Complement C1q enhances homing-related responses of hematopoietic stem/progenitor cells

Abstract

Background: Previously, we reported that the complement cleavage fragments C3a and C5a are important modulators of trafficking of hematopoietic stem/progenitor cells (HSPCs). The aim of this study was to examine a possible role for complement component 1, subcomponent q (C1q) in HSPC migration.

Study design and methods: CD34+ HSPCs isolated from cord blood (CB), bone marrow (BM), and granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood (mPB) were evaluated for the expression of C1q and its receptor for phagocytosis (C1qRp) using reverse transcription-polymerase chain reaction, Western blotting, and fluorescence-activated cell sorting. Chemotactic responses and chemoinvasiveness toward stromal cell-derived factor (SDF)-1 and expression of matrix metalloproteinase (MMP)-9 were also examined after C1q stimulation. Moreover, G-CSF- and zymosan-induced mobilization was evaluated in C1q-deficient mice.

Results: C1q was expressed in CD34+ cells from mPB, but not from CB or steady-state BM; however, stimulation of the latter with G-CSF induced C1q expression. C1qRp receptor was found on BM, CB, and mPB CD34+ cells and more mature ex vivo expanded myeloid and megakaryocytic precursors. Although C1q itself was not a chemoattractant for HSPCs, it primed/enhanced the chemotactic response of CD34+ cells to a low SDF-1 gradient and their chemoinvasion across the reconstituted basement membrane Matrigel and increased secretion of MMP-9 by these cells. Moreover, in in vivo studies C1q-deficient mice were found to be easy G-CSF mobilizers compared to wild-type mice and normal zymosan mobilizers.

Conclusion: We demonstrated that C1q primes the responses of CD34+ HSPCs to an SDF-1 gradient, which may enhance their ability to stay within BM niches, suggesting that the C1q/C1qRp axis contributes to HSPC homing/retention in BM.

Figure 1. C1q is expressed on mobilized (m)PB CD34⁺ cells but not on steady-state BM CD34⁺ cells, but G-CSF induces its expression in the latter

(A) RT-PCR analysis of mRNA transcripts for C1q in CD34⁺ cells from BM, CB and mPB, and in mononuclear cells (MNC) from mPB and CB. GAPDH was used as a housekeeping gene to ensure equivalence of loading. (B) Western blot for C1q in lysates from normal steady-state BM, mPB and CB CD34⁺ cells. An equal amount of protein, as measured by Bradford protein assay (30 μg/sample), was loaded in each lane. (C) Steady-state BM leukocytes from healthy donors (n = 2) were stimulated (with 200 ng/mL G-CSF) or not (control) for 5 days. Cells were stained as described in the Materials and Methods. The left and middle histograms represent the expression of C1q on unstimulated (control) and G-CSF-stimulated cells, respectively. In the right panel, BM cells gated based on the expression of the CD34 antigen were analyzed for C1q expression. The lower line shows unstimulated BM CD34⁺ cells (control) and the upper line G-CSF-stimulated cells.

Figure 2. C1qRp is expressed on HSPC and ex vivo-expanded progenitor cells

(A) CD34⁺ cells from mPB, CB and BM were labeled with mouse isotype IgG control or with mouse anti-human C1qRp and AlexaFluor 488-conjugated goat anti-mouse antibody. (B) Similar staining was carried out on purified CB CD34⁺ cells expanded towards granulocytic, megakaryocytic and erythroid lineages on days 3 and 11 of expansion. Expression of surface lineage markers were evaluated on day 11 of expansion. Shaded histograms show isotype IgG control.

Figure 3. Priming effect of C1q on chemotaxis and trans-Matrigel migration of CD34⁺ cells towards SDF-1

(A) Chemotaxis of CB CD34⁺ cells pre-incubated or not with C1q (1 μg/mL) was evaluated towards media alone, low SDF-1 gradient (10 ng/mL), high SDF-1 gradient (200 ng/mL) or C1q (1 μg/mL). Data are pooled from at least triplicate samples from 3 independent experiments. (B) CD34⁺ cells, pre-incubated or not with C1q and anti-C1q antibody, were allowed to migrate across Matrigel (reconstituted basement membrane) towards media alone (0 ng/mL SDF-1) or towards a low SDF-1 gradient (20 ng/mL). Each experiment was performed using at least 3 chambers for each set of conditions and cell counts were done in duplicate. (C) Zymograms of media conditioned by CD34⁺ cells from cord blood (CB) or mobilized peripheral blood (mPB) in the presence or absence (control) of C1q. Media conditioned by HT-1080 cells was used as standard (Std) to indicate the position of MMP-9 in the gel.

Figure 4. C1q-deficient mice are easy mobilizers but normal zymosan mobilizers

C1q^-/-mice (C1q KO) as well as age- and sex-matched C57BL/6 mice (wt) were mobilized for 3 or 6 days with G-CSF (250 μg/kg s.c. per day; n=5 mice per group; left panels) or 1 h with zymosan (0.5 mg/kg i.v.; n=5 mice per group; right panels). The number of circulating CFU-GM progenitors (A), and white blood cells (WBC) (B) per microliter of PB are shown. * p< 0.05 as compared with wt mice. Data show representative results from three independent experiments.