8-Nitro-cGMP is a promoter of osteoclast differentiation induced by RANKL.

Osteoclasts are multinucleated giant cells differentiated from monocyte-macrophage-lineage cells under stimulation of receptor activator of nuclear factor κ-B (RANK) ligand (RANKL) produced by osteoblasts and osteocytes. Although it has been reported that nitric oxide (NO) and reactive oxygen species (ROS) are involved in this process, the mechanism by which these labile molecules promote osteoclast differentiation are not fully understood. In this study, we investigated the formation and function of 8-nitro-cGMP, a downstream molecule of NO and ROS, in the process of osteoclast differentiation in vitro. 8-Nitro-cGMP was detected in mouse bone marrow macrophages and osteoclasts differentiated from macrophages in the presence of RANKL. Inhibition of NO synthase suppressed the formation of 8-nitro-cGMP as well as RANKL-induced osteoclast differentiation from macrophages. On the other hand, RANKL-induced osteoclast differentiation was promoted by addition of 8-nitro-cGMP to the cultures. In addition, 8-nitro-cGMP enhanced the mRNA expression of RANK, the receptor for RANKL. However, 8-bromo-cGMP, a membrane-permeable derivative of cGMP, did not have an effect on either RANKL-induced osteoclast differentiation or expression of the RANK gene. These results suggest that 8-nitro-cGMP is a novel positive regulator of osteoclast differentiation, which might help to explain the roles of NO and ROS in osteoclast differentiation.

Enhanced in vitro biological activity generated by surface characteristics of anodically oxidized titanium--the contribution of the oxidation effect.

Anodically oxidized titanium surfaces, prepared by spark discharge, have micro-submicron surface topography and nano-scale surface chemistry, such as hydrophilic functional groups or hydroxyl radicals in parallel. The complexity of the surface characteristics makes it difficult to draw a clear conclusion as to which surface characteristic, of anodically oxidized titanium, is critical in each biological event. This study examined the in vitro biological changes, induced by various surface characteristics of anodically oxidized titanium with, or without, release of hydroxyl radicals onto the surface. Anodically oxidized titanium enhanced the expression of genes associated with differentiating osteoblasts and increased the degree of matrix mineralization by these cells in vitro. The phenotypes of cells on the anodically oxidized titanium were the same with, or without, release of hydroxyl radicals. However, the nanomechanical properties of this in vitro mineralized tissue were significantly enhanced on surfaces, with release of hydroxyl radicals by oxidation effects. In addition, the mineralized tissue, produced in the presence of bone morphogenetic protein-2 on bare titanium, had significantly weaker nanomechanical properties, despite there being higher osteogenic gene expression levels. We show that enhanced osteogenic cell differentiation on modified titanium is not a sufficient indicator of enhanced in vitro mineralization. This is based on the inferior mechanical properties of mineralized tissues, without either being cultured on a titanium surface with release of hydroxyl radicals, or being supplemented with lysyl oxidase family members.

Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide.

Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guérin (BCG) strain of Mycobacterium bovis. BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation. Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk. In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation. Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice. Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice. Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection. These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin.

Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption.

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients-namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)-using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Immune response in mice that lack the interferon-gamma receptor.

Interferon-gamma (IFN-gamma) exerts pleiotropic effects, including antiviral activity, stimulation of macrophages and natural killer cells, and increased expression of major histocompatibility complex antigens. Mice without the IFN-gamma receptor had no overt anomalies, and their immune system appeared to develop normally. However, mutant mice had a defective natural resistance, they had increased susceptibility to infection by Listeria monocytogenes and vaccinia virus despite normal cytotoxic and T helper cell responses. Immunoglobulin isotype analysis revealed that IFN-gamma is necessary for a normal antigen-specific immunoglobulin G2a response. These mutant mice offer the possibility for the further elucidation of IFN-gamma-mediated functions by transgenic cell- or tissue-specific reconstitution of a functional receptor.

Involvement of the IRF-1 transcription factor in antiviral responses to interferons.

The mechanisms underlying interferon (IFN)-induced antiviral states are not well understood. Interferon regulatory factor-1 (IRF-1) is an IFN-inducible transcriptional activator, whereas IRF-2 suppresses IRF-1 action. The inhibition of encephalomyocarditis virus (EMCV) replication by IFN-alpha and especially by IFN-gamma was impaired in cells from mice with a null mutation in the IRF-1 gene (IRF-1-/- mice). The IRF-1-/- mice were less resistant than normal mice to EMCV infection, as revealed by accelerated mortality and a larger virus titer in target organs. The absence of IRF-1 did not clearly affect replication of two other types of viruses. Thus, IRF-1 is necessary for the antiviral action of IFNs against some viruses, but IFNs activate multiple activation pathways through diverse target genes to induce the antiviral state.

Requirement for transcription factor IRF-1 in NO synthase induction in macrophages.

Production of nitric oxide (NO) by macrophages is important for the killing of intracellular infectious agents. Interferon (IFN)-gamma and lipopolysaccharide stimulate NO production by transcriptionally up-regulating the inducible NO synthase (iNOS). Macrophages from mice with a targeted disruption of the IFN regulatory factor-1 (IRF-1) gene (IRF-1-/- mice) produced little or no NO and synthesized barely detectable iNOS messenger RNA in response to stimulation. Two adjacent IRF-1 response elements were identified in the iNOS promoter. Infection with Mycobacterium bovis (BCG) was more severe in IRF-1-/- mice than in wild-type mice. Thus, IRF-1 is essential for iNOS activation in murine macrophages.

Nitric oxide in pulp cell growth, differentiation, and mineralization.

Dental preparation sometimes causes transient congestion, edema, and necrosis of the pulp. We hypothesized that nitric oxide (NO) is involved in the pathophysiological changes in pulp after preparation. The mRNA and protein expression of the inducible isoform of NO synthase (iNOS) was examined in murine pulp after dental preparation. The effects of NO on the proliferation, mineralization, and apoptosis of pulp cells were also studied in vitro. We found that not only iNOS, but also mRNAs for alkaline phosphatase and plasma membrane glycoprotein-1, were expressed in the pulp after preparation. NOC-18, an NO donor, suppressed the proliferation of pulp cells without inducing cell death, whereas it promoted the mineralization of cells cultured in the presence of beta-glycerophosphate, ascorbic acid, dexamethasone, and KH(2)PO(4). Under these conditions, NOC-18 induced the apoptosis of pulp cells. These results suggest that NO regulates the growth, apoptosis, and mineralization of pulp cells.

Inhibitors of cyclooxygenase-2 (COX-2) suppressed the proliferation and differentiation of human leukaemia cell lines.

Prostaglandins (PG) are known to play important roles in the proliferation and differentiation of leukaemia cells. The effect of the inhibitors of cyclooxygenase-2 (COX-2), a rate-limiting enzyme for the synthesis of PG, on the proliferation and differentiation of leukaemia cell lines was investigated. COX-2 inhibitors, NS-398 and nabumetone, suppressed the proliferation of U-937 and ML-1 cells by inducing a G0/G1 cell-cycle arrest. Cell-cycle arrest induced by these COX-2 inhibitors was not associated with an upregulation of the cyclin-dependent kinase inhibitors. COX-2 inhibitors also inhibited the differentiation of these cells induced by interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and retinoic acid (RA). Treatment with NS-398 did not suppress the levels of PGs produced by these cells. Although COX-2 antisense oligonucleotide showed a similar inhibitory effect on these cells, its inhibitory effect was smaller than that of NS-398. These results suggest that COX-2 inhibitors may suppress the proliferation and differentiation of leukaemia cells both via COX-2-dependent and -independent pathways.

Generation of nitric oxide and clearance of interferon-gamma after BCG infection are impaired in mice that lack the interferon-gamma receptor.

Mice with a targeted deletion of either the interferon (IFN)-gamma gene or the IFN-gamma receptor gene (IFN-gamma R(0/0) mice) fail to survive infection with the Bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis. Here we show that resident peritoneal macrophages isolated 2 weeks after BCG infection from IFN-gamma R(0/0) mice produced significantly less nitric oxide (NO) than wild-type macrophages. However, the response to lipopolysaccharide (LPS) was not completely abrogated in the IFN-gamma R(0/0) macrophages. BCG infection of wild-type mice led to a marked increase in their urinary nitrite/nitrate levels, as previously described. This increase in urinary nitrite/nitrate was not detected in BCG- infected IFN-gamma R(0/0) mice, indicating that no other cytokine can replace IFN-gamma as a mediator of increased NO synthesis after BCG infection in the intact organism. A comparison of circulating levels of IFN-gamma in BCG-infected animals revealed that sera from IFN-gamma R(0/0) mice contained up to 66-fold more IFN-gamma than sera from identically treated wild-type mice. To determine if the higher levels of circulating IFN-gamma were due to increased IFN-gamma synthesis, we compared the amounts of IFN-gamma mRNA present in the spleens of BCG-infected wild-type and IFN-gamma R(0/0) mice. No increase in IFN-gamma mRNA levels was detected in the spleens from IFN-gamma R(0/0) mice. Since the generation of IFN-gamma protein in cultured spleen cells was also not increased in IFN-gamma R(0/0) mice, we conclude that clearance of IFN-gamma from the circulation is impaired in IFN-gamma R(0/0) mice, thus revealing a heretofore unrecognized important role for the IFN-gamma receptor in the regulation of IFN-gamma levels in the intact organism.

Cytotoxic effect of sodium nitroprusside on cancer cells: involvement of apoptosis and suppression of c-myc and c-myb proto-oncogene expression.

Nitric oxide (NO) is an unstable free radical gas known as an effector molecule of macrophage cytotoxicity against cancer cells. Although several mechanisms of NO-mediated cytotoxicity have been proposed, this phenomenon remains to be characterized in detail. To explore the mechanisms by which NO kills cancer cells, we made use of sodium nitroprusside (SNP), which releases NO in the culture medium. SNP showed a dose-dependent cytotoxic effect on NA cells, an epithelial cancer cell line. When NA cells were killed by SNP, high levels of NO2- (stable end product of NO) were detected in the culture medium. The cell death induced by SNP was mediated by apoptosis, as demonstrated by the presence of nuclear condensation and blebbing of the nuclear membrane, and internucleosomal DNA fragmentation quantified by a specific ELISA. Northern blot analysis revealed that c-myc mRNA expression of NA cells was rapidly reduced by treatment with SNP. RT-PCR analysis showed that c-myb mRNA was expressed in untreated NA cells, and c-myb mRNA level of NA cells was dose-dependently reduced by treatment with SNP. These results indicate that SNP exerts its cytotoxic effect on NA cells through spontaneous release of NO. Cytotoxicity induced by SNP is at least partially mediated via the process known as apoptosis. Our results also suggest that down-regulation of c-myc and c-myb proto-oncogenes might be involved in SNP-induced cytotoxicity.

Involvement of caspases in 5-FU induced apoptosis in an oral cancer cell line.

Although many anticancer drugs have been reported to induce apoptosis in cancer cells, the underlying mechanism remains unclear (1-3). Recent studies have revealed that the caspase family of cysteine proteases have been shown to play an important role in the regulation of several apoptotic processes. Thus, the present study investigated whether apoptosis induced by anticancer drugs is mediated by the activation of caspase cascade. NA cells, a squamous cell carcinoma cell line, were exposed to cisplatin (CDDP) or 5-fluorouracil (5-FU) with or without inhibitors of caspase 1, 3 and 8. Analysis of DNA fragmentation revealed that caspase inhibitors consistently inhibited DNA fragmentation induced by 5-FU. During the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the cell surface. This PS externalization was markedly inhibited by treatment with caspase-8 inhibitor. These findings suggested that 5-FU induced apoptosis was mediated by the activation of a caspase cascade involving caspase 1, 3 and 8.

Biological functions of IFN-gamma and IFN-alpha/beta: lessons from studies in gene knockout mice.

Mice with a targeted disruption in the IFN-gamma receptor gene (IFN-gamma R0/0) provided a useful model to ask to what extent other cytokines could replace IFN-gamma in macrophage activation. In thioglycollate-elicited peritoneal macrophages from wild-typy (WT) mice, TNF enhanced nitric oxide (NO) release in the presence of IFN-gamma, though TNF alone was not effective. In macrophages from IFN-gamma R0/0 mice, which are not responsive to IFN-gamma, TNF completely failed to stimulate NO release. The NO inducing effects of IFN-alpha/beta were indistinguishable in IFN-gamma R0/0 and WT macrophages. The important role of IFN-gamma in the regulation of the induced expression of MHC class II antigen (Ia) was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis, peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from WT mice. BCG infection was not lethal for WT mice whereas all IFN-gamma R0/0 mice died 7-9 weeks after infection. It is well known that BCG infection greatly sensitizes mice to lethal action of LPS. Injection of LPS 2 weeks after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for WT mice. Reduced lethality of LPS correlated with a drastically reduced TNF-alpha production in the IFN-gamma R0/0 mice after BCG infection and LPS challenge. The greatly reduced ability of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection.(ABSTRACT TRUNCATED AT 250 WORDS)

The characteristics of in vitro biological activity of titanium surfaces anodically oxidized in chloride solutions.

Photo-functionalized radical reactions on TiO(2) have been correlated with adsorption of organic impurities and decreasing hydrophilicity of titanium-based biomaterials. Such reactive oxygen species (ROS) spontaneously generated on oxidized titanium surfaces may also have important roles against time-dependent degradation of biological ability and adherent micro-organisms. This study examined in vitro biological ability as a function of time and antimicrobial activity on oxidized titanium surfaces without photo-functionalization. Mechanically polished titanium and thermally oxidized titanium surfaces that had been stored for 4 wks showed adsorbed organic impurities with decreased surface hydrophilicity. Even after the storage period, anodically oxidized titanium surfaces enabled super-hydrophilicity without adsorption of organic impurities, because of the ROS and the hydrophilic functional groups generated on the surfaces. The osteogenic gene expressions of osteoblasts cultured on anodically oxidized titanium surfaces with or without storage were significantly higher than those on thermally oxidized titanium and polished titanium surfaces. Titanium surfaces anodically oxidized in a solution with chloride achieved antimicrobial activity against an oral microorganism due to the amount of ROS generated on the surface. Thus, titanium anodically oxidized in solution with chloride may have potential use for titanium-based internal fixation devices.

Mitogen-activated protein kinases mediate interleukin-1beta-induced receptor activator of nuclear factor-kappaB ligand expression in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Interleukin-1beta-stimulated receptor activator of nuclear factor-kappaB ligand (RANKL) expression in human periodontal ligament cells is partially mediated by endogenous prostaglandin E2, whereas mitogen-activated protein kinases (MAPKs) are implicated in regulating various interleukin-1-responsive genes. We investigated herein the involvement of MAPKs in interleukin-1beta-stimulated RANKL expression in human periodontal ligament cells. MATERIAL AND METHODS: Human periodontal ligament cells were pretreated separately with specific inhibitors of MAPKs, including extracellular signal-regulated kinase, p38 MAPK and c-Jun N-terminal kinase, and subsequently treated with interleukin-1beta. Following each treatment, the phosphorylation of each MAPK, the expression of RANKL, and the production of prostaglandin E2 were determined. RANKL activity was evaluated using an assay to determine the survival of prefusion osteoclasts. RESULTS: Interleukin-1beta induced RANKL expression at the mRNA and protein levels, as well as RANKL activity in human periodontal ligament cells. Interleukin-1beta also activated extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase. Pretreatment with each MAPK inhibitor partially, but significantly, suppressed interleukin-1beta-induced RANKL expression and its activity, as well as prostaglandin E2 production. CONCLUSION: In human periodontal ligament cells, three types of MAPK inhibitor may abrogate RANKL expression and activity induced by interleukin-1beta, directly or indirectly through partial suppression of prostaglandin E2 synthesis. In addition, extracellular signal-regulated kinase, p38 MAPK, and c-Jun N-terminal kinase signals may co-operatively mediate interleukin-1beta-stimulated RANKL expression and its activity in those cells.

Downregulation of c-myc expression by tumor necrosis factor-alpha in combination with transforming growth factor-beta or interferon-gamma with concomitant inhibition of proliferation in human cell lines.

The modulation of cell growth by tumor necrosis factor-alpha (TNF-alpha), or TNF-alpha in combination with transforming growth factor-beta (TGF-beta) or interferon-gamma (IFN-gamma) was investigated. TNF-alpha inhibited the proliferation of U937 cells, a monocytic leukemic cell line, and of NA cells that were established from oral squamous cell carcinoma. TNF-alpha showed a cytolytic effect on NA cells in the presence of actinomycin D. TNF-alpha in combination with TGF-beta and TNF-alpha combined with INF-gamma synergistically inhibited the cell proliferation of U937 and NA cells, respectively. TNF-alpha dose-dependently reduced c-myc mRNA expression of U937 and NA cells within 1 h. The combination of TNF-alpha and TGF-beta in U937 cells and that of TNF-alpha and IFN-gamma in NA cells cooperatively reduced the expression of c-myc mRNA. TNF-alpha had little or no effect on the half-life of c-myc mRNA, indicating that c-myc mRNA expression was reduced at transcriptional level. Cycloheximide did not mediate the inhibition of c-myc gene expression, suggesting that the TNF-alpha action was independent of de novo protein synthesis. These data suggest that the reduction of c-myc gene at transcriptional level by TNF-alpha or TNF-alpha in combination with TGF-beta or IFN-gamma plays a primary role in the inhibition of cell growth at an early stage.

Induction of resistance to TNF cytotoxicity and mitochondrial superoxide dismutase on U-937 cells by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited the cytotoxicity of tumor necrosis factor (TNF) in a human monoblastic leukemic cell line, U-937. Combination of TNF and 1,25(OH)2D3 remarkably increased mitochondrial superoxide dismutase (mSOD) of U-937 cells, TNF alone increased it only slightly and 1,25(OH)2D3 alone did not. The cytosolic SOD (cSOD) activity was not changed by TNF or/and 1,25(OH)2D3. The mSOD activity was not inhibited by 2 mM KCN, suggesting that mSOD should be a manganese SOD (MnSOD). These results suggest that 1,25(OH)2D3 may reduce the susceptibility to TNF cytotoxicity of U-937 cells by enhancing the ability of inducing MnSOD by TNF.

Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines.

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.

Suppression of TNF-stimulated proliferation of diploid fibroblasts and TNF-induced cytotoxicity against transformed fibroblasts by TGF-beta.

Human transforming growth factor-beta (TGF-beta) dose-dependently inhibited proliferation of WI-38 cells, normal human diploid fibroblasts, stimulated by tumor necrosis factor (TNF). Inhibition occurred at 1 ng/ml concentration of TGF-beta. Also, TGF-beta dose-dependently suppressed cytotoxicity of TNF against L-929 cells, murine transformed fibroblasts. The concentration of TNF required for 50% cytolysis of L-929 cells was changed from 30 ng/ml to 350 ng/ml by 10 ng/ml TGF-beta. This suppression was abolished when L-929 cells were treated with actinomycin D or cycloheximide, suggesting that TGF-beta might inhibit the action of TNF via de novo protein synthesis. This response was not due to down regulation of TNF receptors nor to alteration of the affinity of TNF for its receptor.

Generation of nitric oxide and induction of major histocompatibility complex class II antigen in macrophages from mice lacking the interferon gamma receptor.

Availability of mice with a targeted disruption of the interferon gamma (IFN-gamma) receptor gene (IFN-gamma R0/0 mice) made it possible to examine parameters of macrophage activation in the absence of a functional IFN-gamma receptor. We asked to what extent other cytokines could replace IFN-gamma in the induction of nitric oxide or major histocompatibility complex class II antigen (Ia) expression in peritoneal macrophages. In thioglycollate-elicited macrophages from wild-type mice, tumor necrosis factor (TNF) alone was virtually ineffective in inducing release of NO2- (the endproduct of nitric oxide generation), but TNF enhanced NO2- release in the presence of IFN-gamma. In macrophages from IFN-gamma R0/0 mice, which were unresponsive to IFN-gamma, TNF completely failed to stimulate NO2- release. The stimulatory actions of IFN-alpha/beta on NO2- release were indistinguishable in wild-type and IFN-gamma R0/0 macrophages: IFN-alpha/beta was ineffective on its own, showed marginal stimulation of NO2- release in combination with TNF, and was moderately effective in the presence of lipopolysaccharide. The level of constitutive Ia antigen expression was not significantly different in peritoneal macrophages from wild-type and IFN-gamma R0/0 mice. An increased Ia expression was induced by IL-4 and granulocyte-macrophage colony-stimulating factor in both wild-type and IFN-gamma R0/0 macrophages, but the magnitude of this induction was less than with optimal concentrations of IFN-gamma in macrophages from wild-type mice. IFN-alpha/beta showed only a minor stimulatory effect on Ia expression in both wild-type and IFN-gamma R0/0 macrophages. Simultaneous treatment of wild-type macrophages with IFN-alpha/beta and IFN-gamma reduced the IFN-gamma-induced Ia expression in wild-type macrophages, but IFN-alpha/beta did not show an inhibitory effect on IL-4- or granulocyte-macrophage-colony-stimulating factor-induced Ia expression in either wild-type or IFN-gamma R0/0 macrophages. The important role of IFN-gamma in the regulation of the induced expression of major histocompatibility complex class II antigen was confirmed by showing that after systemic infection with the BCG strain of Mycobacterium bovis resident peritoneal macrophages from IFN-gamma R0/0 mice had a lower level of Ia expression than macrophages from wild-type mice. The inability of other cytokines to substitute fully for IFN-gamma in macrophage activation helps to explain the earlier observed decreased resistance of IFN-gamma R0/0 mice to some infections.