Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan

A method for isolating purified populations of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells suitable for culture, using density gradient centrifugation on the polysaccharide material Stractan is described. A nonparenchymal cell digest of liver from either normal rats or rats treated with modest doses of vitamin A is layered on a discontinuous gradient of 6, 8, 12, ind 20% Stractan; lipocytes are separated efficiently from other nonparenchymal cells and are removed from the top of the gradient. Kupffer cells and sinusoidal endothelial cells, which migrate to denser interfaces in the gradient, are further purified by differential plating and selective trypsinization, respectively. Isolated highly viable lipocytes free of contaminants adhere and spread progressively over several days in primary culture and display both intrinsic vitamin A fluorescence and positive immunostaining for desmin. Lipocytes survive for prolonged periods on plain plastic, and collagen synthesis by these cells remains relatively constant for at least 28 days. Based on serial assay of DNA content, lipocytes in primary culture proliferate, beginning 7 days after plating. Kupffer cells and sinusoidal endothelial cells isolated by Stractan density centrifugation likewise retain their typical morphologic and functional characteristics in culture; the purity of these cell isolates has been confirmed by using specific fluorescent markers. This investigation demonstrates that Stractan density gradient centrifugation is an efficient, sensitive, and reproducible method for isolating pure populations of hepatic nonparenchymal cells.     

Cationic lipid-mediated transfection of liver cells in primary culture.

We describe transfection of DNA into parenchymal and individual non-parenchymal cell populations from adult rat liver in early primary culture, using cationic lipid as the carrier. All cell populations were transfectable, although lipid requirements varied by cell type and, for hepatocytes, with the age of the culture. For hepatocytes in early primary culture (2-10 hours after plating), pure DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) was strikingly more effective than commercial formulations (Lipofectin or TransfectACE) containing components in addition to, or other than DOTMA. For hepatocytes fully adapted to culture (approximately 48 hours after plating), pure DOTMA and Lipofectin were similarly effective. Under optimal conditions, about 10% of hepatocytes expressed the transfected reporter gene. CAT expression in hepatocytes doubled from 48 hours to 7 days after transfection. The effect of culture substratum on transfection efficiency also was examined. The presence of basement membrane-like matrix (EHS gel) reduced uptake of the DNA-lipid complex. However, cells in early culture that were transfected on collagen and then replated on EHS gel, displayed significantly greater reporter gene activity than did cells maintained throughout on collagen. In contrast to hepatocytes, non-parenchymal cells (lipocytes, Kupffer cells and endothelial cells, respectively) were transfected most efficiently by Lipofectin; DOTMA alone was inactive. The methods described will facilitate studies of gene regulation in individual liver cell populations.     

Behavior of a cell line derived from normal human hepatocytes on non-physiological and physiological-type substrates: Evidence for enhancement of secretion of liver-specific proteins by a three-dimensional growth pattern

Summary The behavior of a recently described cell line, HH25, derived from normal human hepatocytes, has been investigated on several different substrates—tissue-culture plastic, glass, a thin layer of rat-tail collagen I, and thin layers or thick gels of extracellular matrix derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Cellular morphology, proliferation, and secretion of three hepatocyte-specific proteins (albumin, α₁ acid glycoprotein, and α₁ antitrypsin) have been examined. There were no differences in morphology, proliferation, or differentiated function in the cells on either plastic, glass, collagen, I, or a thin layer of EHS matrix, but on a thick EHS matrix gel the cells altered their morphology (forming three-dimensional colonies with canalicular-like structures) and their production of albumin and α₁ acid glycoprotein was enhanced. This suggests that the enhanced differentiated function is associated with the morphological change (occurring only on the thick EHS gel) rather than with receptor-mediated cell-matrix interactions (which can also occur on the thin layer of EHS matrix). This cell line is therefore a good in vitro cellular model for the investigation of the roles of morphological changes and of cell-cell and cell-matrix interactions in the control of human hepatocyte behavior without the need for an extensive source of primary tissue.     

Characterization of a novel glycoprotein isolated from the basement membrane matrix of the Engelbreth-Holm-Swarm tumor

A previously undescribed protein has been isolated and purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor, a murine tumor that synthesizes an extensive matrix composed of basement membrane molecules. Molecular characterization of the molecule determined that it is a glycoprotein with internal disulfide bonds and an isoelectric point of 6.0. Electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the glycoprotein migrated as a diffuse band with a molecular weight of approximately 72,000-80,000. The amino acid composition was significantly different from known basement membrane components. Polyclonal antibodies that specifically recognize the glycoprotein localized it to the kidney glomerular basement membrane. These antibodies did not cross-react with either known basement membrane components (laminin, type IV collagen, and heparan sulfate proteoglycan), with 70K "culture shock" protein or with components of normal mouse serum (including mouse transferrin, albumin, or alpha-fetoprotein), when analyzed by "Western" immunoblots. Our data indicate that the glycoprotein is synthesized by the EHS tumor cells and is present at relatively high levels in the EHS tumor matrix.     

Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices

The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes. These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate. The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer. The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM). The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably. However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel. This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells. These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.     

Extracellular matrix synthesis is required for the movement of sclerotome and neural crest cells on collagen

During early embryogenesis cells of several different populations disperse by active cell movement from one location to another. Preexisting extracellular materials are major determinants of these dispersal patterns, but the cells are also able to modify their substrata by synthesizing and secreting extracellular matrix molecules as they move. In order to determine the contribution made by these deposited materials, several tissues from the early chick embryo have been cultured in the presence of inhibitors of extracellular matrix synthesis and secretion. The tissues examined were sclerotome cells from differentiated somites and neural crest cells. For comparison, undifferentiated somites were also cultured. The movement of these cells was compared in type I collagen gel culture and in conventional culture on artificial substrata. Inhibitors of collagen synthesis were used (cis-hydroxy proline and L-azetidine-2-carboxylic acid) in addition to a proteoglycan inhibitor (p-nitrophenyl-xylopyranoside) and a secretion inhibitor (monensin). Results indicate that sclerotome cells require collagen synthesis for movement in a collagen matrix. Reversal of the effects of collagen inhibitors, by proline and type II collagen, suggest that sclerotome cells normally condition the type I matrix in order to move in it. Inhibition of proteoglycan synthesis produced the greatest effect on the movement of neural crest cells regardless of the substratum, confirming an important role for these molecules in the crest migratory routes. The attachment of all cells to collagen was highly sensitive to the presence of monensin, which is known to reduce the deposition of glycosaminoglycans and fibronectin. These results suggest that conditioning of the extracellular matrix by newly synthesized material is required for cell attachment and movement during early development.     

Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia.

Human autosomal dominant polycystic kidney disease (ADPKD) epithelia were grown in primary monolayer cultures and their properties compared with intact kidney epithelial cultures derived from individually microdissected normal human kidney proximal convoluted tubules (PCT), proximal straight tubules (PST), and cortical collecting tubules (CCT). In vivo, ADPKD cyst epithelia exhibited a thickened basement membrane, and immunofluorescence demonstrated the presence of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan in basement membranes and type I collagen in the interstitium. ADPKD epithelia grown in culture synthesized and secreted basally a unique, extracellular matrix that took the form of proteinaceous spheroids when the cells were grown on dried, type I collagen. Incorporation of H2[S35O4] into basement membrane extracts was increased more than ten-fold in ADPKD epithelia by comparison to normal PST and CCT. In addition to incorporation into the normal tubular basement membrane 220 kD band, radioactivity was also seen at 175 kD and 150 kD in ADPKD extracts. Growth in culture of cyst-lining ADPKD epithelia was more rapid than normal tubules, and was abnormal since there was no absolute requirement for added extracellular matrix. However, when ADPKD epithelia were grown on different, exogenous matrix protein components, a profound influence on both structure and epithelial cell proliferation was seen. Growth on a complete basement membrane three-dimensional gel derived from the Engelbreth-Holm-Swarm (EHS) sarcoma led to a reduction in the numbers of spheroids and increase in amorphous filaments. Incorporation of [3H]-thymidine into ADPKD epithelia was greater than into normal PCT, PST, and CCT and was also greatly modified by the type of extracellular matrix components provided. In studies using single matrix components, the strongest proliferative response was seen when ADPKD epithelia were plated on type I collagen greater than type IV collagen greater than fibronectin greater than laminin. These findings suggest that the excessive growth of cyst-lining epithelia may be, at least in part, a result of abnormal basement membrane and extracellular matrix production by ADPKD cells.     

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.     

Type I collagen reduces the degradation of basal lamina proteoglycan by mammary epithelial cells

When mouse mammary epithelial cells are cultured on a plastic substratum, no basal lamina forms. When cultured on a type I collagen gel, the rate of glycosaminoglycan (GAG) synthesis is unchanged, but the rate of GAG degradation is markedly reduced and a GAG-rich, basal lamina-like structure accumulates. This effect of collagen was investigated by comparing the culture distribution, nature, and metabolic stability of the 35S-GAG-containing molecules produced by cells on plastic and collagen. During 48 h of labeling with 35SO4, cultures on collagen accumulate 1.4-fold more 35S-GAG per microgram of DNA. In these cultures, most of the extracellular 35S-GAG is immobilized with the lamina and collagen gel, whereas in cultures on plastic all extracellular 35S-GAG is soluble. On both substrata, the cells produce several heparan sulfate-rich 35S-proteoglycan fractions that are distinct by Sepharose CL-4B chromatography. The culture types contain similar amounts of each fraction, except that collagen cultures contain nearly four times more of a fraction that is found largely bound to the lamina and collagen gel. During a chase this proteoglycan fraction is stable in cultures on collagen, but is extensively degraded in cultures on plastic. Thus, collagen-induced formation of a basal lamina correlates with reduced degradation and enhanced accumulation of a specific heparan sulfate-rich proteoglycan fraction. Immobilization and stabilization of basal laminar proteoglycan(s) by interstitial collagen may be a physiological mechanism of basal lamina maintenance and assembly.     

Reconstituted basement-membrane matrix modulates fibroblast activities in vitro

Cellular growth and collagen biosynthesis were compared in dermal calf fibroblasts cultured on plastic or on a reconstituted basement membrane gel, termed matrigel. This matrix, extracted from Engelbreth-Holm-Swarm tumors, consists mainly of laminin, entactin, type IV collagen, and heparan sulfate proteoglycan. The multiplication rate of fibroblasts grown on matrigel was stimulated compared to that of monolayered cells cultured on plastic, and these cells formed multilayers after 4 days. Protein and collagen biosynthesis was reduced in fibroblasts cultured on matrigel. A higher proportion of the newly synthesized collagen (40%) was incorporated to the extracellular matrix in cultures grown on matrigel than in those grown on plastic (14%). Type III collagen was the preferential collagen type deposited on matrigel, and the ratio of type III:type I collagens secreted in the medium was also slightly higher in cultures grown on matrigel. Partially processed collagen was more abundant in fibroblasts grown on matrigel than in cells cultured on plastic. Finally, cells grown on matrigel exhibited a higher catabolic activity than cells grown on plastic. In this experimental model, the reconstituted basement-membrane matrix seems to influence the activities of fibroblasts significantly.     

Hepatic stellate cell (vitamin A-storing cell) and its relative--past, present and future

HSCs (hepatic stellate cells) (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells) exist in the space between parenchymal cells and liver sinusoidal endothelial cells of the hepatic lobule and store 50-80% of vitamin A in the whole body as retinyl palmitate in lipid droplets in the cytoplasm. In physiological conditions, these cells play pivotal roles in the regulation of vitamin A homoeostasis. In pathological conditions, such as hepatic fibrosis or liver cirrhosis, HSCs lose vitamin A and synthesize a large amount of extracellular matrix components including collagen, proteoglycan, glycosaminoglycan and adhesive glycoproteins. Morphology of these cells also changes from the star-shaped SCs (stellate cells) to that of fibroblasts or myofibroblasts. The hepatic SCs are now considered to be targets of therapy of hepatic fibrosis or liver cirrhosis. HSCs are activated by adhering to the parenchymal cells and lose stored vitamin A during hepatic regeneration. Vitamin A-storing cells exist in extrahepatic organs such as the pancreas, lungs, kidneys and intestines. Vitamin A-storing cells in the liver and extrahepatic organs form a cellular system. The research of the vitamin A-storing cells has developed and expanded vigorously. The past, present and future of the research of the vitamin A-storing cells (SCs) will be summarized and discussed in this review.     

THREE-DIMENSIONAL STRUCTURE OF EXTRACELLULAR MATRIX REVERSIBLY REGULATES MORPHOLOGY,PROLIFERATION AND COLLAGEN METABOLISM OF PERISINUSOIDAL STELLATE CELLS (VITAMIN A-STORING CELLS)

The three-dimensional structure of the extracellular substratum was found to regulate reversibly the morphology, proliferation and collagen synthesis of perisinusoidal stellate cells (lipocytes, i.e. fat-storing ‘Ito’ cells). On non-coated polystyrene and type I collagen-coated culture dishes, the cells spread well and extended their cellular processes. On the surface of type I collagen gels, the cells gathered and formed a mesh-like structure. However, in type I collagen gel where the cells were surrounded by type I collagen three-dimensionally, the cells extended their fine cellular processes and resembled the star-shaped stellate cells seenin vivo. The cell proliferation was more prominent in culture dishes coated with type I collagen or in polystyrene culture dishes than on or in type I collagen gels. The collagen synthesis was affected in the same manner. These data indicate that the nature and the three-dimensional structure of the extracellular matrix (ECM) can regulate morphology, proliferation and functions of the perisinusoidal stellate cells. In order to examine the reversibility of these regulations, we liberated cultured cells with trypsin or with purified bacterial collagenase and re-seeded them onto or into each substratum. The cells changed their shape, rate of proliferation and collagen synthesis according to each new substratum. These results indicate that the three-dimensional structure of ECM reversibly regulates the morphology, proliferation rate and functions of the perisinusoidal stellate cells.     

Extracellular matrix is required for the survival and differentiation of transplanted hepatic progenitor cells

Engelbreth-Holm-Swarm (EHS) gel has been reported to maintain the mature hepatocyte phenotypes in primary cultured hepatocytes. We investigated the effect of EHS gel on the differentiation of fetal liver cells, which contain stem/progenitor cells. The isolated fetal liver cells cultured on EHS gel formed a spherical shape and increased liver-specific gene expressions compared with cells cultured on collagen. The hepatic progenitor cells that were transplanted subcutaneously to BALB/c nude mice could survive and express hepatocyte marker alpha-fetoprotein when the cells were suspended with EHS gel. These findings demonstrate that EHS gel supports cytodifferentiation from immature progenitor cells to hepatocytes and maintain its differentiated phenotypes in vitro and in vivo.     

Extracellular matrix and mouse mammary cell function: Comparison of substrata in culture

Summary Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveoluslike structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominately in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.     

Effects of soluble factors and extracellular matrix on DNA synthesis and surfactant gene expression in primary cultures of rat alveolar type II cells

Proliferation and differentiation of epithelial cells are thought to be regulated by soluble factors in extracellular fluid and insoluble components of the extracellular matrix. We have examined the combined effects of soluble factors and an extracellular matrix (EHS matrix) on DNA synthesis, cell proliferation, and surfactant protein gene expression in primary cultures of alveolar type II epithelial cells. Cells on EHS matrix cultured in DMEM containing insulin, cholera toxin, EGF, aFGF, 5% rat serum, and 15-fold concentrated bronchoalveolar lavage fluid (D-GM) formed larger aggregates than cells cultured on the same substratum in DMEM containing 5% rat serum (D-5). Cells cultured in D-GM on EHS matrix incorporated more [³H]-thymidine than cells on the same substratum in D-5, with an eight-fold increase seen on day 4 of culture. This increase in [³H]-thymidine incorporation was accompanied by a labeling index of greater than 65% of the cells. Cell counts showed that exposure of type II cells on EHS matrix to D-GM resulted in increased cell number on day 4 of culture. [³H]-thymidine autoradiography combined with immunostaining with anti-cytokeratin, anti-SP-A, and anti-vimentin antibodies demonstrated that the proliferating cells were epithelial cells that contained SP-A. Type II cells cultured on plastic in D-GM also showed increased [³H]-thymidine incorporation compared to cells cultured in D-5. The level of [³H]-thymidine incorporation by cells on plastic, however, was significantly less than that seen in cells cultured in the same medium on EHS matrix. Type II cells cultured on EHS matrix in D-GM had a decreased abundance of mRNAs for SP-A and SP-C than cells cultured on EHS matrix in D-5 as determined by Northern analysis. This inhibition was reversed by switching from D-GM to D-5 on day 4 and culturing the cells for an additional 4 days. In contrast, SP-B mRNA was increased in response to D-GM. This increase was not reversed by switching from D-GM to D-5 on day 4. These results suggest that the interaction of soluble factors and extracellular matrix components has a strong influence on type II cell proliferation, which were partially associated with the reversible inhibition of lung tissue-specific protein mRNAs. Their dynamic interplay among the type II cell, the extracellular matrix, and growth factors may determine multicellular functions and play an important role in normal lung development and in the repair of the lung epithelium following injury.     

Biosynthesis of sulphated macromolecules by rabbit lens epithelium. II. Relationship to basement membrane formation

Rabbit lens epithelial cells display a similar "cobblestone" morphology and produce the same complement of sulphated macromolecules (also see Heathcote, J.G., and R.W. Orkin, 1984, J. Cell Biol., 99:852-860) whether grown on plastic or glass, dried films of gelatin or type IV collagen with laminin, or on gels of type I collagen. There was no evidence of basement membrane formation by these cells when they were grown on plastic, glass, or dried films. In contrast, cultures that had been grown on gels deposited a discrete basement membrane that followed the contours of the basal surfaces of the cells and in addition, they secreted amorphous basement membrane-like material that diffused into the interstices of the gel and associated with the collagen fibrils of the gel. A significant proportion (approximately 70%) of the heparan sulphate proteoglycan fraction that was secreted into the culture medium (fraction MI) when the cells were grown on plastic became associated with the cell-gel layer in the gel cultures. Further, when basement membrane was isolated by detergent extraction, greater than 90% of the 35S-labeled material present was in this heparan sulphate proteoglycan.     

Surface contact modulation of inflammatory macrophage antibody dependent cytotoxicity and prostanoid release.

Adherence to extracellular matrix proteins modulates the functional and secretory activities of mononuclear phagocytes, although the mechanisms regulating these adherence-dependent changes are poorly understood. In this study, the ability of rat inflammatory peritoneal macrophages (PM) to adhere to an endothelial cell-derived extracellular matrix or a denatured collagen/fibronectin-coated surface and perform antibody dependent cell cytotoxicity (ADCC) and secrete reactive oxygen intermediates was compared with PM adherent to tissue culture plastic. Prostaglandin E2 (PGE2) and thromboxane B2 (TxB2), two major cyclooxygenase products released by inflammatory macrophages, were also measured by PM adherent to the protein coated surfaces. Rat exudate PM were equally adherent to tissue culture plastic or wells coated with either endothelial cell derived matrix or denatured collagen (gelatin)/fibronectin. PM adherent to a denatured collagen/fibronectin-coated wells demonstrated significantly less cytolytic activity (15 +/- 2% lysis) when compared with either tissue culture plastic adherent PM (43 +/- 7% lysis) or PM adherent to extracellular matrix (59 +/- 11% lysis). PM adherent to extracellular matrix released twofold more TxB2 than plastic adherent PM, while PM adherent to denatured collagen/fibronectin released 40% more PGE2 than cells adherent to tissue culture plastic or 80% more PGE2 than PM adherent to the extracellular matrix. PM adherent to denatured collagen/fibronectin release less superoxide anion (27 +/- .9 nmoles/10(6) PM) than PM adherent to either tissue culture plastic (43 +/- 1 nmoles/10(6) PM) or the extracellular matrix (60 +/- 0.5 nmoles/10(6) PM). Furthermore, incubation of plastic adherent PM with exogenous PGE2 reduced superoxide production in a dose-dependent manner. These results demonstrate that the inhibition of ADCC and secretion of reactive oxygen intermediates by PM adherent to a denatured collagen/fibronectin surface correlated with an increased release of the immunosuppressive prostanoid PGE2. Furthermore, the addition of exogenous PGE2 to plastic adherent PM reproduced the depression in ADCC and superoxide anion production observed by PM adherent to a denatured collagen/fibronectin surface. These studies suggest that the increased production and release of PGE2 by inflammatory macrophages adherent to a denatured collagen surface may act to suppress cytotoxic mechanisms and thereby constitutes part of an autocrine feedback mechanism regulating macrophage function during wound injury.