Lipopolysaccharide-induced HIV-1 expression in transgenic mice is mediated by tumor necrosis factor-alpha and interleukin-1, but not by interferon-gamma nor interleukin-6

BACKGROUND: As serum HIV-1 load correlates well with the prognosis of the disease, it is suggested that the viral load is one of the major determinants of the disease progression of AIDS. Accordingly, HIV-1 activation mechanisms were extensively studied in vitro, and involvement of cytokines including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6 and interferon (IFN)-gamma has been suggested in this process. However, so far the roles of these cytokines in the HIV-1 expression in vivo have not been well elucidated because of the lack of appropriate animal disease models. OBJECTIVE: To elucidate the roles of cytokines in HIV-1 activation in vivo. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were used as a model for HIV-1 carriers. In order to examine the possible involvement of cytokines in HIV-1 expression, TNF-alpha-, IL-1-, IL-6- and IFN-gamma-deficient HIV-1 transgenic mice, were produced and HIV-1 expression was analyzed after activation with bacterial lipopolysaccharides (LPS). RESULTS: HIV-1 expression in the transgenic mouse spleen was activated 10- to 20-fold by LPS, and the serum p24 Gag protein levels reached 400 pg/ml, which is nearly equal to the levels that occur in AIDS patients. However, this augmentation was suppressed by 60% in TNF-alpha-deficient mice and by 40% in IL-1alpha/beta-deficient mice. In contrast, no suppression was observed in either IL-6-, IFN-gamma-, IL-1alpha, or IL-1beta-deficient mice. CONCLUSIONS: Results suggest that TNF-alpha and IL-1 play important roles in HIV-1 gene activation and selective suppression of these cytokines could improve clinical prognosis and potentially slow progression of the disease.     

Accumulation of LDL in rat arteries is associated with activation of tumor necrosis factor-alpha expression

Activation of vascular inflammation in response to hyperlipidemia is believed to play an important role during the early stages of atherogenesis. We demonstrate here that exposure of cultured, rat aortic smooth muscle cells to low density lipoprotein (LDL) stimulated tumor necrosis factor-alpha (TNF-alpha) mRNA and protein expression. Oxidative modification of LDL resulted in a reduction of this stimulatory effect. To analyze whether a similar response also occurs in vivo, we used a recently developed model in which the effects of a rapid accumulation of human LDL in rat arteries can be studied. As previously reported, epitopes specific for human apolipoprotein B began to accumulate in the aorta within 2 to 6 hours after injection of 6 mg of human LDL. This was followed by expression of oxidized LDL-specific epitopes after 12 hours. There was no vascular expression of TNF-alpha at baseline or in phosphate-buffered saline-injected control rats. However, 24 hours after injection of native LDL, there was a marked induction of TNF-alpha mRNA and immunoreactivity in the aorta and other large arteries, whereas injection of oxidized LDL was without effect in this respect. Preincubation of LDL with the antioxidant probucol before injection markedly decreased the expression of TNF-alpha immunoreactivity. The present findings support the notion that LDL may activate arterial expression of TNF-alpha and suggest 1 possible mechanism for the inflammatory response in the early stages of atherosclerosis. The role of LDL oxidation in this process remains to be fully elucidated.     

Autoinduction of tumor necrosis factor-alpha in FRTL-5 rat thyroid cells

Tumor necrosis factor-alpha (TNFalpha) may play a role in the development of autoimmune thyroiditis such as Hashimoto's thyroiditis. In the present study, we examined whether TNFalpha induced its own expression in FRTL-5 rat thyroid cells. Lipopolysaccharide (LPS) markedly increased TNFalpha mRNA levels in FRTL-5 cells as assessed by semiquantitative RT-PCR. In addition, LPS-stimulated cells released TNFalpha protein into the culture medium. Similarly, TNFalpha induced its own gene and protein expression in FRTL-5 cells as assessed by RT-PCR and metabolic labeling and immunoprecipitation of TNFalpha. The autoinduction of TNFalpha gene was also observed in TNFalpha-stimulated human thyroid epithelial cells. TNFalpha induction was specific to LPS and TNFalpha since interferon-alpha or amiodarone failed to increase TNFalpha mRNA levels in FRTL-5 cells. Human TNFalpha induced rat TNFalpha gene expression, indicating that type 1 TNF receptor (TNF-R) is involved in the autoinduction. TNFalpha did not increase either type 1 or type 2 TNF-R mRNA levels, suggesting that upregulation of TNF receptors is not involved in the autoinduction of TNFalpha. Although the biological significance of autoinduction of TNFalpha remains unclear, our results suggest that thyroid epithelial cells may participate in the development of autoimmune thyroiditis through production of TNFalpha. Furthermore, inhibition of TNFalpha production in the thyroid may represent a novel approach to mitigating inflammation in autoimmune thyroiditis.     

Induction by tumor necrosis factor-alpha of rapid release of immunoreactive and bioactive luteinizing hormone from rat pituitary cells in vitro

Tumor necrosis factor-alpha (TNF-alpha) is secreted by activated monocytes and other immune cells. This paper reports studies on the effects of TNF-alpha on the releases of pituitary hormones such as luteinizing hormone (LH), follicle-stimulating hormone, prolactin (PRL) and adrenocorticotropic hormone (ACTH). The addition of recombinant human TNF-alpha (rTNF-alpha) to cultures of pituitary cells resulted in significantly increased releases of gonadotropins, PRL, and ACTH for up to 30 min, but not later. rTNF-alpha, like GnRH, also stimulated the release of bioactive LH. In addition, rTNF-alpha induced production of an interleukin-6 (IL-6)-like molecule by pituitary cells. As IL-6 induces the releases of multiple hormones from pituitary cells, our data suggest that rTNF-alpha may stimulate the releases of multiple pituitary hormones through IL-6 production as well as by its direct action on pituitary cells.     

Increased expression of tumor necrosis factor-alpha converting enzyme and tumor necrosis factor-alpha in peripheral blood mononuclear cells in patients with advanced congestive heart failure

BACKGROUND: Tumor necrosis factor-alpha converting enzyme (TACE) has recently been identified as a metalloproteinase-disintegrin, which converts pro-tumor necrosis factor-alpha (TNF-alpha) to the mature form, and is an important mediator in the pathogenesis of CHF. AIMS: In order to establish the importance of TACE in the regulation of TNF-alpha synthesis in peripheral blood mononuclear cells (PBMC), we analyzed mRNAs and protein-positive cells of both TACE and TNF-alpha in PBMC obtained from patients with congestive heart failure (CHF). METHODS AND RESULTS: PBMC were obtained from 46 patients with CHF and 22 controls. PBMC were activated by phorbol 12-myristate 13-acetate and ionomycin and assessed for TACE and TNF-alpha mRNAs by real-time RT-PCR, intracellular TACE and TNF-alpha levels by flow cytometry, and TNF-alpha secretion by supernatant ELISA. Levels of TACE and TNF-alpha mRNAs, intracellular TACE and TNF-alpha, and supernatant TNF-alpha were higher in CHF than in controls (P<0.001). There was a positive correlation between TACE and TNF-alpha levels in CHF patients (mRNA: r=0.60, P<0.001, intracellular protein levels: r=0.76, P<0.001). When the CHF group was divided into two subgroups by NYHA functional class (I and II vs. III and IV), levels of TACE and TNF-alpha were significantly higher in severe CHF patients (NYHA III or IV) than in mild CHF patients (NYHA I or II) (mRNA: P<0.001; intracellular protein levels: P<0.001). CONCLUSION: These results demonstrate that in patients with CHF, and especially those with severe CHF, TACE expression in PBMC increases with TNF-alpha expression. These observations suggest that TACE in PBMC is an important regulator of TNF-alpha maturation, meaning that TACE may be a potential target for the inhibition of cellular TNF-alpha production in CHF.     

Induction of T-kininogen and tumor necrosis factor-alpha by macrophage migration inhibitory factor in vivo

Administration of lipopolysaccharide (LPS) induces inflammation and tissue injuries that occasionally results in disseminated intravascular coagulation (DIC). This process is believed to be mediated by vasoactive molecules such as kinins and leads to endothelial damage and obstruction of the microcirculation. In this study, we evaluated the involvement of T-kininogen and macrophage migration inhibitory factor (MIF) in endotoxin-induced systemic inflammation. T-Kininogen is a protein unique to the rat and known as an acute-phase protein in response to endotoxins. Similarly, MIF functions as a proinflammatory cytokine and glucocorticoid-induced immunoregulator. First, we examined the effects of anti-MIF antibody on Wistar King male rats (ca 400 g) treated with intraperitoneal injection of LPS. At 6 hours after LPS injection (5 mg/kg), the platelet counts had decreased from 85 +/- 12.8 (x 10(4)/microL) to 8.8 +/- 2.6 (x 10(4)/microL). We treated these rats with the anti-rat MIF antibody (5 mg gamma G immunoglobulin [IgG] fraction/kg) 2 hours prior to LPS injection. This treatment prevented the decrease in platelet counts (45.6 +/- 5.6 [x 10(4)/microL]). Next, we examined the potential of MIF for production of T-kininogen. Intraperitoneal injection of rat MIF significantly upregulated the serum content of T-kininogen at the dose of 500 microg MIF/head. These results imply that MIF and T-kininogen might function in concert in the event of endotoxin-induced inflammation.     

Induction of suppurative arthritis in rabbits by Haemophilus endotoxin, tumor necrosis factor-alpha, and interleukin-1 beta

Because Haemophilus influenzae type b (Hib) is the principal cause of suppurative arthritis in young children and its lipooligosaccharide (LOS) is thought to be the main virulence factor, Hib endotoxin was evaluated for its ability to induce synovial inflammation in rabbits. Also, the role of the cytokines tumor necrosis factor-alpha (TNF alpha) and interleukin-1 beta (IL-1 beta) in mediating the synovial inflammatory process was studied. Intraarticular inoculation of 2 pg to 20 ng of Hib LOS produced a dose-dependent increase in concentrations of leukocytes and protein in synovial lavage fluid that was significantly modulated by concomitant administration of rabbit TNF alpha- and rabbit IL-1 beta-specific antibodies. Inoculation of joints with either 10(4) IU of rabbit TNF alpha or 10 ng recombinant rabbit IL-beta induced synovial inflammatory changes similar to those observed after LOS intraarticular challenge. These data provide evidence for the role of Hib LOS in inducing suppurative arthritis and for the critical participation of TNF alpha and IL-1 beta in the initial events of the synovial inflammatory response.     

Induction of matrix metalloproteinases-14 and -2 by cyclical mechanical stretch is mediated by tumor necrosis factor-alpha in cultured human umbilical vein endothelial cells

OBJECTIVE: Mechanical forces have profound effects on endothelial cells. This study was undertaken to examine the hypothesis that tumor necrosis factor-alpha (TNF-alpha) is a potential mediator of stretch-induced effects on matrix metalloproteinase (MMP). METHODS: Human umbilical vein endothelial cells (HUVECs) grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. We used the TNF-alpha monoclonal antibody and c-Jun N-terminal kinase (JNK) inhibitor, SP600125, to investigate the cyclical stretch-induced expression of MMP-14 and -2 in cultured HUVECs. RESULTS: Cyclical mechanical stretch significantly increased protein synthesis and mRNA expression for MMP-14 and -2 from 2 to 24 h. The increased MMP-14 and-2 proteins after stretch were completely blocked after the addition of TNF-alpha monoclonal antibody (5 microg/ml) or SP600125 (20 microM) 30 min before stretch. By zymography, MMP-2 expression was induced by cyclical stretch and was attenuated by TNF-alpha monoclonal antibody and SP600125. Cyclical stretch increased the immunohistochemical labeling of MMP-14 and -2 and significantly increased release of TNF-alpha into the culture media from 120+/-2 to 331+/-2 pg/ml (P<0.001) after stretch for 12 h. Cyclical stretch increased and SP600125 decreased the phosphorylated JNK. Gel-shifting assay showed that DNA-protein binding activity of AP-1 increased after cyclical stretch and TNF-alpha monoclonal antibody and SP600125 abolished the binding activity induced by cyclical stretch. CONCLUSION: These findings indicate that cyclical stretch augments TNF-alpha production and MMP genes expression in HUVECs. TNF-alpha mediates the stretch-induced MMP genes expression, at least in part, through the JNK pathway.     

Elevated tumor necrosis factor-alpha activation of human immunodeficiency virus type 1 subtype C in Southern Africa is associated with an NF-kappaB enhancer gain-of-function

The human immunodeficiency virus type 1 (HIV-1) epidemic within southern Africa is predominantly associated with the HIV-1C subtype. Functional analysis of the enhancer region within the long terminal repeat (LTR) indicates that HIV-1C isolates have >/=3 NF-kappaB binding sites, unlike other subtypes, which have only 1 or 2 sites. A correlation was shown between NF-kappaB enhancer configuration and responsiveness to the proinflammatory cytokine tumor necrosis factor (TNF)-alpha within the context of naturally occurring subtype LTRs, subtype-specific NF-kappaB enhancer regions cloned upstream of an isogenic HXB2 core promoter or a heterologous SV40 minimal promoter, and full-genome subtype clones. In all cases, TNF-alpha activation was correlated with the subtype configuration of the NF-kappaB enhancer. Whether the naturally occurring gain-of-function in the NF-kappaB enhancer of HIV-1C observed in this study can provide a selective advantage for the virus in vivo remains to be determined and warrants further study.     

Regulation of tenascin-C expression by tumor necrosis factor-alpha in cultured human osteoarthritis chondrocytes

Expression of tenascin-C reappears in articular cartilage of persons with osteoarthritis (OA), while it is almost abolished in normal mature cartilage. Tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is upregulated in OA cartilage and is involved in the progression of OA, and stimulates tenascin-C expression in other types of cells. We investigated regulation of tenascin-C expression by TNF-alpha through nuclear factor-alphaB (NF-kappaB) in OA cartilage in vivo and in vitro. METHODS: Human articular cartilages were obtained from patients with OA and immunofluorescence examination of tenascin-C and the activated RelA subunit was performed. Cultured chondrocytes isolated from human OA cartilage were treated with TNF-alpha and with SN50. Activation of RelA subunit of NF-kappaB was examined by immunolabeling. Changes in tenascin-C protein concentrations were determined by immunofluorescence of cells after monensin treatment and Western blot analysis of the cell lysates, and mRNA levels were analyzed by quantitative real-time polymerase chain reaction. RESULTS: Increased intensity of tenascin-C staining was observed in the damaged cartilage compared with normal cartilage. Activated RelA staining in chondrocyte nuclei was prominent in tenascin-C-positive areas of OA cartilage. Treatment of cultured chondrocytes by TNF-alpha induced translocation of activated RelA to the nuclei, followed by upregulation of tenascin-C expression in both mRNA and protein. Treatment with SN50 inhibited increases of RelA and tenascin-C expression in chondrocytes. CONCLUSION: TNF-alpha stimulated tenascin-C expression through NF-kappaB signaling with RelA activation in cultured OA chondrocytes, suggesting involvement of tenascin-C in OA cartilage remodeling.