TNF-alpha In Mice Research Articles

Endogenous TNFα Suppression of Neovascularization in Corneal Stroma in Mice

To examine the role of tumor necrosis factor alpha (TNFalpha) in stromal neovascularization in injured cornea in vivo and in cytokine-enhanced vessel-like endothelial cell tube formation in vitro. An in vitro model of angiogenesis was used to examine the roles of TNFalpha on tube formation by human umbilical vein endothelial cells (HUVECs) cocultured with fibroblasts on induction by transforming growth factor beta1 (TGFbeta1) and vascular endothelial growth factor (VEGF). Central cauterization was used to induce stromal neovascularization in corneas of wild-type (WT) and TNFalpha-null (Tnfalpha(-/-)) mice. At 7, 14, or 21 days of injury, experimental mice were killed, and the eyes were enucleated and subjected to histologic and immunohistochemical examination and real-time reverse transcription-polymerase chain reaction. HUVECs formed a vessel-like tube structure on the fibroblast feeder layer. Adding TGFbeta1, VEGF, or both augmented vessel-like tube formation by HUVECs cocultured with fibroblasts. Adding TNFalpha (5 ng/mL) completely abolished the formation of tube-like structures despite the presence or absence of TGFbeta1 or VEGF in coculture. In vivo, cauterization of the central cornea induced the formation of CD31(+) new vessels surrounding the limbus in WT mice. More prominent central stromal neovascularization accompanied by increased expression of TGFbeta1 and VEGF was found in Tnfalpha(-/-) mice compared with WT mice. In addition to inhibiting TGFbeta1 and VEGF expression by fibroblasts, endogenous TNFalpha may counter the induction effects of TGFbeta1 and VEGF on vascular endothelial cells and may block neovascularization.

Galectin-9 protects mice from the Shwartzman reaction by attracting prostaglandin E 2-producing polymorphonuclear leukocytes

Galectins play a crucial role in the modulation of innate and adaptive immunity. Here we show that galectin-9 (Gal-9) exhibits an anti-inflammatory role in LPS-induced inflammation. Intraperitoneal LPS injection enhances Gal-9 levels as well as promotes the production of pro-inflammatory cytokines, e.g., TNF-alpha, IFN-gamma and IL-12. We found that Gal-9 administration results in the protection of mice from the Shwartzman reaction, and Gal-9-deficient mice became susceptible to the Shwartzman reaction, thus implying the anti-inflammatory activity of Gal-9 against LPS-induced inflammation. Indeed, Gal-9 treatment together with LPS suppresses production of these pro-inflammatory cytokines, while it rather enhances than suppresses IL-4 and IL-10 production. We also found that LPS-induced elevation of TNF-alpha, IFN-gamma, and IL-12 does not occur in Gal-9 transgenic mice. Moreover, Gal-9 induces Gr-1 + cell; probably polymorphonuclear leukocyte (PMN), as well as infiltration in to the peritoneal cavity, causing us to hypothesize PMNs are involved in Gal-9-mediated suppression. The fact that Gal-9 does not suppress LPS-induced TNF-alpha, IFN-gamma and IL-12 production in neutropenic mice, and that it does not protect those mice from the Shwartzman reaction, confirms the involvement of PMN in regulation. PMN attracted by Gal-9 produce PGE 2, which LPS-induced TNF-alpha production from the peritoneal macrophages is suppressed, while PMNs attracted by casein produce less PGE 2 and fail to suppress LPS-induced TNF-alpha production. Our data suggest that Gal-9 regulates LPS-induced inflammation and protects mice from the Shwartzman reaction by attracting PGE 2-producing PMN.

Omega‐3 fatty acids alleviate chemically induced acute hepatitis by suppression of cytokines†‡

Cytokines such as tumor necrosis factor alpha (TNF-alpha) are key factors in liver inflammation. Supplementation with essential omega-3 polyunsaturated fatty acids (n-3 PUFA) has been demonstrated to lower TNF-alpha and IL-1 production in mononuclear cells. An inflammation-dampening effect has been observed with increased omega-3 fatty acid supplementation in several inflammatory diseases. In this study, we used the transgenic fat-1 mouse, expressing a Caenorhabditis elegans desaturase endogenously forming n-3 PUFA from n-6 PUFA, to analyze the effect of an increased n-3 PUFA tissue status in the macrophage-dependent acute D-galactosamine/lipopolysaccaride (D-GalN/LPS) hepatitis model. We show less severe inflammatory liver injury in fat-1 mice with a balanced n-6/n-3 PUFA ratio as evidenced by reduced serum alanine aminotransferase levels and less severe histological liver damage. This decreased inflammatory response was associated with decreased plasma TNF-alpha levels and with reduced hepatic gene expression of TNF-alpha, IL-1beta, IFN-gamma and IL-6 in fat-1 mice, leading to a decreased rate of apoptosis in livers from fat-1 animals, as measured by DAPI-staining. The results of this study offer evidence for an inflammation dampening effect of omega-3 polyunsaturated fatty acids in the context of liver inflammation.

The potential role of fucosylated cationic liposome/NFκB decoy complexes in the treatment of cytokine-related liver disease

Cytokine production by Kupffer cells, which is regulated by NFkappaB, causes severe liver injury in endotoxin syndrome. NFkappaB decoy has been reported to inhibit NFkappaB-mediated transcription. The purpose of this study is to inhibit LPS-induced cytokine production by Kupffer cell-targeted delivery of NFkappaB decoy using fucosylated cationic liposomes (Fuc-liposomes). Cholesten-5-yloxy-N-{4-[(1-imino-2-L-thiofucosyl-ethyl)-amino] butyl-}formamide (Fuc-C4-Chol) was synthesized to prepare Fuc-liposomes. Tissue accumulation, intrahepatic distribution and serum cytokine concentrations were investigated after intravenous injection of Fuc-liposomes/NFkappaB decoy complexes. Intravenously injected Fuc-liposome complexes rapidly and highly accumulated in the liver while little naked NFkappaB decoy accumulated in the liver. An intrahepatic distribution study showed that Fuc-liposome complexes are mainly taken up by non-parenchymal cells. The liver accumulation of Fuc-liposome complexes was inhibited by GdCl(3) pretreatment, which selectively inhibited Kupffer cell uptake. This result suggested that Kupffer cells contribute to liver accumulation. TNFalpha, IFNgamma, ALT and AST serum levels in LPS-infected mice were significantly attenuated by treatment with Fuc-liposome complexes compared with naked NFkappaB decoy. Fuc-liposome complexes also reduced the amount of activated NFkappaB in the liver nuclei. Fuc-liposomes would be a useful carrier for Kupffer cell-selective delivery of NFkappaB decoy by intravenous injection.

Role of TNF‐α in muscularis inflammation and motility disorder in a TNBS‐induced colitis model: clues from TNF‐α‐deficient mice

Macroscopic and histological analysis revealed that the colonic inflammation induced by 2,4,6-trinitrobenzenesulphonic acid (TNBS) was of lower grade in tumour necrosis factor-alpha (TNF-alpha)(-/-) mice than in wild-type mice. Myeloperoxidase activity, an indicator of neutrophilic infiltration, was also low in both the mucosal and smooth muscle layer of the TNF-alpha(-/-) mouse colon. After the induction of inflammation with TNBS, the levels of proinflammatory cytokines, such as TNF-alpha, interleukin-1beta and interleukin-6, were elevated both in the inflamed mucosa and muscle layers in the wild-type mice; however, the productions of these cytokines were greatly reduced in the TNF-alpha(-/-) mouse colon. The contractions of isolated colonic smooth muscle strips induced by several stimulatory agents were significantly decreased after treatment with TNBS in wild-type mice; however, these contractions were scarcely affected in TNF-alpha(-/-) mice. Finally, using the organ culture method, we found that TNF-alpha directly (independent of mucosal inflammation) disturbs the smooth muscle function. These results suggest that TNF-alpha plays an essential role not only in mucosal inflammation but also in muscularis inflammation in the colon of mice with TNBS-induced colitis, and that TNF-alpha directly induces motor dysfunctions by acting on the smooth muscle.

Prevention of acute graft-versus-host disease despite compensatory function of lymphoid organs in vivo

Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer’s patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (>120 d, p<0.0001). Our study shows that multiple priming sites are involved in GVHD initiation, the spleen compensating for the lack of PP and mesenteric LN, and vice versa. In contrast, splenectomy and antibody blocking resulted in a clear survival benefit for all recipients.

Suppression of inflammation by low-dose methotrexate is mediated by adenosine A2A receptor but not A3 receptor activation in thioglycollate-induced peritonitis

Prior studies demonstrate that adenosine, acting at one or more of its receptors, mediates the anti-inflammatory effects of methotrexate in animal models of both acute and chronic inflammation. Both adenosine A2A and A3 receptors contribute to the anti-inflammatory effects of methotrexate treatment in the air pouch model of inflammation, and the regulation of inflammation by these two receptors differs at the cellular level. Because different factors may regulate inflammation at different sites we examined the effect of low-dose weekly methotrexate treatment (0.75 mg/kg/week) in a model of acute peritoneal inflammation in adenosine A2A receptor knockout mice and A3 receptor knockout mice and their wild-type littermates. Following intraperitoneal injection of thioglycollate there was no significant difference in the number or type of leukocytes, tumor necrosis factor alpha (TNF-α) and IL-10 levels that accumulated in the thioglycollate-induced peritoneal exudates in adenosine A2A knockout mice or wild-type control mice. In contrast, there were more leukocytes, TNF-α and IL-10 in the exudates of the adenosine A3 receptor-deficient mice. Low-dose, weekly methotrexate treatment increased the adenosine concentration in the peritoneal exudates of all mice studied, and reduced the leukocyte accumulation in the wild-type mice and A3 receptor knockout mice but not in the A2A receptor knockout mice. Methotrexate reduced exudate levels of TNF-α in the wild-type mice and A3 receptor knockout mice but not the A2A receptor knockout mice. More strikingly, IL-10, a critical regulator of peritoneal inflammation, was increased in the methotrexate-treated wild-type mice and A3 knockout mice but decreased in the A2A knockout mice. Dexamethasone, an agent that suppresses inflammation by a different mechanism, was similarly effective in wild-type mice, A2A mice and A3 knockout mice. These findings provide further evidence that adenosine is a potent regulator of inflammation that mediates the anti-inflammatory effects of methotrexate. Moreover, these data provide strong evidence that the anti-inflammatory effects of methotrexate and adenosine are mediated by different receptors in different inflammatory loci, an observation that may explain why inflammatory diseases of some organs but not of other organs respond to methotrexate therapy.

Prevention of Acute Graft-Versus-Host Disease Despite Compensatory Function of Lymphoid Organs In Vivo.

Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer's patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (>120 d, p<0.0001). Our study shows that multiple priming sites are involved in GVHD initiation, the spleen compensating for the lack of PP and mesenteric LN, and vice versa. In contrast, splenectomy and antibody blocking resulted in a clear survival benefit for all recipients.

DIETARY SUPPLEMENTATION OF SOYBEAN KUNITZ TRYPSIN INHIBITOR REDUCES LIPOPOLYSACCHARIDE-INDUCED LETHALITY IN MOUSE MODEL

We examined the modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean, as intraperitoneal (i.p.) injection and dietary supplements on bacterial lipopolysaccharide (LPS)-induced lethality in mice. We initially examined the suppressing effects of i.p. injection of KTI (50 mg/kg) and BBI (50 mg/kg) on LPS-induced lethality after i.p. injection of LPS. Furthermore, groups of female C57BL/6 were fed a basal diet (control group) or the basal diet supplemented with KTI (50 g/kg) or BBI (50 g/kg). Here, we show that i.p. and daily oral administration of KTI, but not BBI, caused a significant reduction of the LPS-induced lethality; that LPS significantly induced plasma TNF-alpha, IL-1beta, and IL-6 levels in mice after LPS challenge; that concomitant administration of KTI, but not BBI, inhibits the LPS-induced plasma levels of these cytokines; and that KTI, but not BBI, suppressed LPS-induced upregulation of cytokine expression through suppression of phosphorylation of three mitogen-activated protein (MAP) kinase pathways, ERK1/2, JNK, and p38, in peritoneal macrophages. These data allow us to speculate that i.p. injection and dietary supplementation of a soybean KTI may play a role as a potent anti-inflammatory agent by inhibiting activation of MAP kinases, leading to the suppression of cytokine expression.

Hepatic Cytochrome P450 Gene Regulation during Endotoxin-Induced Inflammation in Nuclear Receptor Knockout Mice

Inflammatory agents such as lipopolysaccharide (LPS) down-regulate the hepatic expression of many cytochrome P450 (P450) mRNAs and proteins. Previous studies suggested that suppression of some P450 mRNAs could involve the regulation or modulation of the nuclear receptors peroxisome proliferator-activated receptor alpha (PPARalpha) or pregnane X receptor (PXR). To determine the involvement of these receptors in P450 down-regulation, PPARalpha knockout (KO), PXR KO, and appropriate wild-type (WT) mice were administered either saline or 1 mg/kg LPS. Hepatic mRNA and protein expression of several P450 isoforms, interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF) alpha, alpha1-acid glycoprotein (AGP), and fibrinogen (FBG) were examined 16 h later. LPS administration significantly decreased the hepatic expression of CYP1A2, 2A5, 2C29, 2E1, 3A11, 4A10, and 4A14 mRNAs in both groups of PPARalpha and PXR mice, whereas CYP3A13 mRNA was increased slightly in PPARalpha WT and KO mice, but not in PXR mice. Effects of LPS administration on mouse hepatic P450 proteins (probed using rat P450 2C, 3A, 4A, and 2E antibodies) were consistent with mRNA results in most cases. LPS treatment significantly increased IL-1beta, IL-6, TNFalpha, AGP, and FBG mRNA in both PPARalpha and PXR mice, with the greatest effect observed with TNFalpha. Because decreases in P450 mRNA expression were essentially identical in both WT and KO mice for both nuclear receptors, these data indicate that down-regulation of P450 during inflammation does not require the nuclear receptors PPARalpha and PXR.

Normal liver regeneration and liver cell apoptosis after partial hepatectomy in tumor necrosis factor‐α‐deficient mice

Tumor necrosis factor-alpha (TNF-alpha) is known as a proinflammatory cytokine that has been implicated as a contributing factor in a number of disease processes. TNF-alpha also influences liver repair following hepatotoxic damage, and regeneration following partial hepatectomy (PH). The aim of this study was to assess the mechanism by which TNF-alpha influences liver cell apoptosis and regeneration following PH in TNF-alpha-deficient (TNF-alpha(-/-)) mice. PH was performed in wild mice and TNF-alpha(-/-) mice. In both groups, serum alanine aminotransferase and serum total bilirubin levels comparably peaked at 6 and 48 h after PH, respectively. No differences were observed in hepatocyte proliferation, as determined by mitotic and the proliferating cell nuclear antigen labeling indices, between TNF-alpha(+/+) and TNF-alpha(-/-) mice. Few terminal deoxynucleotidyl transferase nick end-labeling-positive hepatocytes were seen in either type of mice. Nuclear factor-kappa B DNA binding activity in the remaining liver of TNF-alpha(-/-) mice after PH was similar to that of control mice. Ribonuclease protection assay showed that transforming growth factor beta1 mRNA was up-regulated comparably in the livers of the two groups, and that other cytokines were hardly seen in either. Interleukin-6/ signal transducer and activator of transcription-3-dependent pathway was not affected in TNF-alpha(-/-) mice. These findings suggest that TNF-alpha has little influence on liver regeneration and liver cell apoptosis after PH in mice.

Compensatory response of IL-1 gene knockout mice after pulmonary infection with Klebsiella pneumoniae

This study was designed to determine the role of interleukin (IL)-1 in the inflammatory response against experimentally induced pneumonia caused by Klebsiella pneumoniae. The host immune responses of IL-1 gene knockout (IL-1 KO) mice and immunocompetent wild-type (WT) mice were compared after pulmonary infection with K. pneumoniae. There were no significant differences between the survival rates and viable bacterial counts in lungs and blood of IL-1 KO and WT mice after pulmonary infections under different conditions. Histopathological analysis showed a similar inflammatory response in both groups of mice. However, in the early stage of infection, the level of tumour necrosis factor alpha (TNF-alpha) in homogenized lungs of IL-1 KO mice was significantly higher than in WT mice. To determine the role of endogenous TNF-alpha in the recovery of the defence mechanism in IL-1 KO mice, mice were treated with an anti-TNF-alpha mAb before infection with K. pneumoniae. The results revealed a significantly lower survival rate of anti-TNF-alpha mAb-treated IL-1 KO mice than BSA-treated IL-1 KO mice. The data suggest that compensatory production of TNF-alpha in IL-1 KO mice contributes to the host defence against K. pneumoniae infection.

Recombinant human lactoferrin has preventive effects on lipopolysaccharide-induced preterm delivery and production of inflammatory cytokines in mice

The following animal studies have been conducted to investigate whether recombinant human lactoferrin (rh-LF) has the same effect as bovine lactoferrin (b-LF) in the prevention of preterm delivery. Female C3H/HeNCrj mice were pair-mated with male Crj:B6D2F1 mice. On day 15 of gestation, as a model of preterm delivery, a 50 microg/kg intraperitoneal injection of lipopolysaccharide (LPS) was administered twice with a 3-h interval between injections (2:00 and 5:00 PM). One hour prior to each LPS injection (1:00 and 4:00 PM), an intraperitoneal injection of saline, b-LF, or rh-LF (1 mg/body) was administered. In non-LPS-treated controls, an intraperitoneal injection of saline was administered 4 times (1:00, 2:00, 4:00 and 5:00 PM). Body weights and delivery times were recorded. To compare plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) between experimental and other pregnant mice, prepared as above, were sacrificed 6 h after the second LPS injection, and then blood samples were obtained and analyzed. Preterm delivery occurred (16.2+/-0.4 days of gestation) in all LPS-treated mice that were not administered LF. LF significantly prolonged gestation of LPS-treated mice: b-LF+LPS, 17.8+/-0.3 days; rh-LF+LPS, 18.0+/-0.8 days (P<0.05). LF (1 mg/body) significantly suppressed plasma IL-6 in LPS-treated mice:b-LF+LPS, 1060+/-154; rh-LF+ LPSF, 244+/-59; LPS without LF, 1628+/-115 pg/mL (P<0.05). As well, LF (1 mg/body) significantly suppressed plasma TNF-alpha in LPS-treated mice: b-LF+LPS, 88+/-36; rh-LF+LPS, 37+/-30; LPS without LF, 114+/-49 pg/mL (P<0.05). Rh-LF may prolong gestation in LPS-induced preterm delivery in mice, by suppressing LPS-induced plasma IL-6 and TNF-alpha augmentation.

Tetramethoxy Hydroxyflavone p7F Downregulates Inflammatory Mediators via the Inhibition of Nuclear Factor κB

Artemisia has been traditionally used in Korean herbal medicine to clear damp heat and to treat uteritis and jaundice. Flavonoids isolated from Artemisia are also known to possess anti-inflammatory activities. In this study, 5,6,3',5'-tetramethoxy 7,4'-hydroxyflavone (p7F) was isolated from Artemisia absinthium. We examined in vitro and in vivo regulatory functions of p7F on the production of nitric oxide (NO), prostaglandin E(2) (PGE(2)), and tumor necrosis factor-alpha (TNF-alpha) as well as the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and collagen-induced arthritis. p7F inhibited the expression or production of proinflammatory mediators such as COX-2/PGE(2) and iNOS/NO in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. p7F also suppressed the serum level of TNF-alpha in mice treated with collagen and inhibited nuclear factor-kappaB (NF-kappaB) activation as well as NF-kappaB promoter activity in RAW 264.7 cells stimulated with LPS. This compound directly inhibited the intracellular accumulation of reactive oxygen species in hydrogen peroxide-stimulated RAW 264.7 cells. p7F has antioxidant activity and inhibits NF-kappaB activation. Taken together, these results suggest that p7F can be clinically applied to the treatment of inflammatory diseases.

TNF-alpha regulates epithelial expression of MMP-9 and integrin alphavbeta6 during tumour promotion. A role for TNF-alpha in keratinocyte migration?

Mice deficient in TNF-alpha (TNF-alpha(-/-) mice) are resistant to skin carcinogenesis and expression of MMP-9 is inhibited in TNF-alpha(-/-) mice during skin tumour development. In the early stages of tumour promotion, MMP-9 protein initially localized to the follicular epidermis but subsequently began to accumulate in the interfollicular epidermis of wild-type but not TNF-alpha(-/-) mice. Inhibition of TNF-alpha or MMP-9 function reduced keratinocyte migration in vitro. In addition, a deficiency of TNF-alpha delayed re-epithelialization in vivo and this correlated with reduced MMP-9 expression. Collectively, these data suggest that MMP-9 regulates keratinocyte migration in a TNF-alpha-dependent manner. Expression profiling of genes that control cell adhesion and migration revealed markedly lower levels of the integrin subunits alphav and beta6 in TNF-alpha(-/-) compared with wild-type keratinocytes in vitro. alphavbeta6 expression was upregulated by keratinocytes in vitro and during tumour promotion in vivo in a TNF-alpha-dependent manner. Furthermore, alphavbeta6 blockade significantly inhibited keratinocyte migration and TNF-alpha-stimulated MMP-9 expression in vitro. These data illustrate a novel TNF-alpha-dependent mechanism for the control of alphavbeta6 expression and suggest one pathway for TNF-alpha regulation of MMP-9. Increased MMP-9 and alphavbeta6 expression may stimulate epithelial cell migration during tumour formation and may be one mechanism whereby TNF-alpha acts as an endogenous tumour promoter.

Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum.

The impact of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) in the pathology of Parkinson's disease (PD) and in MPTP neurotoxicity remains unclear. Here, male TNF-alpha (-/-) deficient mice and C57bL/6 mice were treated with MPTP (4 x 15 mg/kg, 24 h intervals) and in one series, thalidomide was administered to inhibit TNF-alpha synthesis. Real-time RT-PCR revealed that the striatal mRNA levels of TNF-alpha, of the astrocytic marker glial fibrillary acidic protein (GFAP) and of the marker for activated microglia, macrophage antigen complex-1 (MAC-1), were significantly enhanced after MPTP administration. Thalidomide (50 mg/kg, p.o.) partly protected against the MPTP-induced dopamine (DA) depletion, and TNF-alpha (-/-) mice showed a significant attenuation of striatal DA and DA metabolite loss as well as striatal tyrosine hydroxylase (TH) fiber density, but no difference in nigral TH and DA transporter immunoreactivity. TNF-alpha deficient mice suffered a lower mortality (10%) compared to the high mortality (75%) seen in wild-type mice after acute MPTP treatment (4 x 20 mg/kg, 2 h interval). HPLC measurement of MPP(+) levels revealed no differences in TNF-alpha (-/-), wild-type and thalidomide treated mice. This study demonstrates that TNF-alpha is involved in MPTP toxicity and that inhibition of TNF-alpha response may be a promising target for extending beyond symptomatic treatment and developing anti-parkinsonian drugs for the treatment of the inflammatory processes in PD.

Unexpected role of TNF-alpha in graft versus host reaction (GVHR): donor-derived TNF-alpha suppresses GVHR via inhibition of IFN-gamma-dependent donor type-1 immunity.

Graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation, leading to significant morbidity and mortality. Host-derived TNF-alpha play a role in the induction of allo-reactive donor T cell activation and the pathogenesis of GVHD. On the other hand, the precise role of donor-derived TNF-alpha in GVHD remains unclear. To elucidate this issue, we designed an acute GVHD model using (B6 x D2) F1 recipient mice transferred with spleen cells derived from either wild-type or TNF-alpha(-/-) C57BL/6 mice. Surprisingly, we found that spleen cells from TNF-alpha(-/-) mice induce more severe graft versus host reaction (GVHR) than wild-type spleen cells upon transfer into B6D2F1 mice. Transplantation of TNF-alpha(-/-) mouse spleen cells was associated with enhanced anti-host CTL generation and augmented deletion of host cells. Moreover, mice receiving TNF-alpha(-/-) cells showed significantly higher levels of serum IFN-gamma, which was mainly produced by donor CD8+ T cells. We also demonstrated that TNF-alpha deficiency in donor spleen cells caused a marked elevation of TNF-alpha producing capacity by LPS-stimulated host macrophages. Such enhanced GVHR was completely prevented by using TNF-alpha(-/-)IFN-gamma(-/-) splenic cells. Our findings demonstrate, for the first time, that donor-derived TNF-alpha suppress GVHR by inhibiting IFN-gamma-dependent donor type-1 immunity which is essential for host TNF-alpha elevation.

A highly selective inhibitor of IkappaB kinase, BMS-345541, augments graft survival mediated by suboptimal immunosuppression in a murine model of cardiac graft rejection.

We previously demonstrated in vitro and in vivo that an IkappaB kinase (IKK) inhibitor blocks cytokine production and suppresses immune responses. These results indicate that a potent IKK inhibitor may have the potential of being a novel therapeutic agent for the prevention of graft rejection. The IKK inhibitor BMS-345541 was tested in mice for its ability to inhibit anti-CD3-induced interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha production and T-cell proliferation in an in vivo mixed lymphocyte reaction. BMS-345541 was further tested for its ability to suppress graft rejection in a murine nonvascularized heterotopic cardiac allograft model. BMS-345541 was tested as a single agent and in combination with other immunomodulators for inhibition of T-cell proliferation and graft rejection in vivo. BMS-345541 suppressed, in a dose-dependent manner, the production of both IL-2 and TNF-alpha in mice stimulated with an injection of anti-CD3 antibody. Approximately 70% inhibition of both IL-2 and TNF were observed at a dose of 100 mg/kg. When BMS-345541 was administered at 100 mg/kg as a single agent, in vivo T-cell proliferation was not inhibited. However, when combined with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin (200 microg), a synergistic antiproliferative effect was observed, resulting in 77% inhibition of CD4+ T-cell proliferation. In the murine heterotopic heart transplant model, BMS-345541 did not prolong graft survival when administered at 50 mg/kg as a single agent. However, when administered with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin or cyclosporine A (15 mg/kg), graft survival was significantly increased compared with either agent alone. These results indicate that inhibition of IKK may serve as novel adjunctive therapy for the prevention of graft rejection.

TNF-alpha in vivo stimulates apoptosis in fibroblasts through caspase-8 activation and modulates the expression of pro-apoptotic genes.

Apoptosis of matrix producing cells is common among many inflammatory diseases. The goal of the present study was to examine the apoptotic effects of tumor necrosis factor-alpha (TNF-alpha) on fibroblastic cells in vivo and to investigate the role of different caspases in this process. This was accomplished in vivo by subcutaneous injection of TNF-alpha in mice. The direct effects of TNF-alpha on fibroblast apoptosis were studied in vitro with normal diploid human fibroblasts. The results indicate that TNF-alpha in vivo induces apoptosis of fibroblasts. By RNase protection assay, we demonstrated that TNF-alpha stimulates expression of 12 apoptotic genes. Fluorometric studies demonstrated that TNF-alpha in vivo predominantly increased caspase-8 and -3 activity and by use of specific inhibitors, the activation of caspase-3 was shown to be initiated by caspase-8 with only a minor contribution from caspase-9. Thus, TNF-alpha acts to modulate the expression of many genes that favors apoptosis of fibroblastic cells, which is dependent mostly upon signaling through caspase-8.

Effects of immune-enhancing enteral formula on LPS-induced elevation of plasma IL-6 and TNF-alpha in malnourished mice

Effects of immune-enhancing enteral formula on LPS-induced elevation of plasma IL-6 and TNF-alpha in malnourished mice