Representative immunoblot for p-ERK, t-ERK, p-AKT, t-AKT, and β-actin in lysates of isolated splenic B cells from Eμ-TCL1 mice, stimulated with 100 ng/mL recombinant MIF for 0, 1, 5, 15, 30, 60, and 120 minutes.

MIF and MIF receptor expression in human and murine CLL. (A) Quantitative RT-PCR from purified B cells of healthy donors (n = 10) and of CLL patients (n = 30). Mean ± SEM of MIF mRNA expression in CLL patients was 8.9 ± 2.8 compared with 2.3 ± 1.3 in healthy donors relative to peptidylprolylisomerase A mRNA levels (P = .015, Mann-Whitney U test). (B) Representative immunoblot for MIF and β-actin protein in lysates of B cells from healthy donors (n = 5; > 95% CD19+ cells) and leukemic B cells from CLL patients (n = 10; > 95% CD19+ cells). Overexpression of MIF protein in human CLL cells (P = .0007). (C) MIF plasma levels in 49 patients with CLL and 50 age-matched healthy donors. Significantly elevated MIF plasma levels were found in CLL patients (7353 ± 1618 pg/mL for Binet A, n = 26; and 10 360 ± 2508 pg/mL for Binet B + C, n = 23) compared with healthy controls (105 ± 17 pg/mL; P < .0001). The difference of MIF levels in early stage (Binet A) versus advanced stage (Binet B + C) CLL patients was not significant. (D) MIF protein levels by ELISA in cell culture supernatants of purified CLL cells and CD19+ peripheral blood B cells (n = 5 each) cultured for 0-48 hours. CLL cells release more MIF than healthy B cells (P < .01 by a 2-way ANOVA). (E) Representative immunoblot for MIF, TCL1, and β-actin in lysates of splenocytes from leukemic Eμ-TCL1+ mice (n = 6) and nonleukemic wild-type control animals (n = 3). Overexpression of MIF protein in murine CLL cells (P = .0017). (F) Surface expression of MIF receptors CXCR2, CD44, and CD74 on TCL1+/wt nonleukemic (IgM+CD5−; n = 5) and leukemic (IgM+CD5+; n = 5) murine B cells. Increased receptor expression on leukemic versus nonleukemic B cells seen for CXCR2 and CD44. No difference in CD74 receptor expression in leukemic and nonleukemic B cells. MFI indicates mean fluorescence intensity. (G) Representative immunoblot for p-ERK, t-ERK, p-AKT, t-AKT, and β-actin in lysates of isolated splenic B cells from Eμ-TCL1 mice, stimulated with 100 ng/mL recombinant MIF for 0, 1, 5, 15, 30, 60, and 120 minutes.

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