A new soluble mediator was found in supernatant fluid from cultures of human peripheral blood leukocytes that were stimulated by phytohemagglutinin, or by antigenic material present in human dental plaque deposits. This soluble Jactor produced bone resorption in organ cultures of fetal rat bones as measured by increased release of calcium-45, and also increased the number of active osteoclasts.

Human macrophages produce in culture a factor termed mononuclear cell factor (MCF) that increases the production of collagenase and prostaglandins by isolated adherent rheumatoid synovial cells. A factor with similar biologic activity is also produced by the murine macrophage cell line P388D1. By using a sequential purification scheme involving ammonium sulfate fractionation; chromatography on DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose; and discontinuous polyacrylamide gel electrophoresis, the P388D1 cell-derived, synovial cell-stimulating factor was -copurified with the lymphocyte-activating factor [LAF; interleukin 1 (IL 1)]. The specific activity of the partially purified LAF (IL 1) was approximately 15,000-fold higher than that of the LAF (IL 1) in the original P388D1 cell culture supernatant. On the basis of (i) the copurification of the P388D1 cell-derived LAF (IL 1) and synovial cell-stimulating factors; (ii) the similarity in cell of origin, molecular weight, and phenylglyoxal sensitivity of human MCF and murine LAF (IL 1); and (iii)

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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 ...

Mice that are unresponsive to lipopolysaccharide (LPS) (strain C3H/HeJ) can be rendered LPS-sensitive by the adoptive transfer of bone marrow cells from LPS-sensitive mice (strain C3H/HeN). This model of adoptive transfer was used to evaluate the contribution of lymphoreticular cells to five effects of endotoxin on the host: immunogenicity, adjuvanticity, lethality, induction of interferon, and induction of colony-stimulated factor. C3H//HeJ mice became sensitive to each of these effects after adoptive transfer of bone marrow cells from C3H/HeN mice. The efficacy of transfer was directly proportional to the dose of X-irradiation and inversely proportional to the number of surviving host stem cells. The most effective dose of radiation was 850 rad, and C3H/HeN leads to C3H/HeJx chimeras prepared at this dose were as sensitive to LPS for each parameter tested as were the C3H/HeN donors except for a threefold greater resistance to lethality than LPS-responsive C3H/HeN mice. C3H/HeN mice could also be rendered unresponsive to LPS by the adoptive transfer of C3H/HeJ bone marrow cells. C3H/HeN chimeras were resistant to all of the effects of LPS studied except for the induction of colony-stimulating factor. These results demonstrate that lymphocytes and/or macrophages play a primary role in mediating a number of diverse and seemingly unrelated host responses to endotoxin.