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Inhibition of Immature Erythroid Progenitor Cell Proliferation by Macrophage Inflammatory Protein-1α by Interacting Mainly With a C-C Chemokine Receptor, CCR1

Shao-bo Su, Naofumi Mukaida, Jian-bin Wang, Yi Zhang, Akiyoshi Takami, Sinji Nakao and Kouji Matsushima

Article Figures & Data

Figures

  • Fig. 1.

    CCR1 expression on glycophorin A positive BM nucleated cells. The normal BM nucleated cells were analyzed using a two-color immunofluorescence as described in Materials and Methods. The figure in each quadrant represents the percentage of cells.

  • Fig. 2.

    CFU-E– and BFU-E–derived colony formation by CCR1 positive or negative normal BMMNCs in the presence of Epo plus SCF. The purified CCR1 positive or negative normal BMMNCs were plated in a methylcellulose medium containing Epo and SCF. The numbers of CFU-E (open bar) and BFU-E (closed bar) were determined at day 7 or day 14 of culture, respectively. The data are representative of three independent experiments with quadruplicate determinations.

  • Fig. 3.

    Effects of MIP-α on CFU-E (A) and BFU-E (B) formation by CD34+ BMMNCs. The purified CD34+ BMMNCs were plated in a methylcellulose medium containing growth factor(s), with or without indicated concentrations of recombinant human MIP-α. The number of CFU-E and BFU-E was determined at day 7 and day 14 of culture, respectively. The data are representative of three independent experiments with quadruplicate determinations.

  • Fig. 4.

    Detection of the transcripts of CCR1, CCR4, and CCR5 in CD34+ normal BMMNCs. RT-PCR was performed as described in Materials and Methods. The specificity of the PCR products was confirmed by the determination of their nucleotide sequence (data not shown).

  • Fig. 5.

    The inhibitory effects of MIP-α on BFU-E formation from CD34+ BM cells was partially abrogated by anti-CCR1 antibodies. Purified CD34+ BMMNCs were plated in a methylcellulose medium containing Epo and SCF with (solid bar) or without (open bar) recombinant human MIP-α, or a combination of the growth factors and recombinant human MIP-α with the indicated doses of anti-CCR1 antibodies (diamond bar) or control antibodies (square bar). The number of BFU-E was determined at day 14 of culture. The data are representative of three independent experiments with quadruplicate determinations. * Indicates significant difference from control culture without anti-CCR1 antibody by using one-way ANOVA analysis and multiple comparison by Fisher's method.

Tables

  • Table 1.

    Sequences of Primers Used for RT-PCR on CD34+ BM Cells

    CCR1Sense primerGCGAATTCCATGGAAACTCCAAACACCACA
    Antisense primerGCGGATCCCTAGGCCCCAAAGGCCCTCTCGTT
    CCR4Sense primerGCGAATTCCATGAACCCCACGGATATAGCA
    Antisense primerGCGGATCCCTACTCCCCAAATGCCTTGATGCC
    CCR5Sense primerGCGAATTCCATGGATTATCAAGTGTCAAGT
    Antisense primerGCGGATCCCTAGCGGGCTGCGATTTGCTTCAC
  • Table 2.

    Morphological Analysis of CCR1+or CCR1 BM Cell Populations

    CC Chemokine Receptor 1
    Cell Population+(%)
    ErythroblastMacroerythroblastBasochromatic4.40
    Polychromatic00
    Orthochromatic00
    NormoerythroblastBasochromatic7.40
    Polychromatic11.21.6
    Orthochromatic00
    Mitosis00
    GranulocyteMyeloblast80
    NeutrophilPromyelocyte1.61.6
    Myelocyte0.818
    Band form0.831.6
    Metamyelocyte0.227.8
    Segmented form016
    EosinophilPromyelocyte00
    Myelocyte00
    Band form00
    Metamyelocyte00
    Segmented form00
    Basophil00
    MonocytesPromonocyte00
    Monocyte5.42.2
    Lymphocyte58.81.2
    Plasma cell1.40
    • Bone marrow cells were sorted based on the intensities of CCR1 expression into positive and negative populations. The percentage of each cell type was calculated after morphological examination of at least 500 cells in each cell population. The mean values of results of two independent experiments are shown.

  • Table 3.

    Inhibitory Effects of MIP-1α on BFU-E Formation

    Growth FactorFrequency of BFU-E Formation
    EPO1:288
    EPO + MIP-1α0:288
    EPO + SCF1:19.2
    EPO + SCF + MIP-1α1:57.6
    EPO + SCF + MIP-1α + anti-CCR11:243-150
    EPO + SCF + MIP-1α + anti-GST1:57.6
    • Inhibitory effects of MIP-1α on BFU-E formation from CD34+ cells was partially reversed by anti-CCR1 antibodies. CD34+ cells were seeded at a concentration of 1 cell per well into a well in three 96-well plates in a methylcellulose medium containing different combinations of Epo, SCF, recombinant human MIP-1α, anti-CCR1 or control antibodies as indicated. The number of BFU-E was determined at day 14 after the culture. The data are representative of three independent experiments.

    • F3-150 Significant difference from control culture without anti-CCR1 antibody by using one-way ANOVA analysis and multiple comparison by Fisher's method.