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Recent studies indicate potential roles of monocyte chemotactic protein-1 (MCP-1) in recruitment of monocytes to sites of inflammation. However, their increased expression does not always correlate with monocyte influx, suggesting other possible biological activities for this member of the C-C chemokine family. In view of its potential role in regulating extracellular matrix expression in fibrotic disorders, the effects of MCP-1 on lung fibroblast collagen expression were evaluated. Isolated rat lung fibroblasts were treated with increasing doses of MCP-1 for variable periods of time and examined for effects on collagen synthesis and expression of procollagen ␣ 1 (I) mRNA expression. The results show that MCP-1 was able to stimulate collagen expression in these cells in a dose-dependent manner but required over 24 h for significant elevation to occur. In view of this delayed time course, the possibility of mediation via endogenous transforming growth factor ␤ (TGF␤) was tested by the ability of anti-TGF␤ antibody to inhibit this MCP-1 stimulation of collagen expression. Significant but incomplete inhibition by this antibody was observed. Pretreatment of the cells with antisense but not by sense or missense TGF␤ 1 oligodeoxyribonucleotides caused essentially complete inhibition of this MCP-1 stimulatory effect. Furthermore, MCP-1 treatment was found to also stimulate TGF␤ secretion and mRNA expression, which was also abolished by pretreatment with antisense TGF␤ 1 oligodeoxyribonucleotides. The kinetics of TGF␤ expression indicates that significant increase preceded that for collagen expression. Binding studies using 125 I-labeled MCP-1 indicated the presence of specific and saturable binding sites with a dissociation constant consistent with the dose response curves for stimulation of fibroblast collagen synthesis and TGF␤ activity by MCP-1. These results taken together suggest that MCP-1 stimulates fibroblast collagen expression via specific receptors and endogenous up-regulation of TGF␤ expression. The latter then results in autocrine and/or juxtacrine stimulation of collagen gene expression.Monocyte chemoattractant protein-1 (MCP-1 1

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Sepracell-MN has provided a simple, rapid means of isolating peripheral blood monocytes. However this product is no longer available. Consequentially we have developed a Percoll gradient which matches Sepracell-MN in simplicity and yield of monocytes. Using this Percoll gradient, an average of 7 x 10(6) monocytes with a purity of 83% were obtained from 30-40 ml of blood. These monocytes were at least 97% viable and responded to chemotactic stimuli in comparable numbers to those prepared using Sepracell-MN.

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Bleomycin-induced fibrosis in rodents has been used extensively as a model of human pulmonary fibrosis. The influx of monocytes observed during the early stages of fibrosis is at least partially regulated by the elaboration of chemotactic factors in the lung. Exposure of alveolar macrophages (AM phi) to bleomycin either in vivo or in vitro stimulated secretion of monocyte chemotactic activity (MCA). This MCA has been previously characterized as being primarily due to fibronectin fragments. The present experiments revealed that bleomycin also induced AM phi to secrete a second chemotactic factor, transforming growth factor-beta (TGF-beta). However, the TGF-beta secreted by macrophages was in latent form, since no TGF-beta activity was detected unless AM phi conditioned medium (CM) was acid-activated. After acidification, chemotactic activity in CM from AM phi stimulated with bleomycin in vitro was increased by 3.6, whereas activity in AM phi CM from fibrotic rats increased by 2 and that of a bleomycin-stimulated AM phi cell line increased by 1.6. This acid-activatable chemotactic activity was inhibited by antibody to TGF-beta. Bleomycin-stimulated AM phi s secreted significantly more TGF-beta than did unstimulated controls. Further, in vitro exposure of AM phi to bleomycin induced TGF-beta mRNA expression in a time- and concentration-dependent manner, with maximal mRNA being detected following a 16-h incubation with 1 microgram/ml bleomycin.

Fluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migratiow however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. 5774 macrophages were labeled with either calcein-acetoxymethyl ester (calcein-AM) or 2',7'-bis-(2-carboxyethy1)-5-(and-6)-carbo~uo-rescein, acetomethyl ester (BCECF-AM), then assayed for their ability to migrate to zymosan-activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, comparison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF-AM decreased the number of cells responding to ZAS, while calcein-AM had essentially no effect. Neither probe sigFluorescent probes have been utilized to label a variety of cell types for a wide range of applications including the study of leukocyte migration in vivo, intracellular pH, cell permeability, and cell viability (3,5,7,8). Two such probes which have been used for cell viability and migration studies are 2',7'-bis-( 2-carboxyethyl)5-( and-6-)-carboxyfluorescein, acetomethyl ester (BCECF-AM) and calcein-acetomethyl ester (calcein-AM). When internalized by the cell, these fluorescent molecules are converted by cytoplasmic esterases to a membrane impermeable form (3); the result is cells with brightly fluorescent cytoplasms which can be easily quantified.Recently, BCECF-AM has been used to label leukocytes for in vitro chemotaxis assays (4). This use of fluorescently labeled cells in chemotaxis assays allowed migration to be quantified rapidly using a fluorescent plate reader rather than by the tedious method of visually counting migrated cells ( 2 , 3 ) .The automation of chemotaxis assays was a technologd c a n t l y affected the number of cells migrating to medium alone. The inhibitory effects of BCECF-AM on cell migration increased with probe concentration (0. Abbreviations: BCECF-AM, 2',7'-bis-(2-~arboxyethyl)5-(~d-~)-carboxyfluorescein, acetoxymethyl ester; RBSA, RPMI + 2 mg/ml bovine serum albumin; ZAS, zymosan-activated serum.

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