Macrophages express mRNA for all three T6C chains.
COLLAGEN CATABOLISM SKIN
Total RNA was isolated from human cells including coronary artery SMC, skin fibroblasts (FB), A7 skin melanoma cells (A7), THP-1 monocytes (TPH-1 mono), 6 day-old THP-1 macrophages (TPH-1 macro), and 1-h-old primary monocytes (Primary mono) and 14 day-old primary monocyte-derived macrophages (Primary macro) for RT-PCR. Messenger RNA-specific primers located in two different exons of the corresponding mRNAs were used to amplify cDNA fragments derived from T6C mRNAs (COL6A1, COL6A2, and COL6A3). A, RT-PCR analysis of T6C mRNAs compared with mRNAs of the housekeeping genes GAPDH and SRP14, a 14 M r(K) signal recognition particle. Primary and THP-1 monocytes and macrophages express T6C mRNA and macrophages secrete T6C protein. For these experiments the cells were serum starved in the presence of ascorbic acid and cytokines for 24 h at the concentrations indicated in Figs. In other experiments, macrophages were stimulated with various cytokines (Merck) at different concentrations as indicated. Its presence in the medium is required for proper triple helix formation in the endoplasmic reticulum and thus for the secretion of procollagen peptides ( 21). Ascorbic acid is a cosubstrate for prolyl hydroxylase that catalyzes the posttranslational hydroxylation of proline residues in collagen chains.
1 denote the days on which the cells were harvested. For analyzing T6C production during monocyte-to-macrophage differentiation, the medium was replaced with fresh medium without serum and with 50 μg/ml ascorbic acid for 24 h before harvesting the cells.
Human monocyte-derived primary macrophages were obtained by culturing 10 7 monocytes per 75-cm 2 flask in RPMI 1640 medium (Sigma-Aldrich) containing 20% autologous human serum (PAA Laboratories), 1% nonessential amino acids, 1% sodium pyruvate, and 0.1 mg/ml penicillin/streptomycin/ l-glutamine (Sigma-Aldrich) for 14 days unless stated otherwise. The purity of the monocytes was checked by flow cytometry and exceeded 95%. The procedure for monocyte isolation was approved by the University Hospital Ethics Committee, Münster, Germany. Human monocytes were obtained from consenting healthy volunteers by leukapheresis and countercurrent elutriation as previously described ( 20). We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair.
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