Inducible Levels of Gelatinase B/Matrix Metalloproteinase-9 Gene Expression in Monocytes Are Associated with Marrow Cellularity in Myelodysplastic Syndrome (MDS).

Abstract

Evidence suggests that within the hematopoietic microenvironment (ME) stromal cell function can be modified by activities produced by monocytes/macrophages and that the reciprocal is also true; stroma can influence monocyte function. Critical regulatory molecules produced by stroma are often membrane bound until cleaved by metalloproteinases (MMP); cleavage can serve to either activate or inactivate their functions, making MMPs critical components of hematopoietic regulation. We report here that gene and protein expression of human matrix metalloproteinase-9 (MMP-9) is induced in monocytes in vitro by stromal cell conditioned media (CM). Briefly, flow sorted CD14+ peripheral blood monocytes were cultured for 5 days in the presence or absence of CM from HS-5 stromal cells, and MMP-9 gene expression was determined by real time PCR. Little or no MMP-9 gene expression was detected in CD14+ cells on day 0 prior to culture. In contrast, after 5 days of culture in control media MMP-9 gene expression was increased significantly (p=0.02). However culturing CD14+ cells in CM significantly increased expression another 3 fold (p<0.0001). MMP-9 protein secretion was also increased 12-fold after culture in CM. To identify which factors participate in the induction of MMP-9, the levels of MMP-9 mRNA were determined after CD14+ cells were cultured for 5 days in the presence or absence of 9 different recombinant factors. Factors chosen were those known to be present in CM and known to have their receptors expressed by monocytes. MMP-9 gene and protein expression increased 4 to 8-fold with 0.5ng/mL MCP-1/CCL2 or IL-1b, other factors tested had negligible effects. Immune cytochemical localization of MMP-9 protein in bone marrow biopsies from healthy donors and patients with myelodysplastic syndrome (MDS) indicated that mature myeloid cells including granulocytes and monocytes were stained strongly for MMP-9 protein, whereas stromal cells, fat cells, megakaryocytes, immature myeloid cells, blasts and cells of the erythroid lineage were negative. While levels of CM-induced MMP-9 gene expression among monocytes from 10 normal donors were relatively consistent, there was significant variation in inducible gene expression among CD14+ cells from 25 MDS patients (p= 0.02). Further analysis of MMP-9 gene expression in MDS mononuclear cells indicated that induced levels were negatively correlated with the degree of marrow cellularity (p=0.0002). Although marrow cellularity is a subjective estimate that can vary from one area of bone to another, the strong statistical correlation obtained suggests it may be related directly or indirectly to MMP-9 levels. In conclusion, monocytes can express and secrete MMP-9 in response to factors secreted by stromal cells. We hypothesize that the response of MDS monocytes to stromal signals can be abnormal resulting in unusually high or low levels of MMP-9, and that this may, directly or indirectly, influence marrow cellularity in MDS patients.