Macrophage Tumor Necrosis Factor-alpha Research Articles

IL-12 increases CD80 expression and the stimulatory capacity of bone marrow-derived dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells derived from CD34 bone marrow stem cells. They undergo a series of maturational steps that allow them to stimulate primary T cell responses. Several cytokines are known to contribute to this process. In this study murine DC maturing from bone marrow progenitors under the influence of granulocyte macrophage colony stimulating factor and tumour necrosis factor-alpha were found to produce IL-12 as measured by ELISA and by flow cytometry to detect intracellular cytokine. Administration of additional IL-12 from day 3 to 7 of culture altered the function and phenotype of DC; enhanced stimulation of T cell proliferation by DC in allogeneic mixed leukocyte reactions was associated with an increase in the surface expression of CD80 on DC. These effects were dose dependent, and were consistently seen with IL-12 at 25 ng/ml and were less marked with IL-12 at 50 ng/ml. These results show that IL-12 is both produced by DC and can increase their stimulatory capacity. The findings suggest that there may be an autocrine effect of IL-12 on DC maturation and function.

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production.

In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.

Concentration- and time-dependent upregulation and release of the cytokines MIP-2, KC, TNF, and MIP-1alpha in rat alveolar macrophages by fungal spores implicated in airway inflammation.

Inhalation of fungal spores has been shown to cause primary or secondary infection and respiratory inflammation and diseases such as allergic alveolitis, atopic asthma, and organic dust toxic syndrome, which are rarely reported in the absence of predisposing factors. Biochemical and molecular markers of inflammation were measured in rat bronchial alveolar lavage cells (> 95% macrophages) following stimulation with fungal spores isolated from pathogenic and nonpathogenic fungi that have been implicated in airway inflammation. The results of this study demonstrate that mRNA transcripts for the C-X-C branch of the PF4 superfamily are differentially upregulated over those of the C-C mediators in a time- and concentration-dependent manner. Macrophage inflammatory protein (MIP)-2 and KC were differentially upregulated over the acute phase inflammatory cytokines MIP-1alpha and tumor necrosis factor-alpha (TNF-alpha) in rat alveolar macrophages stimulated with fungal spores from Aspergillus candidus, Aspergillus niger, Eurotium amstelodami, and Cladosporium cladosporioides. Spores from Aspergillus terreus and Penicillium spinulosum failed to stimulate an increase of any cytokine mRNA, whereas those from Aspergillus fumigatus stimulated the upregulation of MIP-2, KC, TNF-alpha, and MIP-1alpha mRNAs. Over time, A. fumigatus stimulated increasing KC production until 24 h, when production levels increased slightly, then leveled off when measurements ceased at 36 h. Latex spheres stimulated modest amounts of MIP-2 and transforming growth factor-beta only. These observations suggest that the inflammatory cytokines MIP-2 and KC may be involved in the inflammation arising from the inhalation of fungal spores in a time- and concentration-dependent manner.

ENDOTOXIN CONTAMINATION MAY BE RESPONSIBLE FOR THE UNEXPLAINED FAILURE OF HUMAN PANCREATIC ISLET TRANSPLANTATION1

Clinical transplantation of human islets has a disappointingly low rate of success. We report here the identification of a possible causative factor: endotoxin present in the collagenase preparations used to disperse the pancreatic tissue before islet purification and transplantation. Supporting evidence includes (1) detection of unexpectedly high levels of endotoxin in most collagenase solutions currently used to digest human pancreases; (2) demonstration that supernatants generated during islet separation are able to induce the inflammatory cytokines interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha) in macrophages; and (3) induction of IL-1, IL-6, and TNF-alpha in the islets during the separation procedure. Cytokine expression was assessed by reverse transcription-polymerase chain reaction and, for TNF-alpha, confirmed by enzyme-linked immunoabsorbent assay. It is proposed that endotoxin and locally induced cytokines carried over with the graft activate the endothelium and promote lymphomonocytic infiltration of grafted islets and surrounding liver tissue favoring primary nonfunction and early rejection. These results also have implications for the numerous experimental procedures that use collagenase, and they point to possible ways to improve islet preparation and transplantation protocols.

L-arginine decreases alveolar macrophage proinflammatory monokine production during acute lung injury by a nitric oxide synthase-dependent mechanism.

Recent clinical reports indicate that inhaled nitric oxide (NO) reduces lung parenchymal inflammation during acute lung injury; however, the mechanism of its protective effects remains incompletely understood. We hypothesized that the provision of substrate for local NO production (L-arginine) would reduce alveolar macrophage proinflammatory monokine production during endotoxin (ETX)-induced acute lung injury. Our purposes were to (1) determine alveolar macrophage tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) production after ETX-induced acute lung injury; (2) determine the effect of L-arginine on alveolar macrophage TNFalpha and IL-1beta production in ETX-induced acute lung injury; and (3) determine whether L-arginine's effects on the alveolar macrophage are mediated by NO. Rats received ETX (0.5 mg/kg intraperitoneal (i.p.)) or vehicle, with or without (1) L-arginine supplementation (300 mg/kg i.p.) and (2) nitric oxide synthase inhibition (N(G)-monomethyl-L-arginine, 30 mg/kg i.p.). Four hours later, alveolar macrophage were harvested by bronchoalveolar lavage and incubated at 10(6) cells/mL + 1 microg/mL phorbol myristase acetate for 24 hours. Cell-free supernatants were collected and assayed (enzyme-linked immunosorbent assay) for TNFalpha and IL-1beta. Sublethal ETX increased alveolar macrophage capacity to produce TNFalpha and IL-1beta (p < 0.05, analysis of variance and Bonferroni/Dunn). L-Arginine decreased alveolar macrophage TNFalpha and IL-1beta release during acute lung injury. Concurrent inhibition of nitric oxide synthase abrogated L-arginine's protective effects, suggesting that L-arginine's anti-inflammatory effects are mediated by NO. (1) L-Arginine is an immunomodulating nutritional supplement; (2) L-arginine decreases alveolar macrophage proinflammatory monokine production during ETX-induced acute lung injury by a nitric oxide synthase-dependent mechanism; and (3) the provision of exogenous substrate for local NO production may reduce inflammation during acute lung injury.

Increased levels of macrophage colony-stimulating factor, gamma interferon, and tumor necrosis factor alpha in sera of patients with Orientia tsutsugamushi infection.

Levels of macrophage colony-stimulating factor (nine of nine patients) and gamma interferon (six of nine patients) in serum were elevated above the range of normal in the acute phase of tsutsugamushi disease. Significant increases in levels of tumor necrosis factor alpha were observed during the convalescent phase in five patients, and they exceeded the levels observed during the acute phase. Hypercytokinemia appeared to be responsible for the emergence of the symptoms of tsutsugamushi disease.

Intratracheal instillation versus intratracheal inhalation: influence of cytokines on inflammatory response.

Our laboratory has developed a method of particle exposure whereby anesthetized rats intratracheally inhale, at a regulated breathing rate and pressure, an aerosolized test material. This method is capable of delivering considerable doses in a short time period and, unlike the commonly used method of intratracheal instillation, does so with an even particle distribution throughout the lung. Early studies comparing the response of male Fischer 344 rats exposed to TiO2 particles of two differing primary particle sizes showed that at similar particle doses animals exposed by the two methods showed differences in response, as measured by bronchoalveolar lavage (BAL) parameters. Building on this, we sought to study the roles that macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor alpha (TNF-alpha), two cytokines thought to have proinflammatory roles in the lung, may play in the differences observed. Increases in MIP-2 protein levels in the lavaged cells, but not the supernatant, were observed in those groups where increased polymorphonuclear cells (PMN) in the lung lavage were found, but not in those where no increase in PMN levels was observed. BAL TNF-alpha levels, measured by enzyme-linked immunosorbent assay, showed no apparent correlation with cellular or biochemical BAL parameters for either particle size or dosing method. Increases in immunocytochemical staining for TNF-alpha, compared to unexposed controls, were observed in several particle-exposed groups. Thus, it appears that increased BAL MIP-2 protein levels, but not TNF-alpha, correlate well with the inflammatory response, as measured by PMN numbers in lavaged cells, for both exposure systems.

Role of iron in NF-kappa B activation and cytokine gene expression by rat hepatic macrophages.

A redox-sensitive nuclear factor, NF-kappa B, induces transcription of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in macrophages. The present study has investigated the role of iron in NF-kappa B activation and TNF-alpha and IL-6 expression by rat hepatic macrophages (HM). As an in vivo model, cholestatic liver injury was induced in rats by ligation of the common bile duct (BDL). During the first 2 wk after BDL, there was an increase in the hepatic level of thiobarbituric acid-reactive substances (TBARS) that was accompanied by the appearance of protein-malondialdehyde adducts in the periportal region. This increase was reduced after 3 wk. TNF-alpha and IL-6 mRNA levels in HM from the BDL rats were increased at 1 and 2 wk and attenuated at 3 wk. Gel mobility shift assay of HM nuclear extracts demonstrated the similar temporal pattern of enhanced NF-kappa B binding activity. Treatment of the BDL animals with 1,2-dimethyl-3-hydroxypyrid-4-one (L-1), a lipophilic iron chelator, suppressed the increases in hepatic TBARS by 64%, plasma alanine aminotransferase by 45%, and HM TNF-alpha and IL-6 mRNA by > 84%. Concomitantly, the HM NF-kappa B binding activity was reduced close to the level observed in sham-operated rats. Treatment of cultured HM with L-1 also blocked lipopolysaccharide-stimulated NF-kappa B activation and TNF-alpha and IL-6 expression at mRNA and protein levels. These results demonstrate that the iron chelator effectively blocks NF-kappa B activation and coordinate TNF-alpha and IL-6 gene upregulation by HM in cholestatic liver injury or under in vitro lipopolysaccharide stimulation. These findings support a pivotal role for iron in activation of NF-kappa B and cytokine gene expression by HM in vitro and in vivo.

Modulatory effect of mycobacterium cell wall extract (Regressin) on lymphocyte blastogenic activity and macrophage cytokine gene transcription in swine.

Mycobacterium cell wall extract (MCWE) (Regressin) contains trehalose dimycolate and muramyl dipeptide, both of which have immunomodulatory properties. The goal of this study was to evaluate the effect of MCWE on the in vitro peripheral blood lymphocyte blastogenic activities to mitogens phytohemagglutinin (PHA) and concanavalin A (ConA) in 6- to 8-week-old piglets. The effect of MCWE on alveolar macrophage tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) gene transcription, as determined by a reverse transcription-PCR assay standardized with the endogenous glyceraldehyde-3-phosphate dehydrogenase gene, was also investigated. The results show enhanced blastogenic lymphocyte activities to mitogens PHA and ConA in MCWE-exposed cell cultures compared to those of control cell cultures. The enhanced blastogenic activity effect of MCWE was dose dependent. The cell background activity (spontaneous [3H]thymidine incorporation) of lymphocyte cultures was also significantly increased in the presence of MCWE, thereby demonstrating a lymphocyte mitogenic effect of MCWE. Cytokine gene transcription analysis showed that the TNF-alpha transcript levels in alveolar macrophage cell cultures stimulated with MCWE for 6 or 16 h were enhanced compared with those in control cell cultures. An enhancement of IL-1beta mRNA levels in cell cultures stimulated for 16 h with MCWE, compared with those in control cell cultures, was also observed. The overall results demonstrate that MCWE can stimulate lymphocyte functional activity and cytokine mRNA expression in swine, thereby indicating its potential use as a clinical immunotherapeutic agent.

CD40 ligation counteracts Fas-induced apoptosis of human dendritic cells.

Dendritic cells (DC) are cells of the hematopoletic system specialized in capturing antigens and initiating T cell-mediated immune responses. We show here that human DC generated in vitro by culturing CD34+ cord blood progenitor cells in granulocyte macrophage colony stimulating factor plus tumor necrosis factor-alpha express the Fas antigen (APO-1, CD95) and undergo apoptosis upon triggering of Fas by mAb. However, only a proportion of the cells die in response to Fas ligation, an observation that may be related to the virtual absence of the bcl-2 protein in about half of the cells. Ligation of DC CD40 by culture on CD40L-transfected fibroblastic cells up-regulates the expression of bcl-2 and, concomitantly, renders DC virtually resistant to Fas-induced apoptosis. Parallel experiments with mature, interdigitating dendritic cells (IDC) isolated from tonsils revealed that IDC express Fas but do not enter into apoptosis following Fas ligation, a finding that may be explained by their high levels of bcl-2. Thus, upon encountering antigen-specific T cells, DC become resistant to Fas-induced apoptosis, as a consequence of CD40 ligation and possibly by mechanisms associated to the up-regulation of bcl-2 protein expression.

Control of major histocompatibility complex class II expression on human monocytes by interleukin-4: regulatory effect of lipopolysaccharide.

Interleukin-4 (IL-4), like interferon-gamma (IFN-gamma), stimulates monocyte major histocompatibility complex (MHC) class II expression and thus, by increasing antigen presentation, has the potential to increase immune reactivity. In this study, activation of human monocytes by lipopolysaccharide (LPS) prevented concomitant IL-4 stimulation of MHC class II expression. However, this was not a general observation for activated monocytes because although the high levels of MHC class II antigen expressed by monocytes stimulated in vitro with IFN-gamma were not further regulated by IL-4, the stimulatory effects of IL-4 persisted on cells activated with granulocyte macrophage colony-stimulating factor and tumour necrosis factor-alpha for enhanced MHC class II expression. MHC class II expression by monocytes cultured for 7 days with macrophage colony-stimulating factor was regulated by IL-4 and LPS in a manner very similar to that detected for freshly isolated monocytes. The inhibitory effect of LPS was not due to LPS-induced production of IL-10 or regulatory prostanoids. Furthermore, IFN-gamma-increased MHC class II expression was suppressed by LPS, suggesting that the regulation was at the level of MHC class II expression per se. This study suggests that during Gram-negative bacterial infections, IL-4 and IFN-gamma may not be able to signal enhanced MHC class II expression and thus, enhanced immune reactivity.

Direct stimulatory effect of insulin-like growth factor-I on monocyte and macrophage tumor necrosis factor-alpha production.

GH has been demonstrated to play a physiological role in the priming of macrophages for tumor necrosis factor-alpha (TNF alpha) synthesis. Although evidence has been presented that GH exerts this effect by an indirect mechanism, the mediators of GH stimulation of TNF alpha synthesis have not been identified. Because insulin-like growth factor-I (IGF-I) is a major mediator of many GH effects, in the present study we investigated the direct in vitro effect of this growth factor on macrophage TNF alpha production. Treatment of murine macrophages with physiological concentrations of IGF-I (0.13-130 nM) enhanced both basal and lipopolysaccharide-stimulated macrophage TNF alpha release and messenger RNA levels. Induction of basal TNF alpha production was also observed after treatment of the cells with supraphysiological concentrations of insulin (130-1300 nM). Exposure of human monocytes to IGF-I led to a similar increase of basal TNF alpha production and messenger RNA expression. Preexposure of macrophages with specific antibodies against IGF-I and IGF-I receptor before IGF-I addition resulted in a complete abrogation of the stimulatory effect of IGF-I on TNF alpha production, indicating that specific binding of IGF-I to its receptor is required for macrophage TNF alpha induction by IGF-I. In contrast to the stimulatory effect of IGF-I, neither GH (0.1-10 micrograms/ml) nor IGF-II (0.13-130 nM) enhanced macrophage TNF alpha release in vitro. To assess the role of the tyrosine kinase system in mediating IGF-I-induced basal TNF alpha production, macrophages were preincubated with the specific tyrosine kinase inhibitors, genistein and tyrphostin A9, before IGF-I exposure. Addition of these compounds resulted in a dose-dependent inhibition of the stimulatory effect of IGF-I on macrophage TNF alpha release, indicating that protein tyrosine kinase activation is required for TNF alpha stimulation by IGF-I. Taken together, these results demonstrate that IGF-I is a monocyte/macrophage activating factor that enhances TNF alpha production, and that such effect is mediated via the IGF-I receptor and involves tyrosine kinase activation.

Direct stimulatory effect of insulin-like growth factor-I on monocyte and macrophage tumor necrosis factor-alpha production

GH has been demonstrated to play a physiological role in the priming of macrophages for tumor necrosis factor-alpha (TNF alpha) synthesis. Although evidence has been presented that GH exerts this effect by an indirect mechanism, the mediators of GH stimulation of TNF alpha synthesis have not been identified. Because insulin-like growth factor-I (IGF-I) is a major mediator of many GH effects, in the present study we investigated the direct in vitro effect of this growth factor on macrophage TNF alpha production. Treatment of murine macrophages with physiological concentrations of IGF-I (0.13-130 nM) enhanced both basal and lipopolysaccharide-stimulated macrophage TNF alpha release and messenger RNA levels. Induction of basal TNF alpha production was also observed after treatment of the cells with supraphysiological concentrations of insulin (130-1300 nM). Exposure of human monocytes to IGF-I led to a similar increase of basal TNF alpha production and messenger RNA expression. Preexposure of macrophages with specific antibodies against IGF-I and IGF-I receptor before IGF-I addition resulted in a complete abrogation of the stimulatory effect of IGF-I on TNF alpha production, indicating that specific binding of IGF-I to its receptor is required for macrophage TNF alpha induction by IGF-I. In contrast to the stimulatory effect of IGF-I, neither GH (0.1-10 micrograms/ml) nor IGF-II (0.13-130 nM) enhanced macrophage TNF alpha release in vitro. To assess the role of the tyrosine kinase system in mediating IGF-I-induced basal TNF alpha production, macrophages were preincubated with the specific tyrosine kinase inhibitors, genistein and tyrphostin A9, before IGF-I exposure. Addition of these compounds resulted in a dose-dependent inhibition of the stimulatory effect of IGF-I on macrophage TNF alpha release, indicating that protein tyrosine kinase activation is required for TNF alpha stimulation by IGF-I. Taken together, these results demonstrate that IGF-I is a monocyte/macrophage activating factor that enhances TNF alpha production, and that such effect is mediated via the IGF-I receptor and involves tyrosine kinase activation.

Pentoxifylline Therapy in Human Immunodeficiency Virus--Seropositive Persons with Tuberculosis: A Randomized, Controlled Trial

Macrophage activation and tumor necrosis factor-alpha (TNF-alpha) production are critical in tuberculosis immunity but may result in increased human immunodeficiency virus (HIV) expression and accelerated HIV disease progression in HIV-infected persons. Pentoxifylline inhibits expression of TNF-alpha and HIV. A double-blind, placebo-controlled study of adjunctive therapy with pentoxifylline (1800 mg/day) as a timed-release formulation was done in Ugandan HIV-infected patients with pulmonary tuberculosis. Subjects had early HIV disease (mean CD4 cell count, 380/microL) and did not receive other antiretroviral drugs. Pentoxifylline resulted in decreased plasma HIV RNA and serum beta 2-microglobulin and, in a subset of moderately anemic patients, improved blood hemoglobin levels. Trends were noted toward reduced TNF-alpha production in vitro and improved performance scores, but these did not reach statistical significance. No effect was noted on body mass, CD4 cell count, or survival. Additional studies of more potent TNF-alpha inhibitors in HIV-positive subjects with tuberculosis are warranted.

Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kappaB to DNA and the subsequent expression of tumor necrosis factor-alpha and interleukin-1beta in macrophages.

A large body of evidence suggests that oxidized LDL (oxLDL) has a role in atherogenesis. One effect is the impact on macrophage function. We have studied the effects of oxLDL and oxysterols on the binding of the transcription factors nuclear factor (NF)-kappaB and AP-1 to DNA. These transcription factors are involved in the regulation of several genes and expressed during activation of macrophages, for example by endotoxin (LPS). OxLDL did not induce binding of NF-kappaB. However, the LPS-induced response to NF-kappaB was substantially reduced after preincubation with oxLDL. Medium and highly oxidized LDL also decreased the constitutive DNA-binding of AP-1. Similar effects on AP-1-binding were seen with the oxysterols, 7beta-hydroxycholesterol, 24- hydroxy-, 25-hydroxy-, and 27-hydroxy-cholesterol. Our data therefore suggest an effect of oxLDL on the DNA-binding of AP-1, which might be mediated by the oxysterol content of oxLDL. A decreased LPS-induced TNF-alpha and IL-1beta mRNA and protein expression were found in macrophages incubated with oxLDL before LPS-exposure. These observations suggest that macrophages that internalize extensively oxidized LDL are suppressed in their response to inflammatory stimulation.

Preexposure of mouse peritoneal macrophages to lipopolysaccharide and other stimuli enhances the nitric oxide response to secondary stimuli.

The aim of this study was to compare the regulation of the production of tumor necrosis factor-alpha (TNF-alpha) and secondary nitric oxide (NO) in macrophages submitted to a sequence of two stimulations. Pre-exposure for 18 h of mouse thioglycollate-elicited peritoneal macrophages to low doses (1-10 ng/ml) of lipopolysaccharide (LPS), in the presence or absence of serum, induces on one hand a desensitization (endotoxin tolerance) for secondary TNF-alpha responses to LPS and, on the other hand, a 4 fold increase (priming) of secondary NO responses. Preexposure to components from Gram-positive bacteria (lipoteichoic acid, peptidoglycan) and to a synthetic lipid structurally related to lipid A (compound M4), induced similar effects. In contrast to the desensitization for TNF-alpha secretion, the priming for NO production was not mimicked by sodium nitroprusside, a generator of NO. The results suggest that concomitant but distinct activation pathways induced by LPS and other agents can be dissociated by serum-independent modulation processes elicited by pre-exposure of the cells to LPS itself, or to other stimuli.

In Vitro Efficacy of Morpholino-modified Antisense Oligomers Directed against Tumor Necrosis Factor-α mRNA

Chemical modification of antisense oligonucleotides to increase nuclease resistance may improve their efficacy within enzyme-rich cellular targets (e.g. macrophages). We evaluated a panel of morpholino antisense oligomers (M-AS) for their ability to inhibit macrophage tumor necrosis factor-alpha (TNF-alpha) release and compared them to phosphodiester (O-AS) and phosphorothioate (S-AS) types of oligonucleotides. M-AS inhibited translation in vitro (rabbit reticulocyte lysate) of target mRNA at concentrations as low as 200 nM (e.g. percent inhibition by M-AS 2 at 0.2, 1.0, and 2.0 microM was 40.9 +/- 5.3%, 50.2 +/- 4.6%, and 57.7 +/- 3.6%, respectively, n = 4, p </= 0.002 versus control). Similarly, M-AS 2 effectively, albeit partially, inhibited TNF-alpha production by LPS-stimulated macrophages (RAW 264.7 cells). Incubation of cells with 25 microM M-AS 2 resulted in 32.6 +/- 2.6% (n = 3, p = 0.002 versus control) decrease in TNF-alpha release. In contrast, S-AS inhibited translation of the target mRNA in the rabbit reticulocyte lysate assay, but not in the cell-based assay. In fact, S-AS nonspecifically augmented TNF-alpha release. O-AS were without effect in either system. Uptake studies with fluorescent M-AS revealed that inhibitory effects were seen despite relatively low cellular uptake (intracellular concentration 30.5 +/- 6.7 nM; efficiency of uptake 0.1%). In contrast, flow cytometric and confocal analysis revealed that S-AS were avidly taken up by RAW 264. 7 cells, confirming that their lack of efficacy was not due to lack of uptake. With improved methods of delivery, M-AS may represent an important therapeutic modality.

Tumor necrosis factor-alpha in macrophages of heart, liver, kidney, and in the pituitary gland.

Tumor necrosis factor-alpha (TNFalpha) is an important mediator in bacterial lipopolysaccharide (LPS)-induced fever and shock. New data on TNFalpha-producing macrophages in heart, pituitary gland, kidneys and liver in correlation with TNFalpha plasma levels are reported here. In adult rabbits, core temperature and TNFalpha plasma levels are significantly increased at 3 and 24 h after treatment with LPS. After a delay of 6-12 h, the number of TNFalpha-containing macrophages, determined by immunohistochemistry, increases more than fivefold in all organs investigated. With the exception of the pituitary gland, the increase in cell number is correlated with the degree of cellular injury, indicating the involvement of TNFalpha in LPS-induced organ damage that is accompanied by the synthesis of the cytokine. Cortisol levels also increase for at least 24 h after LPS treatment, show peak values 6 h after interleukin-1 treatment, and are unchanged after TNFalpha treatment, indicating the different effects of these factors on the hypothalamo-hypophyseal-adrenocortical axis. This study provides evidence that macrophageal TNFalpha of multi-organ origin is involved in LPS-induced tissue injury and supports the concept of a systemic inflammatory response syndrome. We also show for the first time that in the anterior lobe of the pituitary gland TNFalpha is a normal constituent in cells producing growth hormone but not ACTH. Moreover, most cells of the intermediate lobe are positive for TNFalpha.

Interleukin-12 and tumor necrosis factor alpha mediate innate production of gamma interferon by group B Streptococcus-treated splenocytes of severe combined immunodeficiency mice

The existence of interleukin-12-mediated innate immune responses to group B streptococci (GBS) was tested by examining T-lymphocyte-independent gamma interferon (IFN) production in cultured splenocytes from severe combined immunodeficiency mice. Splenocytes were cultured with killed or living GBS for 48 h, and then IFN was measured by enzyme-linked immunosorbent assay. Type III GBS as well as other extracellular bacterial agents of neonatal sepsis (staphylococci and enterococci) induced IFN production, which was enhanced by interleukin-2 and was inhibited by neutralizing antibodies to tumor necrosis factor alpha and to mouse interleukin-12. Interleukin-12 bioactivity was present in conditioned medium from GBS-treated adherent macrophages. Adherent peritoneal macrophages and bone marrow-derived natural killer cells from severe combined immunodeficiency mice cultured separately with GBS did not produce IFN, whereas cocultures did produce IFN. Functional macrophage activation was evident by nitric oxide production in GBS-treated splenocyte cultures. The results show that extracellular pathogens such as GBS, similarly to intracellular microbes, induce macrophage interleukin-12 and tumor necrosis factor alpha, which promote natural killer cell secretion of IFN, which then enhances innate phagocyte resistance mechanisms.

Alcohol Modulates Alveolar Macrophage Tumor Necrosis Factor‐α, Superoxide Anion, and Nitric Oxide Secretion in the Rat

We investigated the effect of alcohol (ethanol) on the ability of the alveolar macrophage to produce tumor necrosis factor-alpha (TNF-alpha), superoxide anion (O2-), and nitric oxide (NO)--three critical components of pulmonary host defense. Male rats were treated with alcohol either acutely (priming dose 175 mg/100 g of body weight, followed by a 7-hr continuous intravenous infusion of 30 mg/100 g of body weight/hr) or chronically (12-14 weeks of feeding ethanol in a liquid diet). Three hours before sacrifice, the rats received an intravenous injection of saline or lipopolysaccharide (LPS; Escherichia coli, 026:B6, 100 micrograms/100 g of body weight). Alveolar macrophages (AMs) were then isolated by bronchoalveolar lavage and assessed for their in vitro capacity to produce TNF-alpha, O2-, and NO spontaneously and in response to different stimuli. Acute alcohol administration suppressed in vitro LPS-stimulated AM TNF-alpha secretion by 52%. AMs from both pair-and alcohol-fed rats secreted TNF-alpha spontaneously in culture. However, the AMs from chronic alcohol-fed group secreted 42-53% less TNF-alpha spontaneously and in response to LPS, interferon-gamma (IFN-gamma) or IFN-gamma + LPS compared with the AMs from pair-fed group. Systemic LPS treatment inhibited in vitro LPS-stimulated AM TNF-alpha secretion (50%) only in the control rats. Acute alcohol administration enhanced significantly in vitro phorbol 12-myristate 13-acetate (PMA)- and opsonized zymosan (OPZ)-induced AM O2- secretion (4-and 1.8-fold, respectively). Systemic LPS treatment primed the AMs from control rats to secrete 83% more O2- in response to PMA but not OPZ; however, in the acute alcohol-treated group, it suppressed both PMA (54%)- and OPZ (66%)-induced AM O2- release (loss of priming effect of LPS). Chronic alcohol feeding suppressed PMA-induced AM O2- secretion (40%) without affecting the OPZ-induced release. Although systemic LPS treatment had no significant effect on PMA or OPZ-induced AM O2- secretion in the pair-fed group, it enhanced the PMA-stimulated AM O2- release (88%) in the alcohol-fed group. AMs recovered from control or acute alcohol-treated rats did not secrete NO spontaneously in vitro. However, AMs from both pair and chronic alcohol-fed rats secreted NO spontaneously with AMs from chronic alcohol-fed group secreting 34% less. Both acute and chronic alcohol treatment inhibited AM NO secretion in response to IFN-gamma, LPS, and IFN-gamma + LPS significantly. Systemic LPS had no effect on AM NO production in response to different in vitro stimuli in any of the treatment groups. These data suggest that (1) both acute and chronic alcohol administration to rats inhibit AM TNF-alpha and NO secretion; (2) acute and chronic alcohol treatment have differential effects on AM O2- secretion; and (3) alcohol-induced alteration in AM TNF-alpha, O2-, and NO secretion may in part explain the increased susceptibility of alcohol-consuming individuals to pulmonary infections.