Collagenase Production by Endotoxin-Activated Macrophages

Abstract

Peritoneal exudate macrophages, when exposed to bacterial lipopolysaccharide in culture, were found to produce collagenase (EC 3.4.24.3). This enzyme was not detected in extracts of the macrophages or in media from nonstimulated macrophage cultures. Lipidcontaining fractions of the lipopolysaccharide, including a glycolipid from the rough mutant of Salmonella minnesota (R595) and lipid A, were potent stimulators of collagenase production. The lipid-free polysaccharide fraction had no effect. Cycloheximide-prevented' the production of collagenase by endotoxin-treated macrophages, suggesting that. it was newly synthesized.Increased levels of collagenase (EC 3.4.24.3) have been found in acutely and chronically inflamed tissues, including healing wounds, rheumatoid joints, and inflamed gingiva, in which connective tissue has been destroyed. Polymorphonuclear leukocytes, which predominate in acute inflammatory reactions, produce collagenase (1). However, after the acute inflammatory process and in chronic inflammatory lesions where collagen breakdown is most evident, relatively few neutrophils are present. Instead, macrophages are present in the tissue in large numbers, suggesting that these cells may play an active role in connective tissue degradation. Indeed, macrophages of alveolar origin have been reported to contain collagenase (2, 3).In the present experiments we found that macrophages obtained from a; peritoneal exudate did not contain or secrete significant amounts of collagenase in vitro. Since it is known that certain activities of macrophages, including respiration (4), phagocytosis (5, 6), and release of some lysosomal enzymes (7,8), increase when macrophages are incubated with a variety of stimulants, we exposed macrophage-rich cell populations to endotoxin in vitro and then assayed the culture media for collagenase activity. Here we report that activation of peritoneal macrophages by endotoxin, particularly the lipid A component, induces these cells to produce an enzyme that lyses reconstituted collagen fibers. MATERIALS AND METHODSMale Hartley guinea pigs (500 g) were injected intraperitoneally with 20 ml of sterile Drakeol (6-VR, Pennsylvania Refining Co., Butler, Pa.) to induce a cellular exudate. Four days later, the peritoneum was lavaged with 150 ml of heparinized (2 U/ml) saline and the cells were concentrated by centrifugation at 250 g. The cells were resuspended and washed twice in Dulbecco-Vogt's medium supplemented with 100 U/ml of penicillin and 100 Mg/ml of streptomycin and 2 Abbreviation: LPS, lipopolysaccharide. mM glutamine. Ten milliliter of this serum-free medium containing 4 X 106 cells per ml were added to 75-cm2 plastic flasks and incubated in an atmosphere of 5% CO2 and air at 370. After 4 or 24 hr the cell cultures were washed three times with Dulbecco's medium to remove nonadherent cells and 10 ml of fresh serum-free medium were added to each flask. The adherent cells obtained in this manner consisted of 94-96% macrophages, as determined by morphology and by the ability ...

Monocyte Membrane Type 1-Matrix Metalloproteinase

“…MMPs have been associated with several pathological conditions such as chronic inflammatory lesions and cancer metastasis (33)(34)(35). Therefore, regulation of signal transduction events that are associated with the production of inflammatory mediators, such as MMPs, has been of considerable interest.…”

Lymphokine-induced production and release of lysosomal enzymes by macrophages.

“…The observations we describe show that stimulated lymphoid cells release products that not only arrest macrophage mobility and lead to cell enlargement, but also induce the production of lysosomal and other cellular enzymes and the release of the hydrolytic enzymes into the extracellular environment. By very similar procedures, collagenase production and release by macrophages have been induced (16). The releasing cells appear to be otherwise viable and healthy by several criteria.…”

“…The releasing cells appear to be otherwise viable and healthy by several criteria. The released enzymes have the capacity to cause extensive pathologic alteration to tissues (13)(14)(15)(16)(17)(18). A similar response can be induced in mononuclear phagocytes exposed to a variety of bacterial and other substances associated with chronic inflammatory lesions (14,15,21).…”

“…The effect of lymphokines on the levels and distribution of various cellular and lysosomal enzymes of mononuclear phagocytes has not been determined. This is an important question because mononuclear phagocytes contain numerous hydrolytic enzymes that are able to alter connective tissue constituents (13)(14)(15)(16)(17), and these cells play an important role in delayed hypersensitivity reactions and other chronic inflammatory lesions (18). We have found that stimulated human peripheral blood lymphocytes release products that not only induce in cultured macrophages the effects described above, but also cause the cells, which are otherwise viable and healthy, to produce and selectively release large quantities of lysosomal enzymes into the extracellular culture environment.…”

Tumour Immunology: Alternative Perspectives

“…The first proteins shown to be actively secreted by macrophages were hydrolytic enzymes [38,39,40]. Together with fibrinolytic activity [41], synthesis of fibronectin [42] and other connective tissue components [43], they give the macrophages the ability to modify the matrix around them.…”