Cultured Rat Peritoneal Macrophages Research Articles

NE Strengthens the Immunosuppression Induced by AlCl3 Through β2-AR/cAMP Pathway in Cultured Rat Peritoneal Macrophages

To investigate the effect of noradrenaline (NE) on the immunosuppression induced by aluminum trichloride (AlCl3), the peritoneal macrophages were cultured with RPMI-1640 medium containing 0.97 mM AlCl3 (1/10 IC50). NE was added to the medium at the final concentrations of 0 (control group, N-C), 0.1 (low-dose group, N-L), 1 (mid-dose group, N-M), and 10 (high-dose group, N-H) nM, respectively. No addition of both AlCl3 and NE serviced as blank group (D-C). Chemotaxis, adhesion, phagocytosis, tumor necrosis factor α (TNF-α) secretion, cyclic adenosine monophosphate (cAMP) content, β2 adrenergic receptors (β2-AR) density, and messenger RNA (mRNA) expression of macrophages were detected. The results showed that AlCl3 reduced the chemotaxis, adhesion, phagocytosis, and TNF-α secretion and increased the cAMP content, β2-AR density, and mRNA expression of peritoneal macrophages. Meanwhile, the chemotaxis, adhesion, phagocytosis, TNF-α secretion, β2-AR density, and mRNA expression were reduced while the cAMP content was increased in NE-treated groups than those in N-C group. The results indicated that NE strengthens the immunosuppression induced by AlCl3 in cultured rat peritoneal macrophages through the β2-AR/cAMP pathway.

Apelin Antagonizes Myocardial Impairment in Sepsis

Background Apelin is a cardiovascular peptide with multiple functions regulating homeostasis of the circulatory system and is the endogenous ligand of angiotensin II receptor like-1 (AGTRL1). Apelin has anti-inflammatory and inhibitory effects on release of inflammatory mediators. We aimed to analyze whether apelin antagonizes myocardial impairment in sepsis by attenuating inflammatory responses. Methods and Results Male rats underwent sepsis by cecal ligation and puncture (CLP) after receiving low- or high-dose apelin for 3 days. Twenty hours later, rats with sepsis showed severe disturbance of hemodynamic features. Reverse transcription-polymerase chain reaction revealed decreased mRNA levels of apelin and AGTRL1 in myocardia of rats with sepsis. Enzyme immune assay detected a lower level of apelin in plasma and myocardia. Western blot analysis revealed decreased level of myocardial AGTRL1 protein. Low- and high-dose apelin administration ameliorated disorders of cardiac function: increased mean arterial blood pressure, attenuated heart rate, elevated +LVdp/dt max and LVdp/dt max, and lowered left ventricular end-diastolic pressure. Rats treated with low- or high-dose apelin showed lower content of plasma monocyte chemoattractant protein 1and interleukin 8. In cultured rat peritoneal macrophages, apelin directly inhibited the production of monocyte chemoattractant protein 1 and interleukin-8 induced by lipopolysaccharide. Conclusions These results suggest that apelin antagonizes cardiac impairment in sepsis by attenuating inflammatory responses and might be a promising therapeutic target for severe sepsis and septic shock.

Endomorphins and ohmefentanyl in the inhibition of immunosuppressant function in rat peritoneal macrophages: An experimental in vitro study

Background: The potential immunosuppressant effects of opioids might have clinical implications. The effects of endomorphins (EMs) and ohmefentanyl (OMF) on cultured rat peritoneal macrophages remain unclear. Objective: The aim of this study was to investigate the immunosuppressant effects of EMs and OMF on cultured rat peritoneal macrophages in vitro. Methods: Purified rat peritoneal macrophages, from healthy adult male Sprague-Dawley rats, were cultured with EM-1 (EM-1 group), EM-2 (EM-2 group), OMF (OMF group), and saline (saline group). We measured the concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-β in supernatant when macrophages were cultured with 10 −6 mol/L of EM-1, EM-2, OMF, or saline for 0, 6, 12, and 24 hours (time-effect relationship) or with 10 −10, 10 −9, 10 −8, 10 −7, and 10 −6 mol/L of these substances for 24 hours (concentration-effect relationship). We also determined the phagocytic and bactericidal activities of macrophages using isotope markers when macrophages were cultured with 10 −6 mol/L of EM-1, EM-2, OMF, or saline for 24 hours. Results: Compared with the saline group, TNF-α concentration decreased significantly in the OMF, EM-2, and EM-1 groups at 12 hours ( P < 0.05, P < 0.05, and P < 0.01, respectively) and at 24 hours ( P < 0.05, P < 0.01, and P < 0.01, respectively). Compared with the saline group, IL-1β concentration decreased signifcantly in the OMF, EM-2, and EM-1 groups at 12 hours ( P < 0.05, P < 0.05, and P < 0.01, respectively) and at 24 hours ( P < 0.05, P < 0.01, and P < 0.01, respectively). Decreased TNF-α and IL-1β concentrations were observed in the supernatant at 24 hours when cultured with 10 −8, 10 −7, and 10 −6 mol/L in the OMF and EM-2 groups (all, P < 0.05) and in the EM-1 group (all, P < 0.01). Compared with the saline group, macrophage phagocytic activity (all, P < 0.05) and macrophage bactericidal activity (all, P < 0.01) were significantly lower in the 3 experimental groups compared with the saline group. Conclusion: In this in vitro experiment, EM-1, EM-2, and OMF inhibited the immunosuppressant function of cultured rat peritoneal macrophages, including decreasing TNF-α and IL-1β concentrations and phagocytic and bactericidal activities.

Fractalkine-Induced MFG-E8 Leads to Enhanced Apoptotic Cell Clearance by Macrophages

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.

Involvement of glial cell line‐derived neurotrophic factor in activation processes of rodent macrophages

The physiological roles of glial cell line-derived neurotrophic factor (GDNF) expressed in the microglia/macrophages of the injured spinal cord have not yet been clarified. mRNA expression of chemokines, including monocyte chemoattractant protein (MCP)-1, was evoked within 1 hr after transection of the spinal cord, and GDNF mRNA expression was similarly up-regulated. Immunohistochemical analysis showed that GDNF was coexpressed with MCP-1 in the CD11b-positive cells. Therefore, we examined further the effects of GDNF on cultured rat peritoneal macrophages. GDNF enhanced the phagocytic activity of the macrophages via GFRalpha-1, glycosylphosphatidylinositol-anchored specific binding site of GDNF, in a c-Ret-independent manner. The influence of autocrine and/or paracrine GDNF synthesis was evaluated by performing activation experiments using macrophages cultured from heterozygous (+/-) GDNF gene-deficient mice or wild-type (+/+) mice. There were no morphological differences dependent on genetic types or stimulators. However, the GDNF mRNA level, but not the MCP-1 or GFRalpha-1 mRNA level, was substantially lower in the mutant macrophages than in the +/+ cells irrespective of stimulation with MCP-1 or lipopolysaccharide (LPS). The phagocytic activity enhanced by MCP-1 or LPS was significantly lower in the mutant cells (+/-) than in the +/+ ones, demonstrating the involvement of endogenous GDNF in the activation processes of macrophages in vitro and suggesting that not only neuroprotective function but also activation of macrophages is effected by the GDNF produced after a spinal cord injury.

Oxidative burst and metallothionein as a scavenger in macrophages.

The role of metallothionein (MT) in the scavenging of superoxide radicals (*O2-) generated by macrophages has been examined. The present work has focused on the effects of added cadmium, a known inducer of MT biosynthesis, on determined amounts of superoxide radicals produced by in vitro cultured rat peritoneal macrophages on their stimulation with phorbol-12-myristate-13-acetate (PMA). The levels of superoxide radicals (*O2-) have been found to decrease when cadmium was added to cells exposed to PMA. However, substantially lower levels of MT have been determined in this case compared to cells untreated with PMA. This effect could be reversed by incubation of the PMA and cadmium-treated cells with a reducing agent, 2-mercaptoethanol (2-ME). Results suggest that *O2- caused thiolate oxidation and subsequent metal loss, thus reducing the cellular MT content as quantified by the silver saturation This conclusion is supported by cell-free experiments in which the oxidation of rabbit MT-I by a xanthine/xanthine-oxidase system could be reversed by its subsequent reduction with 2-ME. The data presented provide direct evidence of the involvement of MT in scavenging superoxide radicals in living cells.

Effects of prostaglandins and nitric oxide on rat macrophage lipid metabolism in culture: implications for arterial wall-leukocyte interplay in atherosclerosis.

Macrophages/foam cells have a pivotal role in atherogenesis although little is known about the way lipid imbalance, a hallmark of atherosclerosis, leads to lipid accumulation in these cells. Modified low-density lipoproteins are associated with macrophage lipid dysfunction in atherosclerosis, but a possible role for altered lipogenesis leading to lipid accumulation remains to be elucidated. Since endothelium-derived nitric oxide (NO) and prostaglandins (PGs) are physiological autacoids whose production may be impaired in atherosclerosis, the effects of these mediators on de novo lipid synthesis in 24-h cultured rat peritoneal macrophages is investigated. In resident (unstimulated) cells, 1 microM PGE2 and the stable analog of PGI2 carbaprostacyclin (cPGI2, 1 microM) deviated the overall [1-14C]acetate from incorporation into cholesterol, free fatty acids and triacylglycerols favoring the formation of phospholipids. In inflammatory (thioglycollate-elicited) macrophages, these eicosanoids likewise reduced 14C-incorporations into all the lipid fractions tested. Also, cPGI2 and PGE2 reduced [4-14C]cholesterol uptake from inflammatory cells but did not interfere in 14C-cholesterol export. The PGE2-derivative PGA2 (10-20 microM) reduced 14C-incorporations into all the lipids in resident cells while it enhanced phospholipid synthesis by up to 129% at the expense of reduced incorporations into the other test lipids. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 1-10 microM), when added to macrophages in the presence of superoxide dismutase (SOD, to avoid the reaction of superoxide with NO), significantly reduced lipogenesis especially in inflammatory cells. These findings suggest that endothelium-derived NO and PGs may be associated with macrophage lipid accumulation by modulating lipogenesis and cholesterol uptake within these cells.

Inhibition of tumor necrosis factor-α production by SK&F 98625, a CoA-independent transacylase inhibitor, in cultured rat peritoneal macrophages

When rat peritoneal macrophages were incubated in medium containing thapsigargin, tumor necrosis factor-α (TNF-α) production was increased time-dependently. In the presence of SK&F 98625, a CoA-independent transacylase inhibitor, the thapsigargin-induced TNF-α production was inhibited dose-dependently. Platelet-activating factor (PAF) and prostaglandin E2 (PGE2) production were also inhibited by SK&F 98625. The SK&F 98625-induced inhibition of TNF-α production was not prevented by addition of PGE2. PAF antagonists such as E6123, L-652,731 and CV-6209 partially inhibited the thapsigargin-induced TNF-α production, suggesting that concurrently produced PAF in thapsigargin-stimulated macrophages up-regulates TNF-α production. The inhibition by SK&F 98625 of thapsigargin-induced TNF-α production might be partly due to the inhibition of PAF production.

The effect of cyclic AMP analogues on cholesteryl ester synthesis and hydrolysis in cultured rat peritoneal macrophages.

The accumulation of large amounts of cholesteryl ester in macrophages which have invaded the artery wall leads to the formation of foam cells, and is one of the early events in the development of atherosclerosis. The activities of both acyl Coenzyme A: cholesterol acyltransferase (ACAT), which is responsible for cholesterol esterification, and cholesteryl ester hydrolase (CEH) have been shown to be increased in foam cells ( 1 ), so that the cholesteryl ester undergoes a continual cycle of formation and hydrolysis ( l,2). Cyclic AMP (CAMP) has been reported to both stimulate (3) and inhibit (4) ACAT activity in the murine macrophage cell line 5774, while in a recent study we were unable to demonstrate any effect (5). CEH activity, on the other hand, has been shown to be increased by dibutyryl cAMP (Bt2cAMP) in two other murine cell lines, P388DI and WEHl (6.7). In the present work, we have investigated the role of cAMP in the regulation of cholesteryl ester metabolism and foam cell formation in cultured rat peritoneal macrophages using cellpermeable cAMP analogues. Macrophages were collected from male Wistar rats (250300g) by peritoneal washing (8) 2 days after intraperitoneal injection of 3ml thioglycollate (3%). Cells from 3-4 rats were pooled for each preparation. After washing, the cells were cultured as monolayers in supplemented Dulbecco's modified Eagle's medium at 37OC in 95% air l 5% CO?. Rat lipoprotein of density < 1.050glml was isolated from rat serum by ultracentrifugation and acetylated by incubation with acetic anhydride (9). Cholesterol esterification was assayed by the incorporation of radioactivity from potassium ('HI =!ca:c in:o cholesteryl esters during a 5h incubation period. Lipids were extracted with chloroform: methanol (2: 1, v:v) and separated by thin layer chromatography. For the determination of CEH activity, the cells were removed from the plates with a plastic scraper, washed with phosphate-buffered saline and disrupted by sonication. The release of I I-'%]oleate from cholesterol I I '%)oleate was then measured in the resulting homogenate (10). The cAMP analogues used were N6 benzoyl cAMP (BEAMP), B-(4-chlorophenylthio) cAMP (CPTcAMP), 8 chloro cAMP (ClcAMP), N6 monobutyryl cAMP (MBcAMP), 8-(6-amino hexyl) amino cAMP (AHA CAMP), 8-piperidino cAMP (PIP CAMP) and dibutyryl cAMP (BtZcAMP). Significance limits were calculated using Student's t test. Cholesterol esterification in the cultured macrophages was unaffected by a range of cAMP analogues at a concentration of

Coinduction of Nitric-oxide Synthase and Arginase I in Cultured Rat Peritoneal Macrophages and Rat Tissues in Vivo by Lipopolysaccharide

Nitric oxide is synthesized by nitric-oxide synthase from arginine, a common substrate of arginase. Rat peritoneal macrophages were cultured in the presence of bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of nitric-oxide synthase (iNOS) and liver-type arginase (arginase I) was analyzed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased to a near maximum at 8-12 h. On the other hand, arginase I mRNA that was undetectable prior to the treatment began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. mRNA for arginase II, an arginase isozyme, was not detected in the LPS-activated peritoneal cells. mRNA for CCAAT/enhancer-binding protein beta (C/EBPbeta), a transactivator of the arginase I gene, was also induced, and the induction was more rapid than that of arginase I mRNA. Changes in iNOS and arginase I mRNAs were also examined in LPS-injected rats in vivo. iNOS mRNA increased rapidly in the lung and spleen, reached a maximum 2-6 h after the LPS treatment, and decreased thereafter. Arginase I mRNA was induced markedly and more slowly in both tissues, reaching a maximum in 12 h. Thus, arginase I appears to have an important role in down-regulating nitric oxide synthesis in murine macrophages by decreasing the availability of arginine, and the induction of arginase I is mediated by C/EBPbeta.

Effects of the naturally occurring arginine analogues indospicine and canavanine on nitric oxide mediated functions in aortic endothelium and peritoneal macrophages.

The ability of the naturally occurring non-protein toxic amino acids indospicine and canavanine to inhibit nitric oxide synthesis was tested in isolated rat aorta and cultured rat peritoneal macrophages. Both compounds inhibited acetylcholine induced relaxation of rat aorta contracted with noradrenaline, a process mediated by nitric oxide generated in vascular endothelium. Nitric oxide is generated in vascular endothelium from arginine by a constitutive nitric oxide synthase. Indospicine and canavanine also increased superoxide mediated reduction of cytochrome c by phorbol myristate acetate stimulated rat peritoneal macrophages. The increase in superoxide under these conditions was due to decreased nitric oxide synthesis. Macrophage synthesis of nitric oxide is mediated by an inducible form of nitric oxide synthase. It is concluded that indospicine and canavanine are inhibitors of constitutive and inducible nitric oxide synthases and it is suggested that the toxicity associated with these compounds could be related to this activity.

Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy.

The role of the glomerular visceral epithelial cell in the physiologic turnover and pathologic breakdown of the glomerular extracellular matrix has remained largely unexplored. In this study a 98-kD neutral proteinase secreted by cultured rat visceral glomerular epithelial cells was shown to be a calcium, zinc-dependent enzyme secreted in latent form. In addition, the protein was heavily glycosylated and demonstrated proteolytic activity against Type I gelatin, Type IV collagen gelatin, and fibronectin. The similarity in molecular mass and substrate specificities to the 92-kD human matrix metalloproteinase-9 (MMP-9, or gelatinase B) suggested the identity of this activity, which was confirmed by immunoprecipitation and Northern blot analysis. The differences in molecular mass (98 vs. 92 kD) were not due to species-specific differences in glycosylation patterns, since cultured rat peritoneal macrophages secreted MMP-9 as a 92-kD enzyme. Furthermore, transfection of the human MMP-9 cDNA into rat glomerular epithelial cells yielded the 98-kD product. Using a specific monoclonal anti-MMP-9 antibody and in situ reverse transcription (ISRT) analysis of MMP-9 mRNA, the expression of this enzyme was evaluated in vivo. Normal rat glomeruli expressed little immunohistochemical or ISRT staining for MMP-9, while in rats with passive Heymann nephritis there was a major increase in MMP-9 protein and mRNA staining within the visceral epithelial cells. The temporal patterns of MMP-9 expression correlated with the period of proteinuria associated with this model, suggesting that a causal relationship may exist between GEC MMP-9 expression and changes in glomerular capillary permeability.

Effects of almitrine detriazinyl metabolite upon lysosomal alterations on rat cultured macrophages.

Phospholipidosis-inducing effects of difluorobenzhydrylpiperadine (DFBP) on cultured rat per-itoneal macrophages were studied as compared with those of several amphiphilic drugs. At 24 hr of exposure to DFBP as well as other amphiphilic drugs, intracytoplasmic acid hematein-stained inclusion bodies were dose-dependently produced in macrophages. Electron microscopy showed lamellar inclusion bodies in macrophages exposed to DFBP. Effective concentrations for 30% positivity of treated cells were: 0.43μM/diethylamino-ethoxyhexestrol, 1.9 μM/perhexiline, 2.8μM/quinacrine, 4.3μM/DFBP, 11μM/chlorcyclizine, and 14 μM/ propranolol. Neither almitrine nor aspirin induced the cytoplasmic inclusion bodies.

Effects of lysosomal protease inhibitors on the degradation of acetylated low density lipoprotein in cultured rat peritoneal macrophages.

The effect of protease inhibitors, leupeptin and pepstatin A, on the metabolism of acetylated low density lipoprotein (acetyl-LDL) was investigated in cultured rat peritoneal macrophages and compared with that of chloroquine. While both leupeptin and pepstatin inhibited the proteolytic degradation of 125I-acetyl-LDL, a combination of both showed an additive effect. Similar to chloroquine, both protease inhibitors diminished [3H] oleate incorporation into cellular cholesteryl[3H] oleate and increased cholesterol content of macrophages. These results suggest that both thiol protease and cathepsin D participate in the physiological degradation of apolipoprotein in macrophages. The inhibition of apolipoprotein degradation seemed to have an effect on cholesterol metabolism in macrophages cultured with acetyl-LDL.

A monokine which binds to class II histocompatibility proteins.

There is a marked decrease in the viability, in vitro, of dense, immature rat thymocytes over a 4 h incubation period. The addition of a monokine of relative molecular weight 36,000 derived from cultured rat peritoneal macrophages which had been previously stimulated with lipopolysaccharide prevented these cells from dying. The early release of this factor, together with preliminary results of its physical and functional properties suggest that it is distinct from the well characterized monokines. The macrophage factor was found to bind in competition with a monoclonal antibody directed against a common determinant on the Ia antigen complex, but also to bind non-competitively with a monoclonal antibody to a strain-specific epitope on Ia. Based on the results of experiments using a fractionated thymic cell model, it would appear that this monokine binds to the Ia protein complex on the cell surface of thymic epithelial cells, causing them in turn to release an activity which is responsible for the survival of thymocytes.

Arachidonic acid metabolism of cultured peritoneal rat macrophages and its manipulation by nonsteroidal antiinflammatory agents

[3H]Arachidonic acid (AA) metabolism by cultured rat peritoneal macrophages (Mø) was examined. Ninety percent of incorporated [3H]AA localized in phospholipids (phosphatidylcholine, 30%; phosphatidylinositol, 23.9%; phosphatidylethanolamine, 23.7%) whereas 8.3% and 1.5% was found in the free fatty acid and neutral lipid fractions. 12-O-tetradecanoate phorbol-13-armacetate (TPA) induced the reduction of label from phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine (42%, 46% and 47%, respectively) and an accumulation of label into free fatty acids (34%) or neutral lipid (61%) fractions. Simultaneously, 12% of the incorporated label was released into the medium as [3H]6-keto prostaglandin (PG)F1α, [3H]thromboxane B2 (TxB2), [3H]PGE2, [3H]hydroxyheptadecanoic acid (HHT), [3H]15-hydroxyeicosatetraenoic acid (HETE), [3H]12-HETE and [3H]AA. Exposure to 0.3–3 μM indomethacin reduced TPA-induced label release into the medium which was distinguished by dose-dependent reductions in all [3H]prostanoids as well as [3H]15- and [3H]12-HETE and a reciprocal increase in [3H]AA. Nordihydroguaiaretic acid (NDGA) altered AA metabolism at concentrations which approached its toxic dose ( > 20 μM). Cells exposed to 10 μM NDGA reduced TPA-induced label release into the medium which was characterized by reductions in [3H]-TxB2, [3H]PGE2 and [3H]HHT, no change in [3H]15-HETE, [3H]12-HETE or [3H]AA and the appearance of [3H]PGF2α. Cellular label redistributions of lipid fractions in cells exposed to NDGA or indomethacin were significantly less than that of control cultures indicating inhibition of acylhydrolase activity. Indomethacin or NDGA, therefore modify AA metabolism of cultured rat Mø by influencing more than one target enzyme.

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro-cathepsins B, H and L to the mature single-chain enzymes was sensitive to a metal chelator,1,10-phenanthroline, and a synthetic metalloendopeptidase substrate, Z-Gly-Leu-NH 2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E-64-d and leupeptin, inhibited the processing of the single-chain forms of cathepsins B, H and L to the two-chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single-chain cathepsins are accomplished by cysteine proteinases in lysosomes.

Tumor necrosis factor/cachectin stimulates peritoneal macrophages, polymorphonuclear neutrophils, and vascular endothelial cells to synthesize and release platelet-activating factor.

Murine tumor necrosis factor (mTNF) stimulates production of platelet-activating factor (PAF) by cultured rat peritoneal macrophages in amounts comparable to those formed during treatment with the calcium ionophore A23187 or phagocytosis of zymosan. The cell-associated PAF that was released into the medium was identical to synthetic PAF, as determined with physicochemical, chromatographic, and enzymatic assays. Furthermore, de novo synthesis of PAF by macrophages was demonstrated by the incorporation of radioactive precursors such as [3H]acetyl-coenzyme A or [3H]2-lyso-PAF. Macrophages incubated with mTNF for 4 h synthesized PAF only during the first h of treatment. At this time, the amount of cell-associated PAF was approximately equal to that released into the medium. The cell-associated PAF decreased afterwards, whereas that in the medium did not correspondingly increase, suggesting that some PAF was being degraded. The response of rat macrophages to different doses of mTNF and human TNF (hTNF) was examined. Maximal synthesis of PAF was obtained with 10 ng/ml of mTNF and 50 ng/ml of hTNF. This finding may be explained by a lower affinity of hTNF for TNF receptors of rat cells. The hTNF stimulated production of PAF by human vascular endothelial cells cultured from the umbilical cord vein. The time course of PAF synthesis was slower than that observed with macrophages, with maximal production between 4 and 6 h of treatment. Optimal synthesis of PAF was obtained with 10 ng/ml of hTNF. Only 20-30% of the PAF synthesized by endothelial cells was released into the medium, even after several hours of incubation. Synthesis of PAF in response to TNF was also detected in rat polymorphonuclear neutrophils, but not in human tumor cells and dermal fibroblasts. Therefore, production of PAF is a specialized response that is transient in macrophages continuously treated with TNF, and that appears to be controlled by unidentified regulatory mechanisms.

Studies on the turnover of glucocerebrosidase in cultured rat peritoneal macrophages and normal human fibroblasts

The kinetics of glucocerebrosidase synthesis and degradation in rat peritoneal macrophages and in human fibroblasts have been studied using conduritol B epoxide (CBE), an irreversible and specific inhibitor of mammalian glucocerebrosidase. In cultured fibroblasts, higher concentrations of CBE and/or longer times were required for inhibition of glucocerebrosidase than were necessary for inhibition of the macrophage enzyme. However, inhibition of activity in cell extracts from both cell types showed identical time and concentration dependence. After the removal of CBE from cultures, enzyme activity returned to normal with a half-time of 48 h for macrophages and 40 h for fibroblasts. The reappearance of enzyme activity was prevented by an inhibitor of protein synthesis. Both the rate of synthesis and degradation of glucocerebrosidase enzyme protein were independent of the presence of CBE. The calculated rate of degradation of glucocerebrosidase was confirmed using metabolically labelled enzyme in cell cultures. The rate of synthesis for macrophages is 1.8 ng enzyme h-1 mg cell protein-1 and the rate of degradation is 1.4% h-1 (0.014 h-1). These values were 2.0 ng h-1 mg-1 and 0.018 h-1 for fibroblasts.

Intralysosomal formation and metabolic fate of N-acetylglucosamine 6-sulfate from keratan sulfate.

The physiological relevance of the ability of beta-N-acetylhexosaminidase A to liberate N-acetylglucosamine 6-sulfate from polymeric keratan sulfate was investigated. Upon intravenous injection into rats of [35S]sulfate-labeled proteokeratan sulfate up to 25% of the radioactivity excreted with the urine were identified as N-acetyl-glucosamine 6-sulfate. Within 24 h, however, excretion of inorganic sulfate rose at the expense of the sulfated monosaccharide. Upon incubation in vitro of liver lysosomes from rats treated with proteokeratan sulfate, inorganic sulfate and minor amounts of sulfated monosaccharide were found in the incubation fluid. Cultured rat peritoneal macrophages ingested proteokeratan sulfate with a clearance rate of 6-9 micrograms X h-1 X mg cell protein-1 and degraded it rapidly. Inorganic sulfate but not N-acetylglucosamine 6-sulfate was delivered to the culture medium. During a chase period the amount of intracellular N-acetylglucosamine 6-sulfate fell, and a corresponding amount of sulfate could be found extracellularly. Significant amount of N-acetylglucosamine 6-sulfate were only found in the culture medium when the cells were challenged with zymosan. These results suggest that N-acetylglucosamine 6-sulfate is a physiological intermediate during the degradation of keratan sulfate, but is usually hydrolyzed intralysosomally by N-acetylglucosamine-6-sulfate sulfatase. Genetic deficiency of the sulfatase in humans therefore results in excessive excretion of the sulfated amino sugar but not of keratan sulfate.