Fifth complement cascade protein (C5) cleavage fragments disrupt the SDF-1/CXCR4 axis: further evidence that innate immunity orchestrates the mobilization of hematopoietic stem/progenitor cells

Abstract

Objective: Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/(desArg)C5a) in human HSPC mobilization.

Materials and methods: C5 cleavage fragments in the plasma were evaluated by enzyme-linked immunosorbent assay using human anti-(desArg)C5a antibody, and expression of the C5a/(desArg)C5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and expression of stromal cell-derived factor-1 (SDF-1)-degrading proteases that perturb retention of HSPC in bone marrow, namely matrix metalloproteinase (MMP)-9, membrane type (MT) 1-MMP, and carboxypeptidase M.

Results: We found that plasma levels of (desArg)C5a are significantly higher in patients who are good mobilizers and correlate with CD34(+) cell and white blood cell counts in mobilized peripheral blood. C5 cleavage fragments did not chemoattract myeloid progenitors (colony-forming unit granulocyte-macrophage), but (desArg)C5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34(+) cells, but appeared on more mature myeloid precursors, monocytes, and granulocytes. Moreover, granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells and polymorphonuclear cells had a significantly higher percentage of cells expressing CD88 than nonmobilized peripheral blood. Furthermore, C5a stimulation of granulocytes and monocytes decreased CXCR4 expression and chemotaxis toward an SDF-1 gradient and increased secretion of MMP-9 and expression of MT1-MMP and carboxypeptidase M.

Conclusion: C5 cleavage fragments not only induce a highly proteolytic microenvironment in human bone marrow, which perturbs retention through the CXCR4/SDF-1 axis, but also strongly chemoattracts granulocytes, promoting their egress into mobilized peripheral blood, which is crucial for subsequent mobilization of HSPC.

Figure 1

_desArgC5a levels correlate with mobilization responses. (A) _desArgC5a in plasma was measured by ELISA, and CD34⁺ cell and WBC counts were evaluated in the G-CSF-mobilized patients, on the day of leukapheresis. Levels of _desArgC5a in the plasma of three selected patients are shown: patient #1, a poor mobilizer, patient #2 intermediate, and patient #3 a very good mobilizer. (B) Positive correlations between _desArgC5a levels and CD34⁺ cell counts and WBC counts and are shown for more plasma samples (n = 9 patients). Samples for ELISA were analyzed in duplicates.

Figure 2

Chemotactic effect of C5a and _desArgC5a on human PB and BM nucleated cells. (A) Migration of PB (left) and BM (right) nucleated cells (NC). (B) Migration of BM CFU-GM progenitors. In physiological concentrations, _desArgC5a but not C5a, strongly chemoattracts both PB and BM nucleated cells. Chemotactic responses to _desArgC5a are several times stronger than those to SDF-1. On the other hand, neither _desArgC5a nor C5a chemoattracted BM CFU-GM progenitor cells. Values are the fold-increases of migrated cells compared to media alone (M). *p < 0.05 as compared with media alone (control). The data shown represents the combined results of three independent experiments carried out in triplicate per group (n = 9).

Figure 3

C5a/_desArgC5a receptor (CD88) expression on myeloid precursors, monocytes and PMN. (A) Isolated BM, mPB and CB CD34⁺ cells were stained with either mouse IgG or anti-CD88 MoAb followed by incubation with goat anti-mouse AlexaFluor-488 secondary antibody. Filled and open histograms correspond to isotype IgG and CD88, respectively. (B) CD88 expression on ex vivo-expanded erythroid, myeloid and megakaryocytic precursors. Cells were expanded as described in Materials and methods and FACS analysis was carried out on days 3, 6 and 11 of expansion. Lineage marker expression was evaluated on day 11 of expansion. (C) FACS analysis of CD88 expression on lymphocytes, monocytes and PMN cells derived from normal (n) and mobilized (m) PB. (D) FACS analysis of CD88 expression on BM leukocytes stimulated or not (control) with G-CSF (100 ng/mL, 48 h). Representative data from three independent experiments are shown.

Figure 3

C5a/_desArgC5a receptor (CD88) expression on myeloid precursors, monocytes and PMN. (A) Isolated BM, mPB and CB CD34⁺ cells were stained with either mouse IgG or anti-CD88 MoAb followed by incubation with goat anti-mouse AlexaFluor-488 secondary antibody. Filled and open histograms correspond to isotype IgG and CD88, respectively. (B) CD88 expression on ex vivo-expanded erythroid, myeloid and megakaryocytic precursors. Cells were expanded as described in Materials and methods and FACS analysis was carried out on days 3, 6 and 11 of expansion. Lineage marker expression was evaluated on day 11 of expansion. (C) FACS analysis of CD88 expression on lymphocytes, monocytes and PMN cells derived from normal (n) and mobilized (m) PB. (D) FACS analysis of CD88 expression on BM leukocytes stimulated or not (control) with G-CSF (100 ng/mL, 48 h). Representative data from three independent experiments are shown.

Figure 4

C5a downregulates surface CXCR4 expression in monocytes and PMN. (A) Definition of subsets of leukocytes (lymphocytes, monocytes and PMN cells) using FSC and SSC analysis. Leukocyte subpopulations were gated using CD45-FITC staining. (B) PB leukocytes were obtained from either normal donors (n = 4) or G-CSF-mobilized patients (n = 6) and co-stained with anti-CXCR4-PECy5; mean percentage of CXCR4 expression is presented. * p ≤ 0.05 (C) Various populations of PB leukocytes were stimulated or not (control) with C5a or G-CSF (100 ng/mL) for 15 h and CXCR4 expression was evaluated by FACS analysis. Cells were stained and gated as described in A. (D) CXCR4 expression in PMN cells after treatment with C5a and anti-C5a antibody. The colors of the histograms (left panel) and bars (right panel) correspond to the percentage of CXCR4 expression in cells labelled with isotype control IgG (black), untreated cells (red), after treatment with C5a (green), and after treatment with C5a and anti-C5a antibody (blue). Representative data from four independent experiments are shown.

Figure 4

C5a downregulates surface CXCR4 expression in monocytes and PMN. (A) Definition of subsets of leukocytes (lymphocytes, monocytes and PMN cells) using FSC and SSC analysis. Leukocyte subpopulations were gated using CD45-FITC staining. (B) PB leukocytes were obtained from either normal donors (n = 4) or G-CSF-mobilized patients (n = 6) and co-stained with anti-CXCR4-PECy5; mean percentage of CXCR4 expression is presented. * p ≤ 0.05 (C) Various populations of PB leukocytes were stimulated or not (control) with C5a or G-CSF (100 ng/mL) for 15 h and CXCR4 expression was evaluated by FACS analysis. Cells were stained and gated as described in A. (D) CXCR4 expression in PMN cells after treatment with C5a and anti-C5a antibody. The colors of the histograms (left panel) and bars (right panel) correspond to the percentage of CXCR4 expression in cells labelled with isotype control IgG (black), untreated cells (red), after treatment with C5a (green), and after treatment with C5a and anti-C5a antibody (blue). Representative data from four independent experiments are shown.

Figure 5

C5a enhances production of proteases. (A) MMP-9 secretion by PMN cells (n = 4) and MNC (n = 3) after stimulation with C5a (100 ng/mL) for 24 h at 37°C as analyzed by zymography. Medium conditioned by fibrosarcoma HT-1080 cells was used as a standard (Std) to indicate the position of soluble MMPs. (B) MT1-MMP gene expression (by semi-quantitative and real-time RT-PCR) and protein expression (by Western blotting) in PMN (n = 4) and MNC (n = 3) in the absence (control) or presence of C5a (100 ng/mL for 24 h at 37°C). * p ≤ 0.05. (C) CPM gene expression in PMN cells (n = 3) and MNC (n = 3) after incubation or not (control) with C5a (100 ng/mL for 24 h at 37°C). GAPDH was used as internal loading control for the RT-PCR. The numbers at the bottom of the gels indicate fold-increase in expression relative to control as determined by densitometric analysis.

Figure 6

Chemotaxis of C5a-treated PMN cells towards SDF-1 in the presence of protease inhibitors. (A) Chemotaxis towards media alone or various concentrations of SDF-1 of normal PMN cells (n = 2) after incubation or not (control) with 100 ng/mL C5a for 15 h. * p ≤ 0.05. (B) Chemotaxis towards SDF-1 (100 ng/mL) of normal PMN cells (n = 2) after incubation or not (control) with C5a (100 ng/mL) in the presence of the MMP inhibitors, epigallocatechin gallate (EGCG, 50 μM) and o-phenanthroline (ophen, 0.5 mM) or neutrophil serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF, 1 mM). Data is representative of two independent experiments done in quadruplicates. * p ≤ 0.05 relative to control.