Transendothelial migration of monocytes followed by their differentiation into macrophages involves interaction of monocytes with subendothelial matrix. The influence of extracellular matrix on monocyte-macrophage differentiation was studied using an in vitro model system with human PBMC maintained on different matrix protein substrata. Upregulation of macrophage specific marker activities such as endocytosis of modified proteins, changes in expression of cell surface antigen, and production of matrix metalloproteinases was studied. Cells maintained on Fibronectin (Fn) showed significantly higher rate of endocytosis and production of MMP2 and MMP9 when compared to other matrix protein substrata. Immunoblot analysis, ELISA, and zymography showed that Fn-dependent upregulation of MMPs was blocked by antibodies to alpha(5)beta(1) integrin indicating that the Fn effect was mediated by integrins. The Fn effect on mo-mPhi was blocked by genistein and herbimycin. As monocytes differentiate to macrophages there was an increase in the rate of production of Fn. These results indicate the influence of the microenvironment of the cell, particularly Fn, on mo-mPhi differentiation and integrin-mediated downstream signaling through focal adhesion kinase and Src type tyrosine kinase is involved in this.

Transendothelial migration of peripheral blood mononuclear cells (PBMCs) and their subsequent interaction with the subendothelial matrix lead to their differentiation to macrophages (mphis). To study whether preexposure of monocytes in circulation to modified proteins influences their differentiation to mphis, an in vitro model system using isolated PBMC in culture was used. The effect of modified proteins such as oxidatively modified LDL (ox-LDL), acetylated and non-enzymatically glycated-BSA (NEG-BSA) on the differentiation process was studied by monitoring the upregulation of mphi specific functions such as endocytosis, production of matrix metalloproteinases (MMPs), expression of surface antigen, activity of beta-glucuronidase and down regulation of monocyte specific myeloperoxidase activity. Rate of endocytosis, production of MMPs and beta-glucuronidase activity were significantly greater in cells treated with modified proteins irrespective of the nature of modification. Both CuSO4 ox-LDL and HOCl ox-LDL increased the rate of expression of the mphi specific functions. FACS analysis showed that the rate of upregulation of mphi specific CD71 and down regulation of monocyte specific CD14 were high in cells supplemented with modified proteins. Studies using PPARgamma antagonist and agonist suggest its involvement in CuSO4 ox-LDL induced monocyte-macrophage (mo-mphi) differentiation whereas the expression of macrophage specific functions in cells exposed to other modified proteins was independent of PPARgamma. PBMC isolated from hypercholesterolemic rabbits in culture expressed mphi specific functions at a faster rate compared to normal controls indicating that these observations are relevant in vivo. These results indicate that preexposure of monocytes to modified proteins promote their differentiation to mphis and may serve as a feed forward type control for clearing modified proteins.

The influence of extracellular matrix (ECM) on monocyte-macrophage (mo-mphi) differentiation was investigated using an in vitro model with human peripheral blood mononuclear cells (PBMC) maintained on different matrix protein substrata. Macrophage specific markers associated with differentiation studied were, (a) endocytosis of modified proteins; (b) appearance of mphi specific matrix metalloproteinases (MMPs); (c) activities of myeloperoxidase (MPO) and beta-D-glucuronidase; (d) changes in the expression of cell surface antigens. As the duration of monocytes in culture increased, a progressive increase in the rate of differentiation was seen as evidenced by mphi specific functions such as endocytosis of 125[I] acetyl BSA and the appearance of gelatinases A and B. Significantly higher rate of endocytosis and production of MMPs were found in monocytes maintained on fibronectin (FN) and COL I than on COL IV (FN > COL I > COL IV) indicating that cells in contact with stromal components differentiate at a faster rate. FACS analysis done on cells maintained in vitro for phenotypic profile characteristic to mo-mphi differentiation showed downregulation of CD14 occurring in a substratum dependent manner viz, (FN > COL IV > COL I) and upregulation of CD71 was high in cells maintained on COL I and COL IV. Intracellular enzymatic activities such as MPO significantly decreased irrespective of matrix substrata, while beta-D-glucuronidase activity increased in a substratum dependent manner (FN > COL I > COL IV). Pretreatment of cells with genistein significantly decreased the secretion of MMPs, particularly MMP 9 in cells maintained on ECM protein (FN) indicating a phosphorylation dependent signalling process in mediating matrix effect. The results of these in vitro studies on mphi specific markers suggest that apart from the diffusible factors, the microenvironment as provided by various matrix proteins particularly FN can modulate mo-mphi differentiation.