Matrix metalloproteinase and tissue inhibitors of metalloproteinase secretion by haematopoietic and stromal precursors and their production in normal and leukaemic long-term marrow cultures.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate the turnover of the extracellular matrix and may modulate the biology of haematopoietic cells. We investigated whether MMPs and TIMPs are produced in long-term marrow cultures (LTMCs) established from normal donors and acute myelogenous leukaemia (AML) patients, and by fibroblast- (F), granulocyte macrophage- (GM) and megakaryocyte- (Meg) colony-forming unit (CFU) and erythroid burst-forming unit (BFU-E)-derived precursor cells. ProMMP-9 levels were highest (> 400 ng/ml) at week 1 of normal LTMC, whereas proMMP-2, TIMP-1, TIMP-2 and TIMP-3 levels peaked (up to 1000 ng/ml) after the establishment of the adherent layer. In LTMC from AML patients, these patterns of secretion were reversed. Moreover, we found that after a 24 h incubation in serum-free media, normal CFU-GM-, BFU-E- and CFU-Meg-derived cells secreted proMMP-9 and CFU-F-derived cells proMMP-2, in contrast to cells from LTMC adherent layer which secreted both active and latent forms of MMP-2 and MMP-9 under serum-free conditions. However, when these adherent cells were incubated in 12.5% fetal calf or horse serum or complete LTMC growth media, active forms of MMP-2 and MMP-9 were no longer detectable, and TIMP levels increased. Hence, we concluded that (i) MMPs/TIMPs are secreted by normal human bone marrow haematopoietic and stromal cells and may play an important role in intercellular cross-talk in haematopoiesis; and (ii) only latent forms of MMPs are present under LTMC conditions, indicating that the specific media used for weekly re-feeding of LTMC can block activation of MMP-2 and MMP-9, maintaining the integrity of the stromal layer and supporting haematopoiesis in vitro.

Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines.

As stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and interleukin-8 (IL-8) are implicated in the homing and mobilization of human hematopoietic progenitors (HPC), we hypothesized that these chemokines mediate the migration of HPC across subendothelial basement membranes by regulating production of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs). Assays for migration across reconstituted basement membrane (Matrigel) and chemotaxis were carried out using CD34(+) cells derived from normal human bone marrow (BM) and mobilized peripheral blood (PB). Secretion of MMPs and TIMPs was evaluated by zymography and reverse zymography and gene expression by RT-PCR. We found that an SDF-1 gradient increases the chemotaxis of BM and PB CD34(+) cells across Matrigel (BM > PB), which is blocked by inhibitors of MMPs (o-phenanthroline, rhTIMP-1, rhTIMP-2, and anti-MMP-9 and anti-MMP-2 antibodies) but enhanced by tumor necrosis factor-alpha (TNF-alpha), a strong stimulator of MMPs. Preincubation of these cells with SDF-1 stimulated the secretion of MMP-2 and MMP-9 in BM and PB CD34(+) cells but of TIMP-1 and TIMP-2 only in PB CD34(+) cells. Preincubation with MIP-1alpha and IL-8 also stimulated the secretion of MMP-9 and MMP-2 (BM > PB), but with respect to TIMPs, the effect was reversed (PB > BM), resulting in trans-Matrigel migration of BM but not of PB CD34(+) cells. We therefore propose that MMPs and TIMPs are involved in 1) SDF-1-induced chemotaxis of human HPC across subendothelial basement membranes, and 2) MIP-1alpha- and IL-8-stimulated migration of HPC.

The limited infectability by R5 HIV of CD34(+) cells from thymus, cord, and peripheral blood and bone marrow is explained by their ability to produce beta-chemokines.

The resistance of human bone marrow (BM) CD34(+) cells to human immunodeficiency virus (HIV) infection is at this point not fully understood. Recently we reported that the chemokines MIP-1alpha, MIP-1beta, and RANTES secreted by BM-derived CD34(+) cells may compete with the macrophagotropic HIV (R5 HIV) strain for the CCR5 coreceptor.In this study we extended our previous observations and examined various lympho-hematopoietic CD34(+) cells isolated from thymus (Th), cord blood (CB), mobilized peripheral blood (mPB), and BM for the expression of beta-chemokines binding to CCR5, i.e., MIP-1alpha, MIP-1beta, RANTES, MCP-2, MCP-3, and MCP-4, and the alpha chemokine SDF-1 (binding to CXCR4) as these chemokines may compete with the R5 and X4 HIV strains, respectively, for entry into cells. We found that Th-, CB-, mPB-, and BM-derived CD34(+) cells express mRNA transcripts for all the beta-chemokines tested but not for SDF-1. Using sensitive ELISA assays we found that although MIP-1alpha and MIP-1beta proteins were secreted by all the lympho-hematopoietic CD34(+) cells tested, RANTES was detectable only in media conditioned by BM- and CB-derived CD34(+) cells and not Th-derived cells. However, media conditioned by BM-, mPB- and Th-derived CD34(+) cells protected the T lymphocytic cell line (PB-1) from infection by the R5 but not the X4 HIV strain. Hence this study demonstrates that beta-chemokines are secreted by lympho-hematopoietic CD34(+) cells originating from various sources and that these endogenously secreted chemokines may limit entry of the R5 HIV strain into the cells by competing for the CCR5 coreceptor.