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Biomedical subjects

C Nathan

Publications and source records attributed to C Nathan.

At least 73 records · Page 4Linked to original sources
PubMed1994

Role of interferon regulatory factor 1 in induction of nitric oxide synthase.

Interferon gamma (IFN-gamma) interacts synergistically with bacterial lipopolysaccharide (LPS) to induce transcription of iNOS, the isoform of nitric oxide synthase whose activity is independent of elevated Ca2+ and exogenous calmodulin. To define a cis-acting element mediating IFN-gamma-dependent synergy, we made deletions in iNOS promoter constructs fused to reporter genes, transfected RAW 264.7 macrophages, and treated the cells with IFN-gamma and/or LPS. This analysis implicated the region from positions -951 to -911, a cluster of four enhancer elements known to bind IFN-gamma-responsive transcription factors, including an interferon regulatory factor binding site (IRF-E) at nucleotides -913 to -923. Site-specific substitution of two conserved nucleotides within IRF-E in the context of the full-length iNOS promoter ablated IFN-gamma's contribution to synergistic enhancement of transcription. Electromobility shift assays performed with a probe containing IRF-E revealed the existence of a complex in nuclei of RAW 264.7 macrophages that was present only after treatment with IFN-gamma, which reacted specifically with anti-IRF-1 immunoglobulin G and which included a species migrating at 40-45 kD, consistent with the apparent molecular weight of murine IRF-1. Thus, the synergistic contribution of IFN-gamma to transcription of iNOS in RAW 264.7 macrophages requires that IRF-1 bind to IRF-E in the iNOS promoter. In conjunction with the work of Kamijo et al. (Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, K. S. Im, T. Kimura, S. Green et al. 1994. Science [Wash. DC]. 263:1612), these findings identify iNOS as the first gene that requires IRF-1 for IFN-gamma-dependent transcriptional regulation.

Amino Acid Oxidoreductases↗
PubMed1994

Inactivation of nitric oxide synthase after prolonged incubation of mouse macrophages with IFN-gamma and bacterial lipopolysaccharide.

Large amounts of nitric oxide (NO) are produced by the inducible isoform of NO synthase (iNOS) in many cell types once the iNOS gene is transcriptionally activated. In primary mouse peritoneal macrophages elicited by thioglycolate broth, expression of iNOS follows treatment with IFN-gamma and is synergistically increased by the addition of bacterial LPS. Expression of iNOS is suppressible at transcriptional and translational levels by certain cytokines and microbial products. The present study describes a novel form of inactivation of iNOS that is post-translational and nondegradative. Mouse peritoneal macrophages cultured in the presence of IFN-gamma alone or IFN-gamma plus LPS rapidly depleted the medium of L-arginine, a substrate for iNOS, and stopped producing NO. Repletion of L-arginine permitted cells treated with IFN-gamma alone to resume NO production for at least 5 days, leading to the release of more NO than macrophages were previously believed capable of generating. L-Arginine repletion also boosted NO production by macrophages cultured for up to 2 to 3 days in the presence of IFN-gamma plus LPS, but thereafter, iNOS was inactive in these cells whether or not L-arginine was repleted. Activity of iNOS could be restored by adding both L-arginine and fresh IFN-gamma with or without LPS, likely reflecting the synthesis of new enzyme. However, the inactivation of iNOS seen late in culture with a single application of IFN-gamma plus LPS could be attributed neither to loss of iNOS protein nor to its autoinactivation by NO. Thus, LPS, a co-inducer of iNOS, causes macrophages to inactivate iNOS about 3 days after the onset of its induction. The mechanism, which remains to be identified, is novel for iNOS, in that it decreases neither its amount nor its apparent molecular mass.

Amino Acid Oxidoreductases↗
PubMed1994

Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase.

The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages.

Amino Acid Oxidoreductases↗
PubMed1994

Mechanism of suppression of nitric oxide synthase expression by interleukin-4 in primary mouse macrophages.

Nitric oxide (NO) contributes to the antitumor, antimicrobial, and immunosuppressive activity of macrophages. An inducible form of NO synthase (iNOS) is responsible for high output generation of nitric oxide by macrophages after stimulation with cytokines and/or lipopolysaccharide (LPS). In the present study, we demonstrate that interleukin 4 (IL-4) suppressed production of NO by primary mouse peritoneal macrophages exposed to IFN-gamma with or without LPS, even while synergizing with IFN-gamma to increase the secretion of TNF-alpha. Suppression of NO production was paralleled by decreases in iNOS enzyme activity and iNOS antigen. IL-4 did not inhibit induction of iNOS mRNA 4-6 h after exposure to IFN-gamma, but strongly reduced iNOS mRNA at later times of stimulation (24-72 h), without increasing its turnover. The conditions for maximal suppression of iNOS expression by IL-4 and the mechanisms of suppression differed from those determined in parallel for transforming growth-factor-beta as described elsewhere. These results illustrate the diversity of phenotypes of macrophages deactivated by different cytokines, and demonstrate that IL-4 has the potential to reduce one component of the anti-tumor, antimicrobial, and immunosuppressive activities of macrophages.

Amino Acid Oxidoreductases↗
PubMed1994

The high-output nitric oxide pathway: role and regulation.

Nitric oxide synthase (NOS) catalyzes the production of nitric oxide (NO), a short-lived radical gas with physiological or pathophysiological roles in nearly every organ system. The inducible NO synthase (iNOS) is a high-output isoform compared to the two constitutive NOSs. The iNOS from murine macrophages tightly binds calmodulin as a subunit, and its activity is not dependent on exogenous calmodulin or elevated calcium. This iNOS is induced at the transcriptional level by bacterial lipopolysaccharide (LPS) and interferon-gamma. The promoter region of the murine iNOS gene contains at least 24 oligonucleotide motifs corresponding to elements involved in the binding of transcription factors in the promoters of other cytokine-inducible genes. Nuclear factor NF-kappa B/c-rel, interacting with cycloheximide-sensitive protein(s) and binding to the NF-kappa Bd site in the iNOS promoter, controls the induction of iNOS by LPS. However, iNOS is also regulated posttranscriptionally. Complex regulation of iNOS at multiple levels may reflect the dual role of iNOS in host defense and autotoxicity.

Amino Acid Oxidoreductases↗
PubMed1994

Cytokine regulation of nitric oxide synthase in mouse retinal pigment epithelial cells in culture.

Nitric oxide is an intercellular signaling molecule whose numerous functions include regulation of vascular tone, mediation of the cytotoxic effects of macrophages and potentiation of synaptic transmission. For some cellular functions, nitric oxide synthesis is mediated by the inducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-gamma and lipopolysaccharide, express inducible nitric oxide synthase. The latter was detected immuno-cytochemically in interferon-gamma-lipopolysaccharide-treated retinal pigment epithelium using rabbit antiserum to a synthetic peptide of mouse nitric oxide synthase. Untreated cultures of retinal pigment epithelium or cultures treated with either interferon-gamma or or lipopolysaccharide alone were not immunoreactive. Induction of iNOS in gamma-interferon-lipopolysaccharide-stimulated retinal pigment epithelial cells was also evidenced by the presence of nitric oxide synthase enzyme activity in lysates of stimulated but not unstimulated retinal pigment epithelial cells. On immunoblots of lysates of stimulated murine retinal pigment epithelial cells, rabbit antiserum to iNOS recognized a 130-kDa protein which comigrated with the inducible nitric oxide synthase of macrophages and which was not detectable in lysates of unstimulated retinal pigment epithelial cells nor in lysates of cells treated with only interferon-gamma or lipopolysaccharide alone. Nitrite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-gamma and lipopolysaccharide, and continued to accumulate in a linear fashion for at least 96 hr. Treatment of cultured retinal pigment epithelium with interferon-gamma, lipopolysaccharide and either basic fibroblast growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensified signals on immunoblots, enhanced accumulation of nitrite and increased iNOS enzyme activity. Conversely, when transforming growth factor-beta was present in the culture medium, gamma-interferon-LPS-induced expression of nitric oxide synthase and NO release were reduced. We conclude that interferon-gamma synergizes with lipopolysaccharide to induce synthesis of inducible nitric oxide synthase and production of nitric oxide by murine retinal pigment epithelium and that this induction can be modulated by basic fibroblast growth factor, epidermal growth factor or transforming growth factor-beta.

Amino Acid Oxidoreductases↗
PubMed1994

Macrophage activation revisited.

Macrophages are extremely adaptable cells, able to modify their behavior in response to diverse signals from other cells and the extracellular matrix. A recent workshop provided insights into current research on these remarkable cells.

Animals↗
PubMed1994

Beta 2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor.

The focal adhesion protein paxillin undergoes tyrosine phosphorylation in response to signals mediated by integrins, neuropeptides and oncogene products, possibly via activation of the focal adhesion-associated kinase, p125FAK. In the present work, tumor necrosis factor-alpha (TNF) stimulated tyrosine phosphorylation of paxillin in human neutrophils. Cell adhesion and participation of the beta 2 integrin CD18 were necessary, but not sufficient, for the response. Adherent neutrophils also tyrosine phosphorylated paxillin in response to phorbol ester, formylmethionyl-leucyl-phenylalanine and opsonized bacteria. In contrast, p125FAK was constitutively tyrosine phosphorylated in a manner unaffected by adherence and/or TNF. Thus, cytokines and microbial products are among the stimuli that can induce the tyrosine phosphorylation of paxillin, and kinases other than p125FAK may be responsible. This is the first identification of paxillin and p125FAK in human cells and neutrophils, and one of the few identifications of a specific protein that undergoes tyrosine phosphorylation in response to any agonist in neutrophils or in response to TNF in any cell.

CD18 Antigens↗
PubMed1993

Inhibition of viral replication by interferon-gamma-induced nitric oxide synthase.

Interferons (IFNs) induce antiviral activity in many cell types. The ability of IFN-gamma to inhibit replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated with the cells' production of nitric oxide (NO). Viral replication was restored in IFN-gamma-treated macrophages exposed to inhibitors of NO synthase. Conversely, epithelial cells with no detectable NO synthesis restricted viral replication when transfected with a complementary DNA encoding inducible NO synthase or treated with organic compounds that generate NO. In mice, an inhibitor of NO synthase converted resolving ectromelia virus infection into fulminant mousepox. Thus, induction of NO synthase can be necessary and sufficient for a substantial antiviral effect of IFN-gamma.

Amino Acid Oxidoreductases↗
PubMed1993

Transcription and translation of inducible nitric oxide synthase in the pancreas of prediabetic BB rats.

The inducible NO synthase (iNOS) was found to be expressed in pancreatic lesions of adult diabetes-prone BB rats. Pancreatic iNOS mRNA was detected by reverse transcriptase PCR in pancreatic RNA of adult diabetes-prone BB rats but not in normal Wistar rats, young diabetes-prone BB rats without insulitis or in diabetes-resistant BB rats. Immunohistochemistry of pancreatic sections using an iNOS-specific antiserum labeled the pancreas of adult diabetes-prone BB rats but not Wistar rats. Parallel staining for ED1-positive macrophages showed restriction of iNOS expression to areas of islet infiltration by macrophages. In conclusion, the data provide direct evidence for enhanced expression of inducible NO synthase in tissue lesions during the development of autoimmune diabetes.

Amino Acid Oxidoreductases↗
PubMed1993

Mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta.

Activated mouse peritoneal macrophages produce nitric oxide (NO) via a nitric oxide synthase that is inducible by interferon gamma (IFN-gamma): iNOS. We have studied the mechanisms by which transforming growth factor beta 1 (TGF-beta) suppresses IFN-gamma-stimulated NO production. TGF-beta treatment reduced iNOS specific activity and iNOS protein in both cytosolic and particulate fractions as assessed by Western blot with monospecific anti-iNOS immunoglobulin G. TGF-beta reduced iNOS mRNA without affecting the transcription of iNOS by decreasing iNOS mRNA stability. Even after iNOS was already expressed, TGF-beta reduced the amount of iNOS protein. This was due to reduction of iNOS mRNA translation and increased degradation of iNOS protein. The potency of TGF-beta as a deactivator of NO production (50% inhibitory concentration, 5.6 +/- 2 pM) may reflect its ability to suppress iNOS expression by three distinct mechanisms: decreased stability and translation of iNOS mRNA, and increased degradation of iNOS protein. This is the first evidence that iNOS is subject to other than transcriptional regulation.

Amino Acid Oxidoreductases↗
PubMed1993

Traces of bacterial lipopolysaccharide suppress IFN-gamma-induced nitric oxide synthase gene expression in primary mouse macrophages.

A nitric oxide synthase (iNOS) inducible by cytokines and microbial products contributes to the cytotoxic and antimicrobial activity of mouse macrophages. Bacterial LPS interacts synergistically with IFN-gamma to induce iNOS when both stimuli are added together. In contrast, we show here that pre-exposure of peritoneal macrophages to low concentrations of LPS suppresses the induction of iNOS when IFN-gamma is added subsequently. Suppression required pretreatment with LPS for at least 8 h and was optimal with LPS concentrations in the range of 50 to 200 pg/ml. Suppression was exerted by smooth and rough forms of LPS from Escherichia coli and by lipid A from Salmonella minnesota, but not by a biologically inactive lipid A from Rhodobacter sphaeorides. Suppression of nitrite accumulation and iNOS enzyme activity by prior exposure of macrophages to LPS could be explained by their markedly decreased content of iNOS protein, as revealed by immunoblot with monospecific anti-iNOS IgG. Messenger RNA for iNOS was affected in a biphasic manner by pretreatment with LPS. Five hours after addition of IFN-gamma, iNOS mRNA levels were unaltered or even enhanced by pretreatment with LPS, but by 24 to 48 h, expression of iNOS mRNA was inhibited strongly enough to account for the reduced levels of iNOS protein. Suppression by LPS did not appear to be mediated by endogenous prostaglandins, transforming growth factor-beta, or TNF-alpha, even though pretreatment with exogenous TNF-alpha was also suppressive. These findings suggest that preactivation of pathways normally contributing to synergistic induction of iNOS may deplete macrophages of factors needed for its expression. Regulation of iNOS in vivo may depend on the relative tempo with which the inflammatory and immune responses evolve.

Amino Acid Oxidoreductases↗
PubMed1993

Modulation of macrophage function by transforming growth factor beta, interleukin-4, and interleukin-10.

The findings reviewed above leave no doubt as to the complexity of actions of TGF-beta, IL-4, and IL-10 on monocytes/macrophages. Along with MDF, whose actions were recently reviewed elsewhere, TGF-beta, IL-4, and IL-10 are the only presently known, purified cytokines that have been shown to have strong macrophage-deactivating effects. However, none of them can be categorized as purely macrophage deactivating since they also exert macrophage-activating effects. In vitro, their effects, both in terms of extent and direction (activating vs. deactivating), are strongly influenced by the stimulation conditions (e.g., triggering signal, cytokine concentration, timing of cytokine addition), the species (mouse vs. human), the source (blood vs. peritoneal, alveolar, colostral) and the state of differentiation/activation of the macrophage (e.g., resting vs. inflammatory). In addition, TGF-beta, as well as IL-4 and IL-10, up- and/or downregulates the function of several cell types other than macrophages, which further hampers our ability to predict, on the basis of in vitro experiments with macrophages, possible effects during an immune response in vivo. Despite this complexity, the highly reductive approach of in vitro studies has revealed important differences in the ability of TGF-beta, IL-4, and IL-10 to modulate the phenotype of monocytes/macrophages. The disparities have been most striking with regard to the secretory function of monocytes/macrophages (see Table 2). First, TGF-beta, IL-4, and IL-10 have a different spectrum of activity. Thus, TGF-beta, but not IL-4 or IL-10, can induce resting human monocytes to produce TNF, IL-1, and IL-6. Second, they affect monokine and RNI and ROI production to a different extent. For example, IL-10 is an approximately 25-fold more potent suppressor of LPS-induced TNF production by mouse macrophages than is TGF-beta. Third, they differ in their ability to overcome additional activating stimuli, so that in the presence of LPS, IL-4, but not TGF-beta or IL-10 suppresses IFN gamma-induced RNI release. Fourth, their macrophage-deactivating effects require different stimulation conditions. Thus, IL-4, but not TGF-beta, interferes with RNI release strongly only after preincubation of the macrophages. Finally, these agents deactivate macrophages by distinct mechanisms. For example, IL-10 causes massive downregulation of TNF mRNA, whereas TGF-beta suppresses TNF release on a translational level. It will be a challenge to define clinical applications for these potent macrophage modulators on the basis of their different spectrum of activities. For TGF-beta and IL-4 such studies have already been initiated.

Animals↗
PubMed1993

Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.

Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; 3 copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-alpha-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.

Amino Acid Oxidoreductases↗
PubMed1993

Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor.

Human neutrophils (PMN) respond to tumor necrosis factor (TNF) by releasing their granules, reorganizing their cytoskeleton, and massively secreting hydrogen peroxide. This response is dependent on adhesion to extracellular matrix proteins and expression of CD11b/CD18 integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. D. Wright. 1989. J. Cell Biol. 109:1341-1349). We investigated the role of tyrosine phosphorylation in the response of PMN to TNF. PMN adherent to protein-coated surfaces but not suspended PMN showed tyrosine phosphorylation of several proteins (approximately 150, approximately 115, approximately 75, and approximately 65 kD) in response to TNF. Tyrosine phosphorylation was evident 5 min after addition of TNF and lasted at least 2 h. The tyrosine kinase inhibitors K252a, genistein and ST638 suppressed tyrosine phosphorylation and blocked hydrogen peroxide production in a reversible manner at low concentrations. Tyrosine kinase inhibitors also blocked the spreading of PMN in response to TNF. Dihydrocytochalasin B did not inhibit tyrosine phosphorylation, but in its presence phosphorylation was rapidly reversed. By immunocytochemistry, the majority of tyrosine phosphoproteins were localized to focal adhesions. Thus TNF-induced tyrosine phosphorylation depends on adhesion of PMN to extracellular matrix proteins, and participates in the transduction of the signals that direct the cells to spread on a biological surface and undergo a respiratory burst.

Carbazoles↗
PubMed1993

Albumin inhibits neutrophil spreading and hydrogen peroxide release by blocking the shedding of CD43 (sialophorin, leukosialin).

Spreading of neutrophils on protein-coated surfaces is a pivotal event in their ability to respond to soluble, physiologic agonists by releasing large amounts of hydrolases and oxidants. Using neutrophils plated on serum-, fibrinogen- or fibronectin-coated surfaces, we investigated the effect of human serum albumin (HSA) on spreading-dependent neutrophil responses. HSA suppressed the respiratory burst of neutrophils in response to tumor necrosis factor-alpha (TNF), complement component C5a or formylated peptide, but not phorbol myristate acetate. HSA was suppressive only if added before the onset of the respiratory burst, and suppression was reversed when HSA was removed. Likewise, HSA selectively and reversibly inhibited TNF-induced cell spreading and the associated fall in cAMP. However, HSA did not hinder TNF-induced cell adherence to the same protein-coated surfaces. We investigated cell surface sialoproteins as modulators of cell spreading and as targets for the anti-spreading action of HSA. Oxidation of the cell surface with periodate followed by reduction with 3H-borohydride and immunoblotting with specific mAbs helped identify the predominant sialoprotein on human neutrophils as CD43 (sialophorin, leukosialin). Treatment of neutrophils with C. perfringens sialidase desialylated CD43, markedly enhanced the ability of the cells to respond to TNF by spreading and undergoing a respiratory burst, and antagonized the ability of HSA to inhibit these responses. TNF-treated, adherent neutrophils shed CD43, and this was blocked by HSA, but not by ovalbumin. Exogenous neutrophil elastase removed CD43 from the neutrophil surface. HSA blocked the actions of both sialidase and elastase on CD43. In contrast, ovalbumin did not block the action of sialidase on CD43, and HSA did not inhibit the ability of sialidase to hydrolyze a synthetic substrate. These results suggested that HSA might bind CD43. In fact, the extracellular portion of CD43 bound to HSA-Sepharose, but not to ovalbumin- or glycylglycine-Sepharose. Finally, two mAbs recognizing different epitopes on CD43 mimicked HSA's inhibitory effects on neutrophil function. Thus, HSA can dissociate attachment of neutrophils from spreading. This dissociation may help neutrophils migrate along a chemotactic gradient, while decreasing their release of oxidants. CD43, a long, rigid molecule with a markedly negative charge, antagonizes neutrophil spreading. HSA appears to inhibit spreading-dependent neutrophil functions by binding to CD43 and interfering with the ability of neutrophils to shed it.

Antigens, CD↗