Tryptophan degradation in transplanted tumor cells undergoing rejection.

The depletion of an essential amino acid, tryptophan, caused by indoleamine 2,3-dioxygenase induction in vitro, has been shown to be due to a mechanism that is used in self-defense against inhaled microorganisms and tumor growth. In this communication, we report the results of measuring dioxygenase activity in the peritoneal exudate cells and tumor cytotoxicity at the transplantation loci after in vivo transplantation of tumor cells into the peritoneal cavity of syngeneic or allogeneic strains of mice. The enzyme was induced only when the tumor cells were being rejected from allogeneic animals and no change was observed when the cells continued to grow in syngeneic animals. Furthermore, when the syngeneic tumor cells in a diffusion chamber were i.p. transplanted simultaneously with i.p. injection of allogeneic tumor cells, the enzyme was induced not only in allografted tumor cells but also in the syngeneic tumor cells. Under these conditions, the tumor cells in the diffusion chamber ceased to grow and 50% of the cells were rejected. To determine the type of cells containing the induced enzyme, the peritoneal exudate cells (tumor cells and host cells--mostly small lymphocytes) were separated into six fractions by sedimentation under gravity and by differential centrifugation. Approximately 80% of total enzyme activity was localized in a tumor-rich fraction (98.9% purity), whereas only 0.2% of the activity was found in a lymphocyte-rich fraction (99.5% purity). The localization of indoleamine 2,3-dioxygenase in the tumor cells was confirmed by complement-dependent lysis with specific antibodies against tumor and host cells.

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis.

The comparison of lysosomal enzymes activities in alveolar and peritoneal macrophages of rat.

Studies were carried out on macrophages isolated from control and thioglycollate injected rats. Intraperitoneal injection of thioglycollate had no effect on the number of harvested alveolar macrophages but caused a marked increase in the number of peritoneal macrophages. The specific activities of all the investigated enzymes were significantly lower in peritoneal macrophages from control rats in comparison to alveolar macrophages. Thioglycollate stimulation of peritoneal macrophages caused increase in activities of all lysosomal hydrolases studied. Cathepsin B, N-acetylglucosaminidase and esterase showed the highest level of stimulation.

Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rat.

Peritoneal exudate produced in rat injected with caseinate contained extracellular phospholipase A2. The activity required Ca2+ ion and had a pH optimum of 9 (Chang, H.W., Kudo, I., Hara, S., Karasawa, K., & Inoue, K. (1986) J. Biochem. 100, 1099-1101). This phospholipase A2 was purified about 14,000-fold to near homogeneity by the sequential use of column chromatography on Sephadex G-75, Toyopearl HW-65, and TSK ODS-120T reverse-phase HPLC. The final preparation showed a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 13,500. The enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). When 1-acyl-2-[1-14C]inoleoyl-sn-glycero-3-phospholipids were used as a substrate, the apparent Km values were 0.027 mM with PE, 0.032 mM with PS, and 0.1 mM with PC, and the Vmax values were 105 mumol/min/mg with PE, 71 mumol/min/mg with PC. The enzyme activity was inhibited by p-bromophenacyl bromide, dithiothreitol, and mepacrine. The amino acid sequence of the NH2-terminal portion and the amino acid composition of the purified enzyme were determined. They were different from those of rat pancreatic phospholipase A2, but very similar to those of phospholipase A2 secreted from rat platelets.

Extracellular phospholipase A2 activity in peritoneal cavity of casein-treated rats.

An extracellular phospholipase A2 was detected in the peritoneal cavity of rats injected intraperitoneally with casein. This activity required Ca2+ ion and had a pH optimum of 9. The increase of phospholipase A2 activity in the peritoneal fluid as a function of time roughly paralleled the increase in the number of polymorphonuclear (PMN) leukocytes found in the peritoneal cavity. Phospholipase A2 activity was also observed in rat PMN homogenates, and its characteristics were similar to those of the enzyme found in cell-free peritoneal fluid fraction. PMN leukocytes may release non-lysozomal phospholipase A2 into the extracellular space as 'inflammation reactions' proceed.

Lysosomal elastase and cathepsin G in beige mice. Neutrophils of beige (Chediak-Higashi) mice selectively lack lysosomal elastase and cathepsin G.

A profound decrease in activities of the two lysosomal serine proteinases, elastase, and cathepsin G, was found in neutrophils of four independent beige mutants. Elastase and cathepsin G activities were assayed with the specific synthetic substrates MeO-Suc-Ala-Ala-Pro-Val-MCA and Suc-Ala-Ala-Pro-Phe-pNA, respectively. The defect is intrinsic to cells of beige mice, since transplantation of bone marrow from normal to mutant mice restored normal proteinase activity, and normal mice transplanted with beige marrow produced neutrophils with a deficiency of proteinase activity. The loss of elastase and cathepsin G activity was confirmed by separation of [3H]diisopropylfluorophosphate-labeled proteins on denaturing gels, which also revealed that other serine proteinases are at normal levels in beige neutrophil extracts. The deficiency of lysosomal proteinase activity appears specific, in that four other common neutrophil lysosomal enzymes, plus the spectrum of major neutrophil proteins are not affected by the beige mutation. The deficiency of proteinase activity is likely not the primary genetic alteration of the beige mutation, since more than one proteinase is affected, and heterozygous F1 mice have normal rather than intermediate levels of both proteinases. The lowered proteinase activity may contribute to the high susceptibility of beige mice and Chediak-Higashi patients to infection.

Degradation of oxalate in rats implanted with immobilized oxalate oxidase.

Accumulation of oxalate leads to hyperoxaluria and calcium oxalate nephrolithiasis in man. Since oxalate is a metabolic end product in mammals, the feasibility of its enzymic degradation has been tested in vivo in rats by administering exogenous oxalate oxidase. Oxalate oxidase, isolated from banana fruit peels, in its native form was found to be non-active at the physiological pH of the recipient animal. However, its functional viability in the recipient animal was ensured by its prior binding with ethylenemaleic anhydride, thus shifting its pH activity curve towards the alkaline range. Rats implanted with dialysis membrane capsules containing such immobilized oxalate oxidase in their peritoneal cavities effectively metabolized intraperitoneally injected [14C]oxalate as well as its precursor [14C]glyoxalate. The implantation of capsules containing coentrapped multienzyme preparations of oxalate oxidase, catalase and peroxidase led to a further degradation of administered [14C]oxalate in rats.

Induction of cell infiltration and acid hydrolase release into the peritoneal cavity of mice.

An injection of thioglycollate into the peritoneal cavity of mice produced a peak of exudate at 4 h, a peak of total leukocytes at 24 h when the predominant cell was the polymorphonuclear neutrophil, and a secondard macrophage phase beginning 2--3 days after thioglycollate. The release of N-acetylglucosaminidase (EC 3.2.1.30) into the peritoneal fluid paralleled the macrophage phase. The amount of enzyme released was related to the dose of thioglycollate. Neither zymosan (400 micrograms) nor endotoxin (30 micrograms) produced a marked inflammatory response when injected into the peritoneal cavity.

Immunoreactive trypsin in serum and peritoneal fluid in acute pancreatitis.

High levels of immunoreactive cathodal trypsin are shown to be present in serum and peritoneal fluid in acute pancreatitis. The immunoreactive material is contained in two fractions with different molecular weights: free trypsinogen and trypsin in complex with protease inhibitors, mainly alpha1-antitrypsin. The presence of trypsin in complex with protease inhibitors indicates the formation of active trypsin during acute pancreatitis.

Characterization of the nonphagocytic adherent cell from the peritoneal cavity of normal and BCG-treated mice.

A distinctive subpopulation of nonphagocytic, tightly adherent cells (NPAC) comprised approximately 6% of the adherent peritoneal cells from untreated mice, and about 18% of those from mice previously given BCG i.p. A separation procedure based on adherence and lack of phagocytosis was devised. Isolated NPAC were morphologically intermediate between small lymphocytes and macrophages. They were positive for nonspecific esterase, negative for peroxidase, positive for surface IgM, and negative for surface IgG1, IgG2 and IgA. When capped, their surface IgM regenerated in vitro. NPAC had demonstrable Fc receptors but not EAC receptors. They resisted killing by an anti-macrophage serum, were negative by immunofluorescence with an anti-T cell reagent, and incorporated increased amounts of thymidine in response to LPS but not to PHA. They were more readily killed with anti-Ia serum and complement than macrophages, but less readily than splenic B cells. NPAC appeared to represent a subpopulation of B lymphocytes which contaminates some preparations previously regarded as "macrophages" and which may be ressponsible for some of the activities previously ascribed to "macrophages".

Production of collagenase by mouse peritoneal macrophages in vitro. Characterization of sites of cleavage of tropocollagen.

Macrophages from mineral oil-stimulated mice produce collagenase at a constant rate over several days in culture. Phagocytosis of latex does not increase production of enzyme. Gel electrophoretic and electron microscopic analyses indicate that the specificity of the macrophage enzyme is similar to that of other previously characterized mammalian collagenases.