Interferon-β induces hepatocyte growth factor in monocytes of multiple sclerosis patients

Abstract

Interferon-β is a first-line therapy used to prevent relapses in relapsing-remitting multiple sclerosis. The clinical benefit of interferon-β in relapsing-remitting multiple sclerosis is attributed to its immunomodulatory effects on inflammatory mediators and T cell reactivity. Here, we evaluated the production of hepatocyte growth factor, a neuroprotective and neuroinflammation-suppressive mediator, by peripheral blood mononuclear cells collected from interferon-β--treated relapsing-remitting multiple sclerosis patients, relapsing remitting multiple sclerosis patients not treated with interferon-β, and healthy volunteers. Using intracellular flow cytometry analysis, increased production of hepatocyte growth factor was observed in circulating CD14(+) monocytes from patients undergoing long-term treatment with interferon-β versus untreated patients. Complementary in vitro studies confirmed that treatment with interferon-β induced rapid and transient transcription of the hepatocyte growth factor gene in CD14(+) monocytes and that intracellular and secreted monocytic hepatocyte growth factor protein levels were markedly stimulated by interferon-β treatment. Additional exploration revealed that "pro-inflammatory" (CD14(+)CD16(+)) monocytes produced similar levels of hepatocyte growth factor in response to interferon-β as "classical" (CD14(+)CD16(-)) monocytes, and that CD14(+) monocytes but not CD4(+) T cells express the hepatocyte growth factor receptor c-Met. Our findings suggest that interferon-β may mediate some of its therapeutic effects in relapsing-remitting multiple sclerosis through the induction of hepatocyte growth factor by blood monocytes by coupling immune regulation and neuroprotection.

Figure 1. IFN–β induces HGF production by PBMCs.

Human PBMCs or T cells were treated with IFN–β for the indicated time and dose. Culture supernatants were collected and HGF levels were evaluated using ELISA. (A) IFN–β increased release of HGF by human PBMCs in a time– and dose–dependent manner. (B) IFN–β did not stimulate HGF production by MACS–sorted CD4⁺ T cells. Data are expressed as means and standard deviations for triplicate wells of one representative experiment. **, p<0.01; ***, p<0.001, as determined by Student’s t test). (C) IFN–β did not induce cell–associated HGF levels by peripheral CD4⁺ T cells, as determined by flow cytometry. Cells were labeled with monoclonal anti–human HGF antibody or isotype control antibody and anti–human CD4 antibody. Representative histograms depict monoclonal anti–human HGF antibody (unfilled histogram) and isotype control antibody (filled histogram). Data are representative of three independent experiments.

Figure 2. IFN–β stimulates in vitro HGF expression and the release of mature bioactive HGF by monocytes.

(A) IFN–β increased HGF secretion by human MACS–separated monocytes from PBMCs in a dose-dependent manner, as determined by ELISA analysis. ***The mean value was significantly different from the control (medium alone) as determined by Student’s t test (p<0.0001). (B, C) HGF α–subunit (mature bioactive HGF protein) levels increased in monocytes in a time–dependent (B) and dose–dependent (C) manner in response to IFN–β treatment, as shown by Western Blot analysis. Cytosolic and plasma membrane proteins from MACS–separated monocytes were separated by SDS–PAGE and revealed using an anti–human HGF monoclonal antibody. The molecular mass of HGF is indicated. (D) IFN–β induces monocytic HGF gene expression, as determined by quantitative real–time PCR. Cytokine mRNA levels from MACS–separated monocytes were normalized with respect to the level of human β–actin. The results presented are representative of at least three different experiments.

Figure 3. IFN–β increased cell–associated HGF in both human (A) CD14⁺CD16⁻ “classical” and (B) CD14⁺CD16⁺ “pro-inflammatory” monocytes in a time– and-dose–dependent manner.

CD14⁺ monocytes were treated with IFN–β for the indicated time and dose, and cell–associated HGF was measured by flow cytometry. Cells were labeled with an antibody against anti–human HGF or isotype control antibody together with anti–CD14 and anti–CD16 antibodies. Histograms depict monoclonal anti–human HGF antibody (unfilled histograms) and isotype control antibody (filled histogram). Histograms depict representative data from two independent experiments. The values shown are the percentages of HGF positive cells as defined by fluorescence intensity greater than the control values of untreated cells.

Figure 4. c–Met protein is expressed on CD14⁺ monocytes, CD19⁺ B lymphocytes, but not T lymphocytes.

Surface expression of c–Met was evaluated on CD4, CD8, CD14, CD16, CD19, and CD56 PBMC subpopulations by six-color flow cytometry. Cells were labeled with monoclonal anti–human c–Met antibody or isotype control antibody and specific antibodies for CD4, CD8, CD14, CD16, CD19 and CD56. Histograms depict monoclonal anti-human c–Met antibody (unfilled histogram) and isotype control antibody (filled histogram).

Figure 5. IFN–β–treatment increased levels of cell–associated HGF in monocytes from RRMS patients.

(A) Flow cytometry for HGF was performed on CD14⁺ monocytes from the three groups (healthy control, untreated RRMS patients, and IFN–β−treated RRMS patients). Monocytes were stained for surface CD14 antigen and cell–associated HGF. Data show representative histogram overlays of isotype (filled histogram) and HGF–stained cells (unfilled histogram). (B) Cell-associated HGF levels in CD14⁺ cells were higher in healthy controls and IFN–β–treated RRMS patients. Surface expression was measured by flow cytometry and calculated as the mean corrected fluorescence index (MFI) ratio. Background HGF expression was assessed by measuring the fluorescence of cells incubated with a nonspecific isotype control antibody similarly labeled. The MFI for control anti–HGF antibody isotype staining was divided with the HGF MFI of monocytes. (C) Median serum HGF levels were similar in all three groups. ELISA for HGF was performed on sera from the three groups (healthy control, untreated RRMS patients, and IFN–β−treated RRMS patients). For both monocyte and serum HGF level analysis, each circle represents a single individual and the lines show the medians. Difference in median levels between groups was examined by Kruskal-Wallis test followed by Mann-Whitney U test due to a non-Gaussian distribution of values. *, p<0.05 and ***, p<0.001.