Mary A. Misukonis

The effects of static and intermittent compression on nitric oxide production in articular cartilage explants

Nitric oxide (NO) production and NO synthase (NOS) expression are increased in osteoarthritis and rheumatoid arthritis, suggesting that NO may play a role in the destruction of articular cartilage. To test the hypothesis that mechanical stress may increase NO production by chondrocytes, we measured the effects of physiological levels of static and intermittent compression on NOS activity, NO production, and NOS antigen expression by porcine articular cartilage explants. Static compression significantly increased NO production at 0.1 MPa stress for 24 h (P < 0.05). Intermittent compression at 0.5 Hz for 6 h followed by 18 h recovery also increased NO production and NOS activity at 1.0 MPa stress (P < 0.05). Intermittent compression at 0.5 Hz for 24 h at a magnitude of 0.1 or 0.5 MPa caused an increase in NO production and NOS activity (P < 0.05). Immunoblot analysis showed stress‐induced upregulation of NOS2, but not NOSI or NOS3. There was no loss in cell viability following any of the loading regimens. Addition of 2 mM 1400 W (a specific NOS2 inhibitor) reduced NO production by 51% with no loss of cell viability. These findings indicate that NO production by chondrocytes is influenced by mechanical compression in vitro and suggest that biome‐chanical factors may in part regulate NO production in vivo.

Macrophages and infertility: oviductal macrophages as potential mediators of infertility.

Human peritoneal macrophages have previously been shown to phagocytize normal sperm. We had hypothesized that if macrophages were present in the distal oviducts, they could interfere with fertilization by phagocytizing sperm in vivo. The present study was designed to determine whether functional macrophages are present in the human oviducts, and to determine the relationship between oviductal and peritoneal macrophages. Forty patients undergoing laparotomy for sterilization or evaluation of infertility or other gynecologic factors were studied. Infertile patients with endometriosis had more peritoneal macrophages than did fertile normal women or infertile women with distal or proximal tubal obstruction. Oviductal macrophages were observed in all patients. The oviductal macrophages were indistinguishable from the peritoneal macrophages, as judged by similar morphologic features, adherence to plastic, phagocytosis of polystyrene spheres and IgG-coated erythrocytes, and presence of peroxidase and alpha-naphthylbutyrate esterase. Patients with endometriosis had the highest numbers of oviductal macrophages, while those patients with distal tubal obstruction had extremely few oviductal macrophages. The results suggest that oviductal macrophages may arise from peritoneal macrophages that migrate into the oviducts.

Elevated Nitric Oxide Production in Children with Malarial Anemia: Hemozoin-Induced Nitric Oxide Synthase Type 2 Transcripts and Nitric Oxide in Blood Mononuclear Cells

ABSTRACT Experiments outlined here investigate the role of nitric oxide (NO) in the pathogenesis of Plasmodium falciparum-induced malarial anemia (MA). The results show that ex vivo and in vitro NO synthase (NOS) activity in peripheral blood mononuclear cells (PBMCs) is significantly elevated in children with MA and inversely associated with hemoglobin levels. Additional experiments using PBMCs from non-malaria-exposed donors demonstrate that physiologic amounts of P. falciparum-derived hemozoin augment NOS type 2 (NOS2) transcripts and NO production. Results of these experiments illustrate that elevated NO production in children with MA is associated with decreased hemoglobin concentrations and that hemozoin can induce NOS2-derived NO formation in cultured blood mononuclear cells.

Nitric Oxide Synthase Type 2 Promoter Polymorphisms, Nitric Oxide Production, and Disease Severity in Tanzanian Children with Malaria

Nitric oxide (NO) plays an important role in host resistance to infection with a variety of organisms. Two recent reports from Gabon and Gambia identified associations of malaria disease severity with the inducible NO synthase (NOS2) promoter G-954C and short allele (<11 repeats) pentanucleotide microsatellite polymorphisms, respectively. It was postulated that there would be a correlation of these polymorphisms with malaria disease severity and with measures of NO production in our cohort of Tanzanian children with malaria. In Tanzanian children, 15% were heterozygous or homozygous for the G-954C polymorphism, and 13% had the short-allele microsatellite polymorphism. There was no significant correlation of either polymorphism with disease severity or with measures of NO production and NOS2 expression. Black and white Americans differed significantly in the frequencies of these polymorphisms. The various association of these gene polymorphisms with malaria severity in different populations underscores the complexity of host resistance to malaria.

Reduction of NOS2 overexpression in rheumatoid arthritis patients treated with anti-tumor necrosis factor α monoclonal antibody (cA2)

OBJECTIVE Peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) have increased expression of nitric oxide synthase type 2 (NOS2) protein and enhanced formation of nitric oxide (NO) that correlate with disease activity. NO may play a role in the inflammation of RA. Treatment of RA patients with a chimeric monoclonal antibody against tumor necrosis factor alpha (TNFalpha; cA2) results in clinical improvement in the majority of patients. The present study was designed to determine if cA2 therapy decreases PBMC NOS2 protein expression and NOS enzyme activity in RA patients. METHODS RA patients receiving background oral methotrexate participated in a double-blind, placebo-controlled clinical trial in which they were randomly assigned to receive a single infusion of either placebo or cA2 at 5, 10, or 20 mg/kg. NOS2 protein and NOS enzyme activity were measured in PBMC at baseline and 4 weeks following cA2 therapy. These results were compared with the degree of clinical change in disease activity. RESULTS At baseline, elevated levels of NOS2 protein and NOS enzyme activity were more frequently detected in PBMC from RA patients than in those from healthy controls. Treatment of the RA patients with cA2 significantly reduced NOS2 protein expression and NOS enzyme activity. Changes in NOS activity following treatment correlated significantly with changes in the number of tender joints. CONCLUSION These results indicate that TNFalpha likely plays an important role in enhancing NOS2 expression in RA, and that the antiinflammatory effects of cA2 treatment may be mediated by a reduction of NO overproduction.

Nitric oxide modulation of the growth and differentiation of freshly isolated acute non-lymphocytic leukemia cells

Freshly isolated acute non-lymphocytic leukemia (ANLL) cells were treated with the nitric oxide (NO)-liberating compounds sodium nitroprusside or S-nitrosoacetyl penicillamine and analyzed for viability, growth, and differentiation at 3-5 days. NO decreased the viability and the growth of freshly isolated ANLL cells in vitro. NO treatment significantly increased expression of CD14 in blast cells from patients with M5 ANLL, and increased at least one differentiation parameter in M4 or M5 cells. It had little or no effect on parameters of differentiation in other ANLL cells. We conclude that in vitro culture with NO decreases the growth and viability of most freshly isolated ANLL cells. NO also induces the differentiation of ANLL cells with a monocytic phenotype.

IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived non-dividing CD5+ B cells. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells may be dependent on the autocrine production of nitric oxide by inducible nitric oxide synthase (NOS2). We performed this study to determine whether cytokine factors that prevent spontaneous in vitroapoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity. B-CLL cells expressed NOS enzyme activity and NOS2 protein and mRNA. IL-4 and IFN-γ increased B-CLL cell NOS2 enzyme activity and protein expression during in vitro culture. IFN-γ, but not IL-4, increased NOS2 mRNA expression in cultured B-CLL cells suggesting that IL-4-mediated changes of NOS2 protein expression occurred at the post-transcriptional level. We were unable to detect increased concentrations of nitrite or nitrate (NOx) as surrogate markers of NO production in B-CLL cell cultures treated with IL-4 or IFN-γ. IL-4 and IFN-γ diminished NOS inhibitor-induced B-CLL cell death. In summary, we found that B-CLL cells expressed NOS2 and that IL-4 and IFN-γ increased B-CLL NOS2 expression. Cytokine-mediated expression of NOS2 by B-CLL cells may promote their survival, and therapeutic strategies that target NOS2 or quench NO may be beneficial in patients with B-CLL.

Host Response to Infection: the Role of CpG DNA in Induction of Cyclooxygenase 2 and Nitric Oxide Synthase 2 in Murine Macrophages

ABSTRACT Depending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E2 (PGE2) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 μg of CpG DNA/ml increased NO and PGE2production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE2 production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE2 production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells convertedl-arginine to l-citrulline, but the NOS inhibitorNG-monomethyl-l-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE2 production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, andN-iminoethyl-l-lysine effectively blocked NO production and increased the production of PGE2 in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE2occurring in an interdependent manner.

Nitric oxide production and mononuclear cell nitric oxide synthase activity in malaria-tolerant Papuan adults.

ABSTRACT Individuals living in regions of intense malaria transmission exhibit natural immunity that allows them to be without fever and other symptoms for most of the time despite frequent parasitization. Although this tolerance of parasitemia appears to be more effective in children than in adults (as evidenced by lower parasitemia fever thresholds with age), adults do exhibit a degree of tolerance but the mechanism(s) underlying this are unclear. Asymptomatic malaria-exposed children have higher levels of nitric oxide (NO) than children with severe disease, and NO has been proposed as a mediator of malarial tolerance. However, the ability of highly malaria-exposed asymptomatic adults to generate high-level basal NO is unknown, as is the relationship between NO and malaria tolerance in adults. The relationship between NO and malaria parasitemia was therefore determined in asymptomatic adults from Papua, Indonesia. Adults with Plasmodium falciparum parasitemia had markedly increased basal systemic NO production relative to aparasitemic Papuan controls, who in turn produced more NO than healthy controls from a region without malaria. Immunoglobulin E levels were universally elevated in malaria-exposed Papuan subjects, suggesting that the prevalence of intestinal parasitosis may be high and that nonmalarial infection may also contribute to high basal NO production. Basal peripheral blood mononuclear cell (PBMC) NO synthase activity was elevated in Papuans but poorly correlated with systemic NO production, suggesting that NO production in this setting arises not only from PBMCs but also from other tissue and cellular sources. NO production was associated with and may contribute to malaria tolerance in Papuan adults.

Synovial Mononuclear Phagocytes in Rheumatoid Arthritis and Osteoarthritis: Quantitative and Functional Aspects

Macrophages are normal constituents of synovial tissue, and in inflammatory synovitis the number of synovial macrophages increases. Synovial macrophages and their secretory products are important in initiating, propagating, and maintaining the synovial inflammation in rheumatoid arthritis (RA). The purpose of this study was to determine the absolute numbers of macrophages in synovia resected from patients with RA and osteoarthritis (OA) and to determine their abilities to produce and/or functionally express tumor necrosis factor (TNF), interleukin-1 (IL-1), and tissue factor (thromboplastin). Results demonstrate that synovial tissue from RA patients (as compared to that from OA patients) weighed more, contained more cells, more macrophages, and more multinucleated giant cells (macrophage polykaryons). Also, isolated cells from both OA and RA patients had tissue factor activity and could produce TNF and IL-1 with in vitro culture, but these parameters were not different in cells from OA and RA patients. RA patients receiving glucocorticoid treatment for their arthritis had fewer total synovial cells than did patients not on glucocorticoids, but treatment with nonsteroidal anti-inflammatory agents did not alter cell numbers. Patient treatment with glucocorticoids or non-steroidal anti-inflammatory drugs did not influence the ability of their isolated cells to produce TNF or IL-1.