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Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.

Tumor necrosis factor-alpha (TNF-alpha) is well recognized for its role in mediating innate immune responses. However, the mechanisms of TNF-alpha that influence the adaptive immune response to virus infections are not well understood. In this study, we have investigated the role of TNF-alpha in activating the cellular and humoral responses to systemic viral challenge with recombinant replication-defective adenovirus (rAd). Evaluation of T cell function in TNF-alpha-deficient (TNFKO) mice revealed impaired virus-specific proliferation of T cells derived from the draining lymph nodes of the liver. Analysis of dendritic cells (DC) isolated from local draining lymph nodes after systemic challenge showed that DC from TNFKO mice were relatively immature compared with those from strain-matched wild-type mice. In vitro, TNF-alpha was required to mature DC efficiently during virus-mediated stimulation. Adoptive transfer of primed, mature DC into TNFKO mice restored T cell responses and reconstituted anti-adenovirus antibody responses. Thus, TNF-alpha plays a significant role in the maturation of DC after adenovirus challenge both in vitro and in vivo, highlighting the importance of this innate cytokine in activating adaptive immunity to viral challenge.

The contribution of granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic and immunoregulatory cytokine, to resistance to blood-stage malaria was investigated by infecting GM-CSF-deficient (knockout [KO]) mice with Plasmodium chabaudi AS. KO mice were more susceptible to infection than wild-type (WT) mice, as evidenced by higher peak parasitemia, recurrent recrudescent parasitemia, and high mortality. P. chabaudi AS-infected KO mice had impaired splenomegaly and lower leukocytosis but equivalent levels of anemia compared to infected WT mice. Both bone marrow and splenic erythropoiesis were normal in infected KO mice. However, granulocyte-macrophage colony formation was significantly decreased in these tissues of uninfected and infected KO mice, and the numbers of macrophages in the spleen and peritoneal cavity were significantly lower than in infected WT mice. Serum levels of gamma interferon (IFN-gamma) were found to be significantly higher in uninfected KO mice, and the level of this cytokine was not increased during infection. In contrast, IFN-gamma levels were significantly above normal levels in infected WT mice. During infection, tumor necrosis factor alpha (TNF-alpha) levels were significantly increased in KO mice and were significantly higher than TNF-alpha levels in infected WT mice. Our results indicate that GM-CSF contributes to resistance to P. chabaudi AS infection and that it is involved in the development of splenomegaly, leukocytosis, and granulocyte-macrophage hematopoiesis. GM-CSF may also regulate IFN-gamma and TNF-alpha production and activity in response to infection. The abnormal responses seen in infected KO mice may be due to the lack of GM-CSF during development, to the lack of GM-CSF in the infected mature mice, or to both.

Background: Tumor necrosis factor-alpha (TNF) is thought to act as a stimulator for initiating hepatocyte proliferation after partial hepatectomy (PH). At the same time, TNF induces a series of inflammatory responses that may be detrimental for the liver and other remote organs. The purpose of this study was to investigate the effect of TNF on the pathophysiologic state after PH., Methods: Wild-type (TNF+/+) and TNF-deficient (TNF-/-) mice underwent 70% PH. Hepatocyte proliferation was assessed by bromodeoxyuridine labeling and mitotic index. Liver function was evaluated by alanine aminotransferase (ALT) and total bilirubin levels in serum after PH. Myeloperoxidase activity in the liver and lung was measured as a marker for neutrophil activation., Results: No differences were observed in liver regeneration or hepatocyte proliferation between TNF+/+ and TNF-/- mice. The survival of TNF-/- mice on day 1 after PH was significantly higher than that of TNF+/+ mice, but both groups had similar survival thereafter. The ALT level was significantly higher in TNF+/+ mice 6 hours after PH and myeloperoxidase activities in both liver and lung were markedly elevated in TNF+/+ mice compared with TNF-/- mice., Conclusions: These findings demonstrate that TNF gene-depleted mice do not demonstrate delayed liver regeneration but do suppress neutrophil activation after PH compared with results in wild-type (TNF +/+) mice.

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF-/mdx). Body mass and muscle mass of the TNF-/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.

Tumor necrosis factor (TNF)-deficient mice were challenged with Leishmania donovani to characterize TNF in the response of visceral intracellular infection to antileishmanial chemotherapy. In wild-type controls (i) liver infection peaked at week 2 and resolved, (ii) discrete liver granulomas developed at weeks 2 to 4 and involuted, and (iii) leishmanicidal responses to antimony (Sb), amphotericin B (AmB), and miltefosine were intact. In TNF knockout (KO) mice (i) initial liver infection was unrestrained, plateaued, and then declined somewhat by week 6, (ii) an absent early granulomatous reaction abruptly accelerated with striking tissue inflammation, widespread hepatic necrosis, and 100% mortality by week 10, and (iii) while the initial response to AmB and miltefosine was intact, killing induced by Sb therapy was reduced by >50%. Although initial AmB treatment during weeks 2 to 3 killed 98% of liver parasites, 75% of AmB-treated KO mice subsequently relapsed and died by week 12; however, additional maintenance AmB preserved long-term survival. These results for a model of visceral infection indicate that endogenous TNF is required early on to control intracellular L. donovani, support granuloma development, and mediate optimal initial effects of Sb and prevent relapse after ordinarily curative AmB treatment. A compensatory, TNF-independent antileishmanial mechanism developed in TNF KO mice; however, its effect was uncontrolled fatal inflammation. Chemotherapeutic elimination of the parasite stimulus reversed the hyperinflammatory response and preserved survival.

T cell production by the thymus, thymic size, cellularity and output all decrease drastically after puberty. Among the candidates that may mediate this decrease are the sex steroids: hypersecretion or pharmacological administration of these hormones has long been known to induce thymic hypocellularity, and their depletion yields thymic hypercellularity. Here we show that a typical sex steroid, testosterone, specifically targets CD8+CD4+ double-positive (DP) thymocytes for apoptosis via TNF-alpha. Anti-TNF-alpha monoclonal antibodies abrogated testosterone-induced DP apoptosis, and TNF-alpha-/- DP thymocytes were largely resistant to testosterone-mediated apoptosis in vivo. Testosterone accomplished this effect by upregulating TNF-alpha production and by simultaneously sensitizing DP thymocytes to TNF-alpha. Thus, TNF-alpha is the critical mediator of sex steroid-induced apoptosis in thymocytes, and its manipulation should provide a point of intervention to modulate T cell production in sex hormone disorders.

Mice with combined lymphotoxin-alpha (LTalpha) and tumor necrosis factor (TNF) deficiencies show defects in the structure of peripheral lymphoid organs such as spleen, lymph nodes, and gut-associated lymphoid tissues. To identify genes associated with this defective phenotype in spleen, we applied a gene profiling approach, including subtractive cloning and gene array hybridizations, to mice with combined TNF/LT deficiency. The differentially expressed genes identified by these techniques was then evaluated by Northern blot analysis for splenic expression in knockout mice with single LTalpha or single TNF deficiency. Most of the genes detected in this analysis are directly or indirectly associated with disrupted LT and not TNF signaling.

Tumour necrosis factor-alpha (TNF) plays a central role in the recruitment and activation of mononuclear cells in mycobacterial infection. In the absence of type 1 TNF receptor, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) infection of mice is not contained, leading to fatal disease. Because type 1 TNF receptor binds both TNF and lymphotoxin-a, we used TNF-deficient mice to determine the specific role of TNF in the host resistance to BCG infection. The bacterial burden of the lungs of TNF-deficient mice was substantially increased and the mice succumbed to pneumonia between 8 and 12 weeks with a defective granuloma response. Atypical granulomas developed by 4 weeks expressing low levels of MHC class II, intracellular adhesion molecule (ICAM-1), CD11b and CD11c. Macrophages showed little signs of activation and had low levels of acid phosphatase activity and inducible nitric oxide synthase (INOS) expression. Despite the defective cellular recruitment, the chemokines, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1alpha), were increased in broncho-alveolar lavage fluid of TNF-deficient mice. The defective host response was corrected by the transplantation of normal bone marrow cells into irradiated TNF-deficient mice. These results demonstrate that TNF derived from hemopoietic cells rather than from mesenchymal origin are essential for a normal host response to BCG infection. Furthermore, TNF dependent expression of adhesion molecules may be essential for the recruitment of mononuclear cells for the formation of bactericidal BCG granulomas.

Apoptosis is one of the key regulatory mechanisms in tissue modeling and development. In the thymus, 95-98% of all thymocytes die by apoptosis because they failed to express a TCR with an optimal affinity for the selecting intrathymic peptide-MHC complexes. We studied the possible role of two prominent nerve growth factor (NGF-TNF) family member systems, Fas ligand (FasL)-Fas receptor (FasR) and TNF-alpha-TNFR, in apoptosis of murine CD8+4+ double-positive (DP) thymocytes induced via TCR-CD3- and cAMP-mediated signaling. TCR-CD3epsilon-mediated apoptosis of DP thymocytes was found not to be dependent on either of the two systems. The FasL-FasR system was also found to be dispensable for the cAMP-mediated apoptosis. By contrast, cAMP agonists (dibutyryl-cAMP and forskolin) induced apoptosis via TNF-alpha, as evidenced by 1) the ability of anti-TNF-alpha mAbs to abrogate cAMP analogue-induced DP apoptosis in a dose-dependent manner; and 2) increased resistance of DP thymocytes from TNF-alpha-/- and TNFR I-/-II-/- animals to cAMP agonist-mediated apoptosis. cAMP agonists induced DP thymocyte death by a combination of two mechanisms: first, they induced selective up-regulation of TNF-alpha production, and, second, they sensitized DP thymocytes to TNF-alpha. The latter effect may be due to the down-regulation of TNFR-associated factor 2 protein. These results identify TNF-alpha as the critical mediator of cAMP-induced apoptosis in thymocytes and provide a molecular explanation for how the cAMP stimulators, including the sex steroids, may modulate T cell production output, as observed under physiological and pharmacological conditions.

To examine the hypothesis that tumor necrosis factor (TNF) alpha is an essential cytokine in carcinogenesis, we conducted two-stage carcinogenesis experiments with an initiator, 7,12-dimethylbenz(a)anthracene (DMBA), plus either of two tumor promoters, okadaic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the skin of TNF-alpha-deficient (TNF-/-) mice. TNF-/- mice treated with DMBA plus okadaic acid developed no tumors for up to 19 weeks, and at 20 weeks, the percentage of tumor-bearing TNF-/- mice was 10%, whereas the percentage of tumor-bearing TNF+/+ mice was 100%. In TNF-/- mice treated with DMBA plus TPA, tumor onset was delayed 4 weeks, and the time to development of small tumors in 100% of mice was 9 weeks later than that seen in TNF+/+ CD-1 mice. The average number of tumors in TPA-treated TNF-/- mice was 2.8, compared with 11.8 for TNF+/+ CD-1 mice. To understand the residual tumor-promoting activity in TNF-/- mice, we also investigated the possible significance of interleukin (IL) 1 as an additional cytokine in tumor promotion. A single application of TPA and okadaic acid increased IL-1alpha and IL-1beta gene expression in TNF-/- mice. All of our results demonstrate that TNF-alpha is the key cytokine for tumor promotion in mouse skin and, very possibly, for carcinogenesis in humans as well.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling.

The present study evaluated behavioral and histopathological outcome after controlled cortical impact (CCI) brain injury in mice deficient in tumor necrosis factor [TNF(-/-)] and their wild-type (wt) littermates. Mice were subjected to CCI brain injury [TNF(-/-), n = 10; wt, n = 10] or served as uninjured controls [TNF(-/-), n = 10; wt, n = 10] and were evaluated for deficits in memory retention at 7 days postinjury. Although both brain-injured wt and TNF(-/-) mice exhibited significant memory dysfunction compared to uninjured controls (P < 0.02), the deficits in memory retention in injured TNF(-/-) mice were significantly less severe than in injured wt mice (P < 0.02). A second group of mice was subjected to CCI brain injury [TNF(-/-), n = 20; wt, n = 20] or served as uninjured controls [TNF(-/-), n = 15; wt, n = 15] and were evaluated over a 4-week period for neurological motor function. In the acute posttraumatic period (48 h postinjury), brain-injured TNF(-/-) mice were significantly less impaired than injured wt mice on composite neuroscore (P < 0.001), rotarod (P < 0.05), and beam balance (P < 0. 02) tests. However, wt mice recovered from brain injury by 2-3 weeks postinjury, whereas TNF(-/-) mice continued to demonstrate persistent motor deficits up to 4 weeks postinjury. Histopathological analysis at 2 and 4 weeks postinjury revealed that brain-injured TNF(-/-) mice had significantly more cortical tissue loss than wt mice (P < 0.02). Our results suggest that although the presence of TNF in the acute posttraumatic period may be deleterious, this cytokine may play a role in facilitating long-term behavioral recovery and histological repair after brain injury.

The cellular and humoral immune responses to adenovirus (Ad) remain a major barrier to Ad-mediated gene therapy. We recently reported that mice deficient in tumor necrosis factor alpha (TNF-alpha) or Fas (APO-1, CD95) have prolonged expression of an Ad transgene expressing a foreign protein in the liver. To determine whether blockade of TNF-alpha or Fas would have the same effect in normal mice, we created transgenes that expressed soluble murine CD8 or CD8 fused to the extracellular regions of TNF receptor 1 (TNFR) or Fas and inserted into the left-end region of first-generation (E1/E3-) Ad vectors. Consistent with the results observed in TNF-deficient mice, expression of the TNFR-CD8 fusion protein was prolonged in vivo compared to that of control proteins. Not only did expression of TNFR-CD8 persist in the liver and the lung, but when coadministered with another first-generation vector, the protein provided "transprotection" for the companion vector and transgene. In addition, TNFR-CD8 attenuated the humoral immune response to the Ad. Together, these findings demonstrate that blockade of TNF-alpha is likely to be useful in extending the expression of an Ad-encoded transgene in a gene therapy application.

Mice lacking the RelA (p65) subunit of NF-kappaB die between days 14 and 15 of embryogenesis because of massive liver destruction. Fibroblasts and macrophages isolated from relA-/- embryos were found to be highly sensitive to tumor necrosis factor (TNF) cytotoxicity, raising the possibility that endogenous TNF is the cause of liver cell apoptosis. To test this idea, we generated mice lacking both TNF and RelA. Embryogenesis proceeds normally in such mice, and TNF/RelA double-deficient mice are viable and have normal livers. Thus, the RelA-mediated antiapoptotic signal that protects normal cells from TNF injury in vitro can be shown to be operative in vivo.

Previous studies demonstrated that Plasmodium yoelii 17XL, a lethal strain of rodent malaria, causes a syndrome in SW mice that resembles human cerebral malaria. The mouse brain pathology is characterized by cytoadherence of parasitized erythrocytes. Here, the possible mechanisms mediating cerebral malaria in this model were studied and the results were compared with a nonlethal strain of this parasite, P. yoelii 17XNL (nonlethal), which does not cause cerebral malaria. Immunostaining for intercellular adhesion molecule-1 (ICAM-1) revealed an increase in expression of this protein in the small venules and capillaries of the brains of infected mice that increased with time after infection. Staining was more pronounced during the lethal infection than the nonlethal infection. Some staining with monoclonal antibody to vascular cell adhesion molecule-1 was also observed, but it was quantitatively less than ICAM-1 staining and was limited to larger venules. During the lethal infection, levels of tumor necrosis factor-alpha (TNF-alpha) increased rapidly, peaking on day 4. In contrast, mice infected with nonlethal P. yoelii had a slower serum TNF-alpha response that peaked on day 10, prior to the maximum parasitemia. In addition, mice with a targeted disruption of the TNF-alpha gene (TNF-alpha-/- mice) were infected with the lethal and nonlethal strains of P. yoelii 17X. The TNF-alpha-/- mice infected with the nonlethal parasite had significantly higher levels of parasitemia than controls, whereas TNF-alpha-/- mice infected with the lethal strain had slightly higher levels of infected erythrocytes but were equally susceptible to death from this infection. Thus, TNF-alpha does not appear to be essential in mediating death. These results demonstrate that P. yoelii 17XL infection has features in common with human cerebral malaria and suggest that this model may be useful in testing strategies to alleviate this syndrome.

Granulocyte-macrophage colony-stimulating factor-deficient (GM-CSF-/-) mice produce far lower serum levels of IFN-gamma in response to LPS than GM-CSF+/+ mice. CD4+ and CD8+ T cells from LPS-injected GM-CSF-/- mice showed a deficiency in IFN-gamma production and proliferative activity in response to IL-2 and IL-12, whereas IFN-gamma production by NK cells was not compromised. These defects of T cells were reversed by administration of GM-CSF in vivo, but not by supplementation with GM-CSF in vitro. GM-CSF-/- mice do not have an intrinsic defect in IFN-gamma production, because IL-12 injection induces the same high levels of IFN-gamma in GM-CSF-/- and GM-CSF+/+ mice. To investigate the inhibitory effect of LPS on GM-CSF-/- T cells and the indirect restorative activity of GM-CSF, we tested the action of supernatants from cultured dendritic cells (DC). A factor or factors in the DC supernatant normalized serum IFN-gamma levels and T cell responses in LPS-injected GM-CSF-/- mice. IL-18 reproduced some but not all of these in vivo and in vitro effects of DC supernatants. Our results indicate that GM-CSF is important in protecting T cells from inhibitory signals generated during immunization or exposure to LPS, and that this effect of GM-CSF is indirect and mediated by factors produced by DC.

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) gamma production. IFN-gamma, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-gamma inhibits production of chemokines (macrophage inflammatory proteins MIP-1alpha and MIP-1beta). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-gamma priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-gamma-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF+/+ and TNF-/- mice injected with Corynebacterium parvum were compared. TNF-/- mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF-/- mice compared with TNF+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF-/- mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-gamma inhibition of chemokine production and inhibition of IFN-gamma-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by localized areas of demyelination. Although the etiology and pathogenesis of MS remain largely unknown, it is generally assumed that immune responses to myelin antigens contribute to the disease process. The exact sequence of events, as well as the molecular mediators that lead to myelin destruction, is yet to be defined. As a potent mediator of inflammation, the cytopathic cytokine, tumor necrosis factor (TNF) has been considered to be a strong candidate in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). However, its role in immune-mediated demyelination remains to be elucidated. To determine the contribution of TNF to the pathogenesis of the MS-like disease provoked by the myelin oligodendrocyte glycoprotein (MOG), we have tested mice with an homologous disruption of the gene encoding TNF. Here we report that upon immunization with MOG, mice lacking TNF develop severe neurological impairment with high mortality and extensive inflammation and demyelination. We show further that inactivation of the TNF gene converts MOG-resistant mice to a state of high susceptibility. Furthermore, treatment with TNF dramatically reduces disease severity in both TNF-/- mice and in other TNF+/+ mice highly susceptible to the MOG-induced disease. These findings indicate that TNF is not essential for the induction and expression of inflammatory and demyelinating lesions, and that it may limit the extent and duration of severe CNS pathology.

Cytokines, in particular tumor necrosis factor-alpha (TNF-alpha), have significant effects on energy metabolism and appetite although their mechanisms of action are largely unknown. Here, we examined whether TNF-alpha modulates the production of leptin, the recently identified fat-specific energy balance hormone, in cultured adipocytes and in mice. TNF-alpha treatment of 3T3-L1 adipocytes resulted in rapid stimulation of leptin accumulation in the media, with a maximum effect at 6 h. This stimulation was insensitive to cycloheximide, a protein synthesis inhibitor, but was completely inhibited by the secretion inhibitor brefeldin A, indicating a posttranslational effect. Treatment of mice with TNF-alpha also caused a similar increase in plasma leptin levels. Finally, in obese TNF-alpha-deficient mice, circulating leptin levels were significantly lower, whereas adipose tissue leptin was higher compared with obese wild-type animals. These data provide evidence that TNF-alpha can act directly on adipocytes to regulate the release of a preformed pool of leptin. Furthermore, they suggest that the elevated adipose tissue expression of TNF-alpha that occurs in obesity may contribute to obesity-related hyperleptinemia.

Immunological functions were analyzed in mice lacking granulocyte/macrophage colony-stimulating factor (GM-CSF). The response of splenic T cells to allo-antigens, anti-mouse CD3 mAb, interleukin 2 (IL-2), or concanavalin A was comparable in GM-CSF +/+ and GM-CSF -/- mice. To investigate the responses of CD8(+) and CD4+ T cells against exogenous antigens, mice were immunized with ovalbumin peptide or with keyhole limpet hemocyanin (KLH). Cytotoxic CD8+ T cells with specificity for ovalbumin peptide could not be induced in GM-CSF -/- mice. After immunization with KLH, there was a delay in IgG generation, particularly IgG2a, in GM-CSF -/- mice. Purified CD4+ T cells from GM-CSF -/- mice immunized with KLH showed impaired proliferative responses and produced low amounts of interferon-gamma (IFN-gamma) and IL-4 when KLH-pulsed B cells or spleen cells were used as antigen presenting cells (APC). When enriched dendritic cells (DC) were used as APC, CD4+ T cells from GM-CSF -/- mice proliferated as well as those from GM-CSF +/+ mice and produced high amounts of IFN-gamma and IL-4. To analyze the rescue effect of DC on CD4(+) T cells, supernatants from (i) CD4(+) T cells cultured with KLH-pulsed DC or (ii) DC cultured with recombinant GM-CSF were transferred to cultures of CD4(+) T cells and KLH-pulsed spleen cells from GM-CSF -/- mice. Supernatants from both DC sources contained a factor or factors that restored proliferative responses and IFN-gamma production of CD4(+) T cells from GM-CSF -/- mice.

Obesity is highly associated with insulin resistance and is the biggest risk factor for non-insulin-dependent diabetes mellitus. The molecular basis of this common syndrome, however, is poorly understood. It has been suggested that tumour necrosis factor (TNF)-alpha is a candidate mediator of insulin resistance in obesity, as it is overexpressed in the adipose tissues of rodents and humans and it blocks the action of insulin in cultured cells and whole animals. To investigate the role of TNF-alpha in obesity and insulin resistance, we have generated obese mice with a targeted null mutation in the gene encoding TNF-alpha and those encoding the two receptors for TNF-alpha. The absence of TNF-alpha resulted in significantly improved insulin sensitivity in both diet-induced obesity and that resulting for the ob/ob model of obesity. The TNFalpha-deficient obese mice had lower levels of circulating free fatty acids, and were protected from the obesity-related reduction in the insulin receptor signalling in muscle and fat tissues. These results indicate that TNF-alpha is an important mediator of insulin resistance in obesity through its effects on several important sites of insulin action.

Adenovirus (Ad) gene transfer vectors are rapidly cleared from infected hepatocytes in mice. To determine which effector mechanisms are responsible for elimination of the Ad vectors, we infected mice that were genetically compromised in immune effector pathways [perforin, Fas, or tumor necrosis factor alpha (TNF-alpha)] with the Ad vector, Ad5-chloramphenicol acetyl transferase (CAT). Mice were sacrificed at 7-60 days postinfection, and the levels of CAT expression in the liver determined by a quantitative enzymatic assay. When the livers of infected mice were harvested 28 days postinfection, the levels of CAT expression revealed that the effectors most important for the elimination of the Ad vector were TNF-alpha > Fas > perforin. TNF-alpha did not have a curative effect on infected hepatocytes, as the administration of TNF-alpha to infected severe combined immunodeficient mice or to infected cultures in vitro had no specific effect on virus persistence. However, TNF-alpha-deficient mice demonstrated a striking reduction in the leukocytic infiltration early on in the infection, suggesting that TNF-alpha deficiency resulted in impaired recruitment of inflammatory cells to the site of inflammation. In addition, the TNF-deficient mice had a significantly reduced humoral immune response to virus infection. These results demonstrate a dominant role of TNF-alpha in elimination of Ad gene transfer vectors. This result is particularly important because viral proteins that disable TNF-alpha function have been removed from most Ad vectors, rendering them highly susceptible to TNF-alpha-mediated elimination.

The contribution of granulocyte-macrophage CSF (GM-CSF) to endotoxin-mediated septic shock has been assessed by treating GM-CSF-deficient mice with LPS. Hypothermia and loss in body weight were markedly attenuated in LPS-treated GM-CSF-deficient mice compared with similarly treated control mice; moreover, the levels of circulating IFN-gamma, IL-1alpha, and IL-6 were lower in LPS-treated GM-CSF-deficient mice than LPS-treated control mice. Intriguingly, the peak levels of TNF-alpha in response to LPS treatment were the same in the serum of GM-CSF-deficient mice and control mice, although in GM-CSF-deficient mice, TNF-alpha persisted longer. Activation of macrophages by LPS, resulting in expression of cytokines including TNF-alpha and IL-1, is thought to underlie endotoxin-mediated effects. Accordingly, the response of peritoneal macrophages from GM-CSF-deficient mice to LPS was studied in vitro. LPS-stimulated peritoneal macrophages from GM-CSF-deficient mice produced significantly less IL-1alpha and nitric oxide than macrophages from wild-type mice, although there was no difference in TNF-alpha production. Collectively, these observations indicate that GM-CSF contributes to cytokine production in LPS-mediated septic shock, and that the attenuated production of these secondary cytokines (IFN-gamma, IL-1alpha, and IL-6) may contribute to the endotoxin-resistant phenotype of GM-CSF-deficient mice.

Although tumor necrosis factor (TNF) initially came to prominence because of its anti-tumor activity, most attention is now focused on its proinflammatory actions. TNF appears to play a critical role in both early and late events involved in inflammation, from localizing the noxious agent and amplifying the cellular and mediator responses at the local site and systemically, to editing (e.g., apoptosis) injured cells or effete immune cells and repairing inflammatory damage. We have generated mice deficient in TNF (TNF-/- mice) and have begun to examine the multiple functions attributed to TNF. TNF-/- mice develop normally and have no gross structural or morphological abnormalities. As predicted, they are highly susceptible to challenge with an infectious agent (Candida albicans), are resistant to the lethality of minute doses of lipopolysaccharide (LPS) following D-galactosamine treatment, have a deficiency in granuloma development, and do not form germinal centers after immunization. Phagocytic activity of macrophages appears relatively normal, as do T cell functions, as measured by proliferation, cytokine release, and cytotoxicity. B cell response to thymus-independent antigens is normal, but the Ig response to thymus-dependent antigen is reduced. Surprisingly, cytokine production induced by LPS appears essentially intact, with the exception of reduced colony-stimulating factor activity. Other unexpected findings coming from our initial analysis are as follows. (i) TNF has low toxicity in TNF-/- mice. (ii) TNF-/- mice show an anomalous late response to heat-killed Corynebacterium parvum. In contrast to the prompt response (granuloma formation, hepatosplenomegaly) and subsequent resolution phase in C. parvum-injected TNF+/+ mice, similarly treated TNF-/- mice show little or no initial response, but then develop a vigorous, disorganized inflammatory response leading to death. These results suggest that TNF has an essential homeostatic role in limiting the extent and duration of an inflammatory process-i.e., an anti-inflammatory function. (iii) In contrast to the expectation that TNF+/+ mice and TNF+/- mice would have identical phenotypes, TNF+/- mice showed increased susceptibility to high-dose LPS lethality, increased susceptibility to Candida challenge, and delayed resolution of the C. parvum-induced inflammatory process, indicating a strong gene dose requirement for different actions of TNF.

Tumor necrosis factor (TNF) promotes diverse responses in endothelial cells that are important to the host response to infections and malignancies; however, less is known of the postreceptor events important to TNF action in endothelial cells than in many other cell types. Since phosphorylation cascades are implicated in cytokine signaling, the effects of the protein kinase inhibitor dimethylaminopurine (DMAP) on TNF action in bovine aortic endothelial cells (BAEC) were investigated. In BAEC, TNF promotes phosphorylation of eukaryotic initiation factor 4E (eIF-4E), c-Jun N-terminal kinase (JNK) and ceramide-activated protein kinase activities, Jun-b expression, prostacyclin production, and, when protein synthesis is inhibited, cytotoxicity. DMAP abrogated or significantly attenuated each of these responses to TNF, without affecting the specific binding of TNF to its receptors. Histamine, another agent active in the endothelium, promotes phosphorylation of elongation factor-2 (EF-2) and prostacyclin production, but not phosphorylation of eIF-4E in BAEC. Histamine-stimulated EF-2 phosphorylation was not inhibited and prostacyclin production was unaffected by DMAP. These observations demonstrate that a distinct signal transduction cascade, which can be selectively inhibited by DMAP, promotes the response of BAEC to TNF. Thus, we have identified a reagent, DMAP, that may be useful for characterizing the TNF signal transduction pathway.

The initiation of mRNA translation is regulated by the reversible phosphorylation of several initiation factors. We report here that tumor necrosis factor-alpha (TNF) rapidly stimulates phosphorylation of one such factor, an mRNA cap binding protein, in several cell types which are important in vitro models of TNF action. This protein has been purified, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor 4E. These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the various actions of TNF in diverse cell types.

A population of latent (cryptic) receptors for tumor necrosis factor-alpha (TNF) has been characterized in the rat liver plasma membrane (PM). 125I-TNF bound to high (Kd = 1.51 +/- 0.35 nM) and low (Kd = 13.58 +/- 1.45 nM) affinity receptors in PM. Solubilization of PM with 1% Triton X-100 prior to incubation with 125I-TNF increased both high affinity (from 0.33 +/- 0.04 to 1.67 +/- 0.05 pmol/mg of protein) and low affinity (from 1.92 +/- 0.16 to 7.57 +/- 0.50 pmol/mg of protein) TNF binding without affecting the affinities for TNF. Digestion of intact PM with chymotrypsin abolished most of the TNF binding capacity of PM. However, substantial binding activity was recovered by solubilization of chymotrypsin-treated PM with 1% Triton X-100, suggesting the presence of a large latent pool of TNF receptors. The affinities of the high and low affinity sites recovered from chymotrypsin-treated membranes were similar to those of intact PM. Affinity labeling of receptors whether from PM, solubilized PM, or membranes digested with chymotrypsin and then solubilized resulted in cross-linking of 125I-TNF into Mr 130,000, 90,000, and 66,000 complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, 125I-TNF binding to control and TNF-pretreated membranes was assayed. Specific binding was increased by pretreatment with TNF (p less than 0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF.

Hepatocytes from male rats were incubated with [32P]Pi for 40 min at 37 degrees C, thereby equilibrating the cellular ATP pool with 32P. Subsequent exposure to bovine growth hormone for 10 additional min did not change the specific activity of cellular [gamma-32P]ATP. Two-dimensional gel electrophoresis or chromatofocusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to fractionate phosphoproteins solubilized from control or hormone-stimulated cells. Stimulation of hepatocytes with 5 nM growth hormone for 10 min at 37 degrees C affected the phosphorylation of a number of proteins including an Mr 46,000 species of pI 4.7 whose phosphorylation was augmented (2.65 +/- 0.50)-fold. A significant fraction of the maximal effect of growth hormone on phosphorylation of the Mr 46,000 species was elicited by 1-5% receptor occupancy. Bovine growth hormone, which binds to somatogenic receptors with great specificity, or recombinant human growth hormone, which is not contaminated with other hormones, affected phosphorylation of hepatic proteins similarly. The Mr 46,000 phosphoprotein was isolated in a fraction enriched in cytosol after centrifugation of cellular homogenates. Phosphorylation of the Mr 46,000 phosphoprotein was also increased (1.75 +/- 0.35)-fold and (2.15 +/- 0.50)-fold by insulin and glucagon, respectively. These observations are consistent with the possibility that selective changes in the phosphorylation state of cellular proteins may mediate growth hormone actions in cells.

In situ hybridization of radiolabeled fibrinogen cDNAs to human and rat metaphase chromosomes has shown that the genes encoding the A alpha, B beta, and gamma fibrinogen subunits are syntenic in both species. Our data localize the human fibrinogen gene cluster to band q31 on chromosome 4, thereby confirming and extending previous map assignments of these genes in man. We have also assigned these genes to the q31----q34 region of rat chromosome 2. This is the first map assignment of these genes in the rat and also the first report to clearly establish linkage of the B beta subunit gene to the A alpha and gamma genes in this species.

Tumor necrosis factor alpha (TNF-alpha) stimulated the phosphorylation of a 28-kDa protein (p28) in the ME-180 line of human cervical carcinoma cells. The effect of TNF-alpha on the phosphorylation state of p28 was rapid (4-fold increase within 15 min) and persistent, remaining above the basal level for at least 2 hr. The specific binding of 125I-labeled TNF-alpha to cell-surface binding sites, the stimulation of p28 phosphorylation by TNF-alpha, and the inhibition of cell proliferation by TNF-alpha occurred with nearly identical dose-response relationships. Two-dimensional SDS/PAGE resolved p28 into two isoforms having pI values of 6.2 and 6.1. A phosphorylated cap-binding protein was substantially enriched from lysates of control or TNF-alpha-treated ME-180 cells by affinity chromatography with 7-methylguanosine 5'-triphosphate-Sepharose. The phosphoprotein recovered from this procedure was the substrate for TNF-alpha-promoted phosphorylation, p28. Thus, TNF-alpha stimulates the phosphorylation of this mRNA cap-binding protein, which may be involved in the transduction of TNF-alpha-receptor binding into cellular responses.