Regulation of the expression of cyclooxygenases and production of prostaglandin I2 and E2 in human coronary artery endothelial cells by curcumin.

Curcumin regulates prostaglandin (PG) synthesis in a variety of cells. PGE2 and PGI2 are generated from arachidonic acid (AA) by cyclooxygenases 1 and 2 (COX-1 and COX-2) and the synthase (PGES and PGI2S) pathways. This study evaluates the in vitro effect of curcumin on the expression of COX-1, COX-2, PGI2S and microsomal PGES-1 (mPGES-1), and the production of PGE2 and PGI2 in human coronary artery endothelial cells (HCAEC). HCAEC monolayers were incubated with curcumin and the expression of mRNA, protein and the production of PGI2 and PGE2 were quantified. Incubation of HCAEC with curcumin led to a time and concentration-dependent increases in COX-2 mRNA with a small but significant decrease in COX-1 mRNA expression. Curcumin also stimulated the expression of PGI2S and mPGES-1 mRNA. Although curcumin stimulated COX-2, PGI2S and mPGES-1 gene expression, it failed to increase PGI2 or PGE2 production. Interestingly, supplementation of the culture medium with AA increased prostanoid production by both quiescent and curcumin-treated cells. However, in comparison to the quiescent cells, the prostanoid production by curcumin-treated cells was markedly enhanced as AA concentrations in the medium were increased, and the enhanced prostanoid production was blocked by the presence of COX-2 specific inhibitor. Taken together, these results suggest that curcumin regulates prostanoid homeostasis in HCAEC by modulating multiple steps including the expression of COX-1, COX-2, PGI2S and mPGES-1.

Increased aortic atherosclerotic plaque development in female apolipoprotein E-null mice is associated with elevated thromboxane A2 and decreased prostacyclin production.

The production of thromboxane A(2) (TXA(2)) and prostacyclin (prostaglandin I(2), PGI(2)) is known to be increased in patients with atherosclerosis. In this study, we evaluated the influence of gender on TXA(2) and PGI(2) production, and their association with the progression of atherosclerosis in apolipoprotein E-null (ApoE(-/-)) mice maintained on a high fat diet for 3 months. En face analyses of aortas showed marked increases in plaque formation in female ApoE(-/-) mice. Quantification of the hematoxylin/eosin (H&E) stained cross sections of the aortic arch revealed 3 to 4-fold higher plaque thickness in female ApoE(-/-) mice. Analyses of 24-hours urine samples for 11-dehydro TXB(2) and 2, 3-dinor-6-keto PGF(1a) indicated that female ApoE(-/-) mice produce up to 15-fold more TXA(2) and 50% less PGI(2) than the age matched males. Interestingly, the serum cholesterol levels in ApoE(-/-) females were 20% lower than males on the high fat regimen. No gender-associated changes in the number of T lymphocytes, mast cells and macrophages were evident in the lesion areas of ApoE(-/-) mice. The results suggest that the markedly elevated TXA(2) production and reduced PGI(2) production are gender-related proatherogenic risk factors in female ApoE(-/-) mice.

Interleukin-6 production by endothelial cells via stimulation of protease-activated receptors is amplified by endotoxin and tumor necrosis factor-alpha.

Human endothelial cells respond to extracellular proteases, endotoxin (lipopolysaccharide, LPS), and inflammatory cytokines. Endothelial cells express several protease-activated receptors (PAR), including the thrombin-activated receptors PAR-1 and PAR-3 and a thrombin-independent, protease-activated receptor, PAR-2. To examine the potential cooperation between PAR and inflammatory stimuli, we investigated the effects of the PAR-1 agonist peptide Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and PAR-2 agonist peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro with SFLLRN or SLIGKV in the presence and absence of LPS or tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) levels in the culture supernatants were assayed. Both SFLLRN and SLIGKV induced detectable levels of IL-6 production in a dose-dependent fashion, with the PAR-1 receptor agonist being more potent. In the presence of all stimulatory concentrations of LPS or TNF-alpha tested, both peptides were found to further enhance IL-6 production. The effects of SFLLRN and SLIGKV were specific, as related peptides with identical amino acid compositions, but lacking in consensus sequences, were biologically inactive either alone or in the presence of LPS. Both the direct and the amplifying effects of PAR agonist peptides on IL-6 production were pertussis toxin sensitive and caused an increase in the intracellular levels of calcium, implicating G-proteins and calcium mobilization in these pathways. Furthermore, the amplifying effect of LPS or TNF-alpha on PAR-mediated cytokine production was associated with corresponding increases in nuclear NF-kappaB proteins. The results demonstrate significant potentiation of PAR-induced signaling by LPS and TNF-alpha and indicate the potential cooperation of proteases and inflammatory stimuli in amplifying vascular inflammation.

Histamine-induced production of interleukin-6 and interleukin-8 by human coronary artery endothelial cells is enhanced by endotoxin and tumor necrosis factor-alpha.

In this study, we tested the synergy between histamine and LPS, and histamine and TNF-alpha, on endothelial cell production of interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). Human coronary artery endothelial cells (HCAEC) were cultured in vitro with histamine (0.1 to 1000 microM) in the presence or absence of LPS or TNF-alpha for 24 h, and the secreted IL-6, IL-8 and MCP-1 were quantified. Unactivated HCAEC produced minimal levels of IL-6, IL-8, or MCP-1. The incubation of HCAEC with histamine resulted in low level induction of IL-6 and IL-8 production, which was dose-dependent and attained a plateau at a concentration of 10 microM. On the other hand, histamine failed to induce MCP-1 production. Stimulation of HCAEC with LPS or TNF-alpha caused dose-dependent increase in cytokine production. In the presence of all stimulatory concentrations of LPS and TNF-alpha tested, histamine was shown to further enhance IL-6 and IL-8 production. The effect of histamine on endothelial cell production of cytokines was completely inhibited by the H-1 receptor antagonist, diphenhydramine, and not by the H-2 antagonist, famotidine. Electrophoretic mobility shift assays of nuclear proteins extracted from HCAEC treated with histamine and LPS, or histamine and TNF-alpha, revealed amplified translocation of NF-kappaB proteins to the nuclei. Since both LPS and TNF-alpha potentiated histamine-induced cytokine production, it is possible that these activators stimulate H-1 receptor expression and/or augment the signal transduction pathways leading to the expression of IL-6 and IL-8. These results indicate the importance of synergy between histamine and other inflammatory stimuli on endothelial cell activation and implicate their cooperative participation in vascular leak and inflammation.

Endotoxin and mast cell granule proteases synergistically activate human coronary artery endothelial cells to generate interleukin-6 and interleukin-8.

Mast cells (MC) are strategically located along blood vessels and, on activation, exocytose granules that contain many vasoactive mediators. Endothelial cell (EC) activation, which includes the production of such cytokines as interleukin-6 (IL-6) and IL-8, is a key event in vascular inflammation. In this study, the effects of purified MC granules (MCG) on the production of IL-6 and IL-8 by human coronary artery EC (HCAEC) were examined. HCAEC were cocultured with MCG in the presence or absence of lipopolysaccharide (LPS), and IL-6 and IL-8 levels in the culture medium were assayed by ELISA. Unactivated HCAEC produced only low levels of IL-6 or IL-8, and the addition of MCG alone resulted in little or no increase in production of these cytokines. LPS-activated HCAEC produced significant amounts of IL-6 and IL-8 in a dose-dependent and time-dependent fashion, which was amplified 2-3-fold by MCG at EC/MC ratios of 16:1-2:1. Scanning electron microscopy revealed direct communication between MCG and HCAEC. The enhancement of IL-6 and IL-8 production by MCG was abrogated when MCG were pretreated with the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). These results demonstrate that MCG interaction with HCAEC causes amplification of endotoxin-stimulated cytokine production via serine proteases present in MCG. The synergistic activation of EC by endotoxin and MCG proteases emphasizes the role of MC in amplifying vascular inflammation.

The interface between bone and tendon at an insertion site: a study of the quadriceps tendon insertion.

Traumatic avulsions of ligament or tendon insertions rarely occur at the actual interface with bone, which suggests that this attachment is strong or otherwise protected from injury by the structure of the insertion complex. In this study we describe the terminal extent of quadriceps tendon fibres where they insert into the patellae of adult rabbits, humans, dogs and sheep. Specimens were examined by scanning electron microscopy (SEM) and light microscopy (LM). To facilitate tracing of tendon fibres the specimens were decalcified for SEM, and polarised light microscopy (PLM) was used in the LM segment of the study. By SEM it was possible to identify mature bone by the presence of osteocytes and a lamellar organisation. PLM and SEM showed that, unlike tendon fibres elsewhere, those in the calcified fibrocartilage were not crimped. No specific cement line was identified by SEM. Tendon fibres interdigitated among separate bone lamellar systems, (osteons or marrow spaces), but did not merge with the collagen systems of individual lamellae. The interdigitation was more extensive and the margin between tendon and bone was less distinct in the anterior third of the insertion. The segment of calcified tendon which interdigitated with bone stained less intensely blue and was less cellular than the more proximal calcified fibrocartilage zone adjacent to the tidemark. Lamellar collagen fibres of the bony trabeculae in the anterior patella were unusually parallel and longitudinal in orientation, making distinction of interposed tendon fibres difficult on LM and PLM sections. LM, SEM and transmission electron microscopy of rabbit patellae at birth revealed that anterior quadriceps tendon fibres extended over the patella in a fibrous cellular layer. By 2 wk of age, this layer had acquired chondroid features (i.e. cell lacunae and metachromasia) and contained vessels extending from patellar marrow. At 6 wk of age, part of this fibrocartilaginous layer was replaced by mature bone and osteoid. In the young adult animal, the quadriceps tension interdigitates extensively with the patellar bone. This segment of the insertion is perhaps the remnant of calcified fibrocartilage which has been remodelled by bone formation.

Potential pitfall of the EndoButton.

A clinical and cadaveric example show the EndoButton (Acufex Microsurgical Inc, Mansfield, MA), used for anterior cruciate ligament endoscopic fixation, flipping outside the extensor mechanism or vastus lateralis rather than flipping directly outside the lateral femoral cortex. This pitfall was caused by overdrilling the femoral socket beyond the recommended 6 mm and overadvancing the EndoButton beyond the required depth to flip the EndoButton. Overdrilling the femoral socket to a depth of 10 mm still allows the EndoButton to rest properly on the cortex without soft tissue interposition. Increasing angles of knee flexion at the time of Endobutton placement decrease the safe distance beyond the lateral femoral cortex for flipping without soft tissue interposition. There is also potential to flip the EndoButton within the substance of the vastus lateralis, but the flipping action is blunted and not discrete.

Effect of mast cell granules on the gene expression of nitric oxide synthase and tumour necrosis factor-alpha in macrophages.

Previous studies have shown that mast cell granules (MCG) inhibit numerous macrophage functions including tumour cytotoxicity, superoxide and nitric oxide (NO) production, and FCgamma2a receptor-mediated phagocytosis. In this study, the effect of MCG on macrophage TNF alpha and nitric oxide synthase (iNOS) mRNA expression, and the production and fate of TNF alpha were examined. Upon activation with LPS+IFN gamma, macrophages expressed both TNF alpha and iNOS mRNA and produced both TNF alpha and NO. Co-incubation of LPS+IFN gamma-activated macrophages with MCG resulted in dose-dependent inhibition of iNOS mRNA expression. TNF alpha production in the activated macrophages was decreased by MCG, which was associated with a reduction in TNF alpha mRNA expression. MCG were also capable of degrading both macrophage-generated and recombinant TNF alpha. The direct effect of MCG on TNF alpha was partially reversed by a mixture of protease inhibitors. These results demonstrate that MCG decrease the production of NO and TNF alpha by inhibiting macrophage iNOS and TNF alpha gene expression. Furthermore, MCG post-transcriptionally alter TNF alpha levels via proteolytic degradation.

Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules.

The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression.

Mast cell granules potentiate endotoxin-induced interleukin-6 production by endothelial cells.

Mast cells are constituent cells of vascular tissue and their numbers are increased in atherosclerotic vessels. To gain insight into the role of mast cells in vascular inflammation, the effect of mast cell granules (MCG) on endothelial cell production of interleukin-6 (IL-6) was examined. Human umbilical vein endothelial cells (HUVEC) were cultured with lipopolysaccharide (LPS) in the presence or absence of rat peritoneal MCG and IL-6 production was assayed by enzyme-linked immunosorbent assay. The interaction of MCG with HUVEC in culture was examined by electron microscopy (EM). The EM studies revealed that MCG are internalized by HUVEC and appear intact even after 24 h in culture. Unactivated HUVEC produced little or no IL-6 either in the presence or absence of MCG. Treatment of HUVEC with LPS stimulated IL-6 production in a dose- and time-dependent fashion. Addition of MCG to LPS-activated HUVEC-resulted in the potentiation of IL-6 production at all LPS doses. MCG-induced enhancement of IL-6 production was evident even at a mast cell-to-endothelial cell ratio of 1:32. The enhancement of IL-6 production by MCG was also seen when tumor necrosis factor alpha was used as an activator. Although potentiation was evident when MCG were added 6 h before or after LPS stimulation, the maximum effect was noted when MCG and LPS were added simultaneously. MCG-mediated enhancement of IL-6 production was abrogated by pretreating MCG with protease inhibitors. Although MCG proteases potentiate IL-6 production by HUVEC, they do not degrade secreted IL-6. These results demonstrate that MCG interact with endothelial cells and modulate the production of an important inflammatory cytokine.

Macrophage function in mice with a mutation at the microphthalmia (mi) locus.

Microphthalmic (mi/mi) mice are unpigmented, osteopetrotic, mast cell deficient, and exhibit diminished gastric acid secretion and natural killer cell activity. In order to assess the effect of this mutation on macrophage function, we studied superoxide (O2-) and nitric oxide (NO) production, superoxide dismutase (SOD) activity, phagocytic capacity, and tumor cell killing by peritoneal macrophages from mi/mi mice and normal (+/+) litter mates. Macrophages from mi/mi mice, upon activation with phorbol myristate acetate (PMA), generated significantly higher amounts of O2- than did macrophages from their +/+ litter mates. The phagocyte respiratory burst as assessed by nitroblue tetrazolium (NBT) reduction assay also displayed a 2-fold enhancement in mi/mi macrophages. The assay of SOD activity revealed significantly lower levels in mi/mi macrophages than in the wild type. Furthermore, in comparison with controls, macrophages from mi/mi mice demonstrated significantly higher levels of NO production and increased capacity to lyse tumor cells upon activation with lipopolysaccharide (LPS) or gamma-interferon (IFN-gamma). The mi mutation, therefore, is associated with reduced macrophage SOD activity, increased O2- and NO production, and enhanced capacity for tumor cell killing. The molecular mechanisms for these changes have not been identified.

Fibronectin lacking the ED-B domain is a major structural component of tracheal cartilage.

Fibronectin is a highly conserved dimeric glycoprotein found in high concentrations in plasma and widely distributed in low concentrations in the extracellular matrix of tissues. The protein is the product of a single gene, but multiple splicing variants are expressed that show tissue specificity. Three exons (IIIA, IIIB, and V) can be alternatively spliced to produce different fibronectin isoforms. We report here that fibronectin is a remarkably abundant component of the extracellular matrix of bovine tracheal cartilage, increasing with age to more than 20% of the tissue, dry weight. This matrix form of fibronectin is inextractable by 4 M guanidine HCl, indicating that it is a covalently cross-linked structural component. By protein sequence analysis, the main molecular form of fibronectin in bovine tracheal cartilage was shown to lack the ED-B domain encoded by exon IIIB.

Dexamethasone inhibits nitric oxide-mediated cytotoxicity via effects on both macrophages and target cells.

In order to evaluate the mode of action of dexamethasone (DEX) on macrophage-mediated cytotoxicity and to understand its association with nitric oxide (NO) production, the effect of DEX on macrophage- and spermine NONOate-mediated cytotoxicity was studied. DEX caused 100% inhibition of cytotoxicity by LPS- and IFN gamma-activated macrophages whereas it caused only partial inhibition of NO production. Inhibition of macrophage-mediated cytotoxicity by DEX was not reversed by supplementation of rTNF alpha. The partial inhibition of NO production by DEX was due to partial inhibition of iNOS mRNA expression. Incubation of macrophages with DEX for up to 24 h prior to activation did not cause further inhibition of NO production. DEX failed to inhibit NO production if added 6 h after addition of LPS and IFN gamma. Addition of P815 cells after the onset of NO production resulted in partial restoration of cytotoxicity in DEX-treated macrophages. Incubation of P815 cells with spermine NONOate, a synthetic NO donor, resulted in P815 cell lysis, which was dose-dependent, had a lag phase of 3 h and was blocked by hemoglobin. DEX also inhibited spermine NONOate-mediated tumor cell lysis, indicating that DEX may have a protective effect on tumor targets. These results indicate that DEX inhibits macrophage-mediated cytotoxicity by decreasing NO production and by inhibiting the cytotoxic effects of NO on the target cells.

Direct activation of murine peritoneal macrophages for nitric oxide production and tumor cell killing by interferon-gamma.

Interferon-gamma (IFN-gamma) is known to prime macrophages for tumor cell lysis and nitric oxide (NO) production as measured by enhanced sensitivity to lipopolysaccharide (LPS). In the present study, the ability of IFN-gamma to directly activate peritoneal macrophages from C57BL/6 and Balb/c mice for tumor cytotoxicity and NO production was evaluated. Macrophage-mediated tumor cell killing was measured by an 18 h 51Cr release assay using P815 mastocytoma cells as targets. Concurrent NO production was measured as nitrite in the supernatants of macrophage cultures. Incubation of macrophages with IFN-gamma resulted in activation of macrophages for tumor cell lysis. IFN-gamma alone also activated macrophages for NO production under identical conditions. Addition of LPS along with IFN-gamma resulted in synergism in the activation of macrophages for both cytolysis and NO production. LPS contamination of the IFN-gamma preparation was absent as evidenced by the following criteria: (1) the IFN-gamma preparation as well as the reagents used were shown to be free of LPS contamination based on LAL endotoxin tests (sensitivity 25 pg/ml), (2) the ability of IFN-gamma to activated macrophages was not abrogated by prior treatment of the cytokine with polymyxin B, whereas the effect of LPS was inhibited (70-100%) under similar conditions, (3) pretreatment of the IFN-gamma preparation with a specific endotoxin neutralizing protein did not abrogate the ability of IFN-gamma to induce macrophage activation, and (4) heat treatment of solutions containing IFN-gamma alone or IFN-gamma+LPS abolished only the effect of IFN-gamma, not that of LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Phagocytosis of mast cell granules results in decreased macrophage superoxide production.

The mechanism by which phagocytosed mast cell granules (MCGs) inhibit macrophage superoxide production has not been defined. In this study, rat peritoneal macrophages were co-incubated with either isolated intact MCGs or MCG-sonicate, and their respiratory burst capacity and morphology were studied. Co-incubation of macrophages with either intact MCGs or MCG-sonicate resulted in a dose-dependent inhibition of superoxide- mediated cytochrome c reduction. This inhibitory effect was evident within 5 min of incubation and with MCG-sonicate was completely reversed when macrophages were washed prior to activation with PMA. In the case of intact MCGs, the inhibitory effect was only partially reversed by washing after a prolonged co-incubation time. Electron microscopic analyses revealed that MCGs were rapidly phagocytosed by macrophages and were subsequently disintegrated within the phagolysosomes. Assay of MCGs for superoxide dismutase (SOD) revealed the presence of significant activity of this enzyme. A comparison of normal macrophages and those containing phagocytosed MCGs did not reveal a significant difference in total SOD activity. It is speculated that, although there was no significant increase in total SOD activity in macrophages containing phagocytosed MCGs, the phagocytosed MCGs might cause a transient increase in SOD activity within the phagolysosomes. This transient rise in SOD results in scavenging of the newly generated superoxide. Alternatively, MCG inhibition of NADPH oxidase would explain the reported observations.

Effect of superoxide exposure on albumin permeability of isolated rat glomeruli.

Reactive oxygen radicals generated by mesangial cells or by resident or infiltrating macrophages may contribute to glomerular injury in glomerulonephritis. To determine whether superoxide anions have an effect on the glomerular barrier to macromolecules, we studied the responses of isolated glomeruli after exposure to superoxide generated by xanthine and xanthine oxidase or by activated macrophages. Glomerular volumetric responses to oncotic gradients of bovine serum albumin or an impermeant neutral dextran (mol wt 252 kd0 were used to calculate the albumin reflection coefficient (sigma albumin) and convectional permeability to albumin (1-sigma albumin) of control and superoxide-treated glomeruli. Albumin permeability of control glomeruli was not different from 0 (0.02 +/- 0.01, N = 50). After 10 minutes of exposure of glomeruli to superoxide generated by xanthine oxidase, albumin permeability was increased to 0.06 +/- 0.01 (N = 50). Albumin permeability did not increase further after incubations with xanthine and xanthine oxidase for up to 60 minutes. The increase in albumin permeability was prevented by superoxide dismutase (-0.02 +/- 0.01, N = 48) but was not affected by catalase or by indomethacin pretreatment. Coincubation of glomeruli with activated macrophages also increased albumin permeability to a maximum of 0.80 +/- 0.05 (N = 17). Albumin permeability was not increased by incubation of glomeruli with phorbol myristate acetate alone or with macrophages in the absence of phorbol myristate acetate. The effect of activated macrophages on albumin permeability, like that of superoxide generated chemically, was prevented by superoxide dismutase but not by catalase or indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

Bleomycin injury of the lung in a mast-cell-deficient model.

Lung mast cell hyperplasia and fibrosis is induced by bleomycin lung injury. The role of the mast cell in this process of injury and resultant fibrosis is unclear. Mutant mi/mi mice, profoundly mast-cell-deficient, were treated with intraperitoneal bleomycin and demonstrated minimal acute inflammatory and chronic fibrotic responses. Lung histamine values determined at 14 and 42 days after bleomycin injury in mi/mi mice were not increased compared to untreated mi/mi animals. However, lung histamine levels in normal mice demonstrated a 300% increase over controls on Day 14 after bleomycin injury, and then returned to baseline by Day 42. The mi/mi BAL cell recovery at 2 weeks after injury and lung hydroxyproline levels at 4 weeks after injury were not altered from baseline. The normal litter mates, in contrast, demonstrated significant increases compared to controls in both of these parameters (p < 0.01, p < 0.04). Although the mi/mi mouse is also deficient in basophils, natural killer cells and functional osteoclasts, there is no evidence of lowered pulmonary defense mechanism and neutrophils and alveolar macrophages are present in normal numbers. This investigation supports the hypothesis that the mast cell contributes to bleomycin-induced lung injury and fibrosis.

Mast cell granules inhibit macrophage-mediated lysis of mastocytoma cells (P815) and nitric oxide production.

The effects of mast cell granules (MCGs) on macrophage-mediated lysis of P815 mastocytoma cells and nitric oxide (NO) production were studied. Murine peritoneal macrophages exhibited tumor cell killing and NO production only when activated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma). Coincubation of macrophages with MCGs during LPS activation dose-dependently inhibited macrophage-mediated tumor cell lysis. The MCG effect was not due to inactivation or removal of LPS by MCG. The inhibitory effect was also not due to histamine or serotonin present in the MCGs. The granules were not toxic to macrophages or P815 mastocytoma cells. The effect of MCGs on macrophage-mediated tumor cell killing was evident whether MCGs were added before or after a 4-h exposure of macrophages to LPS. However, the inhibitory effect was not seen if MCGs were added after macrophages had been exposed to LPS for 24 h. To assess whether MCGs could inhibit a non-LPS trigger, MCGs were tested on macrophages activated with IFN-gamma. In these experiments, MCGs dose-dependently inhibited macrophage-mediated tumor cell killing induced by IFN-gamma, LPS, or IFN-gamma plus LPS. Furthermore, in parallel experiments, MCGs significantly inhibited macrophage NO production induced by LPS, IFN-gamma, or IFN-gamma plus LPS. Pretreatment of MCGs with diisopropylfluorophosphate, a serine protease inhibitor, only partially abrogated the effects of MCGs. The results demonstrate that MCGs inhibit both LPS- and IFN-gamma-induced macrophage killing of P815 cells and the inhibition is associated with the decrease of NO production.

Comparison of normal and rachitic rat matrix vesicles.

Accumulated information about MVs in PO4-deficient rachitic rats shows them to be normal in most aspects. Both normal and rachitic MVs show the same ultrastructural features and selective spatial distribution in the growth plate, and both contain a nearly identical array of major proteins. Rachitic and normal MVs show the same avidity to calcify in vivo and in vitro. A slightly greater specific activity of ALP in rachitic MVs may enhance their calcifiability. We conclude that rachitic rat MVs are essentially 'normal' and can be used as an adequate model to study the mechanism of biological calcification.

Presence and specific concentration of carbonic anhydrase II in matrix vesicles.

Matrix vesicles were isolated from the epiphyseal growth plates of normal weanling rats, and the presence of carbonic anhydrase II was demonstrated by Western blotting and ultrastructural immunolocalization using the immunogold technique. Total carbonic anhydrase activity was assayed and showed a statistically significant increase in matrix vesicles as compared to normal rat chondrocytes derived from the same growth plates. These results are the first to establish the presence of carbonic anhydrase in matrix vesicles.