Tumor necrosis factor (TNF)-alpha and TNF receptors in viral pathogenesis

Tumor necrosis factor-alpha (TNF-alpha) and TNF receptors (TNFR) are members of the growing TNF ligand and receptor families that are involved in immune regulation. The present report will focus on the role of the prototypic ligand TNF and its two receptors, TNFR1 and TNFR2, in viral pathogenesis. Although TNF was reported years ago to modulate viral infections, recent findings on the molecular pathways involved in TNFR signaling have allowed a better understanding of the molecular interactions between cellular and viral factors within the infected cell. The interactions of viral proteins with intracellular components downstream of the TNFR have highlighted at the molecular level how viruses can manipulate the cellular machinery to escape the immune response and to favor the spread of the infection. We will review here the role of TNF and TNFR in immune response and the role of TNF and TNFR signaling in viral pathogenesis.     

Nerve/glial antigen (NG) 2 is a crucial regulator of intercellular adhesion molecule (ICAM)-1 expression

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses.     

Tumor necrosis factor (TNF)-alpha induces leptin production through the p55 TNF receptor

Tumor necrosis factor (TNF)-alpha acts directly on adipocytes to increase production of the lipostatic factor, leptin. However, which TNF receptor (TNFR) mediates this response is not known. To answer this question, leptin was measured in plasma of wild-type (WT), p55, and p75 TNFR knockout (KO) mice injected intraperitoneally with murine TNF-alpha and in supernatants from cultured WT, p55, and p75 TNFR KO adipocytes incubated with TNF-alpha. Leptin also was measured in supernatants from C3H/HeOuJ mouse adipocytes cultured with blocking antibodies to each TNFR and TNF-alpha as well as in supernatants from adipocytes incubated with either human or murine TNF-alpha, which activate either one or both TNFR, respectively. The results using all four strategies show that the induction of leptin production by TNF-alpha requires activation of the p55 TNFR and that although activation of the p75 TNFR alone cannot cause leptin production, its presence affects the capability of TNF-alpha to induce leptin production through the p55 TNFR. These results provide new information on the interplay between cells of the immune system and adipocytes.     

Tumor necrosis factor receptor-associated factor (TRAF) 2 and its role in TNF signaling

Tumor necrosis factor (TNF) is the prototypic member of the TNF ligand family and has a key role in the regulation of inflammatory processes. TNF exerts its functions by interaction with the death domain-containing TNF-receptor 1 (TNF-R1) and the non-death domain-containing TNF-receptor 2 (TNF-R2), both members of a receptor family complementary to the TNF ligand family. Due to the prototypic features of the TNF receptors and their importance for the regulation of inflammation, the signal transduction mechanisms utilized by these receptors have been extensively studied. Several proteins that interact directly or indirectly with the cytoplasmic domains of TNF-R1 and TNF-R2 have been identified in the recent years giving ideas how these receptors are connected to the apoptotic pathway and the signaling cascades leading to activation of NF-kappaB and JNK. Of special interest are TNF receptor-associated factor (TRAF) 1 and 2, which defines a novel group of adaptor proteins involved in signal transduction by most members of the TNF receptor family, of IL-1 receptor and IL-17 receptor as well as some members of the TOLL-like receptor family. TRAF 2 is currently the best-characterized TRAF family member, having a key role in mediating TNF-R1-induced activation of NF-kappaB and JNK. Moreover, recent studies suggest that TRAF 2 represents an integration point for pro- and antiapoptotic signals. This review focuses on the molecular mechanisms that underlay signal initiation by TNF-R1 and TNF-R2, with particular consideration of the role of TRAF 2, and highlights the importance of this molecule for the integration of such antagonizing pathways as death induction and NF-kappaB-mediated surviving signals.     

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.     

Interleukin -1beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor (TNF) in plasma and pleural fluid of pneumonia, lung cancer and tuberculous pleuritis

Interleukins IL-1beta, IL-6 and TNF are increased in plasma of patients with severe infections and septic shock. Our objective was the evaluation of IL-1beta, IL-6 and TNF in plasma and exudates of pleural fluid and their contribution to the diagnosis. We studied 44 patients, 27 men and 17 women with mean age 66.81 +/- 11.75 years; 16 with pneumonia and parapneumonic effusion, 14 with primary lung cancer and pleural effusion and 14 with tuberculous pleuritis. We measured IL-1beta, IL-6 and TNF in serum and pleural fluid with ELISA. In patients with pneumonia and parapneumonic effusion the mean value of IL-1beta IL-6 and TNF in plasma was 9.05, 19.24 and 21.34 pg/ml and in pleural fluid 10.34, 32.19 and 25.30 pg/ml. In patients with lung cancer the mean values of IL-1beta, IL-6 and TNF were 5.33, 11.74 and 11.51 pg/ml and 6.70, 13.13, 20.89 pg/ml, respectively. In those with tuberculous pleuritis the respective mean values were 10.33, 49.94, 21.27 pg/ml and 14, 56.59, 23.58 pg/ml. In conclusion, IL-1beta and IL-6 were found increased in plasma and tuberculous pleural fluid, indicating an inflammatory status.     

Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production. However, there is little information on the physiological regulation and roles of the cloned N-SMase enzyme, nSMase2. In this study, nSMase2 was found to translocate acutely to the plasma membrane of A549 epithelial cells in response to tumor necrosis factor alpha (TNF-alpha) in a time- and dose-dependent manner. Additionally, TNF-alpha increased N-SMase activity rapidly and transiently both endogenously and in cells overexpressing nSMase2. Furthermore, the translocation of nSMase2 was regulated by p38-alpha MAPK, but not ERK or JNK, and the increase in endogenous N-SMase activity was abrogated by p38 MAPK inhibition. In addition, both p38-alpha MAPK and nSMase2 were implicated in the TNF-alpha-stimulated up-regulation of the adhesion proteins vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM), but this was largely independent of NF-kappaB activation. These data reveal p38 MAPK as an upstream regulator of nSMase2 and indicate a role for nSMase2 in pro-inflammatory responses induced by TNF-alpha as a regulator of adhesion proteins.     

Prognostic prediction and diagnostic role of intercellular adhesion molecule-1 (ICAM1) expression in clear cell renal cell carcinoma

The intercellular adhesion molecule-1 (ICAM1) has been reported to function in multiple malignancies, but its effect on clear cell renal cell carcinoma (ccRCC) hasn’t been discussed yet. This study aimed to identify the potential role of ICAM1 in prognostic prediction and early diagnosis of ccRCC. ICAM1 expression was inspected by immunohistochemistry and correlated with clinicopathologic variables. Association between protein expression and cancer-specific survival (CSS) of ccRCC patients was evaluated and the value of area under the receiver operating characteristics (ROC) curve (AUC) was calculated to measure the protein’s diagnostic accuracy. ICAM1 was positively immunostained in 83.2 % of 173 ccRCC tissues, but negatively immunostained in all the para-cancerous normal epitheliums of renal tubules. High ICAM1 expression was significantly related to male sex (P = 0.00241), T3/T4 stage (P = 0.02249), non-N0M0 stage (P = 0.03797) and positive renal pelvis invasion (P = 0.04227). Kaplan–Meier survival analysis illustrated that high ICAM1 expression was significantly correlated to a decreased CSS (P = 0.00006). Multivariate Cox analysis indicated that ICAM1 was an independent predictor for CSS of patients (P = 0.00451). Furthermore, the AUC value of ICAM1 in diagnosing ccRCC was 0.916 (P < 0.00001). In conclusion, high ICAM1 expression on tumor cells indicates a poor outcome of patients and ICAM1 is likely to be an independent predictor for the prognosis of ccRCC. Moreover, ICAM1 has a high AUC value and may be a potential and useful diagnostic marker.     

The effects of pentoxifylline on lipopolysaccharide (LPS) fever, plasma interleukin 6 (IL 6), and tumor necrosis factor (TNF) in the rat

The purpose of these studies was to test whether pentoxifylline, a drug that can inhibit the production and action of cytokines hypothesized to be endogenous pyrogens (for example, interleukin 1 and tumor necrosis factor [TNF]), is antipyretic. We also tested the effects of pentoxifylline on plasma activities of interleukin 6 (IL 6) and TNF in response to an injection of a fever-inducing dose of lipopolysaccharide (LPS). Our results showed that a high dose of pentoxifylline (200 mg/kg) caused hypothermia in control rats and blocked LPS fever, while a low dose (50 mg/kg) did not have these effects. Injection of the high dose of pentoxifylline in control rats caused a rise in plasma IL 6 but not in plasma TNF. However, the peak levels of plasma IL 6 and TNF activities following an injection of LPS were significantly reduced by pretreatment with pentoxifylline. Overall, the data are consistent with the hypothesis that pentoxifylline is an antipyretic drug, which may act at least in part by inhibiting the secretion of pyrogenic cytokines.     

A mitogenic factor, released by stimulated human mononuclear cells and distinct from interleukin 2 (IL 2), B cell growth factor (BCGF), and interleukin 1 (IL 1)

Two lymphocyte mitogenic factors, interleukin 2 (IL 2) and blastogenic factor (BF), are generated concomitantly in human mixed lymphocyte cultures (MLC). The latter mitogenic factor is directly mitogenic for unstimulated lymphocytes, whereas the former mitogenic factor acts only on previously activated lymphocytes. Both factors had a m.w. range, as determined by gel filtration, of 18,000 to 30,000. Thus, these two factors were inseparable on the basis of m.w. size. However, BF and IL 2 were separable during ion exchange chromatography on the DEAE cellulose and phenyl-Sepharose chromatography. In addition, BF activity in the supernatants of MLC reached a maximum after day 5, whereas IL 2 activity peaked at day 3, thus distinguishing BF from IL 2 kinetically. These results clearly indicate that BF activity is mediated by molecules distinct from IL 2. The biochemical relationship between B cell growth factor (BCGF) and BF was also examined. Because BF was readily separable from BCGF by Con A-Sepharose chromatography, BF is distinguishable from BCGF. No augmentation of PHA-stimulated C3H mouse thymocyte proliferation was associated with the preparation of partially purified BF, demonstrating that BF and IL 1 are distinct molecules. Taken together, these results indicate that BF is clearly distinct from IL 2, BCGF, and IL 1. BF-containing MLC supernatants have direct mitogenic activity on both T and B cells. Both T and B cell blastogenic activities copurified during ammonium sulfate precipitation, gel filtration, DEAE cellulose ion exchange chromatography, and hydrophobic chromatography. Thus, these two activities appear to be biochemically inseparable. Monoclonal anti-Tac, that has been suggested to recognize the receptor for human IL 2, was highly inhibitory to the T cell response to the phenyl-Sepharose preparations of BF (IL 2-free). In contrast, this antibody had minimal or no effect on BF-induced B cell proliferation. However, when MLC supernatants were absorbed with a cloned IL 2-dependent T cell line, only IL 2 activity, but not BF activity, was removed, demonstrating that BF and IL 2 have different binding specificities. The precise mechanism(s) by which anti-Tac inhibits BF-induced proliferation of T cells is unknown at present. Additionally, during the course of these experiments, we observed that Con A-Sepharose chromatography could be used as a simple one-step method of separating BCGF from IL 2.     

Tumor necrosis factor-alpha-converting enzyme (TACE/ADAM-17) mediates the ectodomain cleavage of intercellular adhesion molecule-1 (ICAM-1)

Ectodomain shedding has emerged as an important regulatory step in the function of transmembrane proteins. Intercellular adhesion molecule-1 (ICAM-1), an adhesion receptor that mediates inflammatory and immune responses, undergoes shedding in the presence of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA). The shedding of ICAM-1 in ICAM-1-transfected 293 cells upon PMA stimulation and in endothelial cells upon tumor necrosis factor-alpha stimulation was blocked by metalloproteinase inhibitors, whereas serine protease inhibitors were ineffective. p-Aminophenylmercuric acetate, a mercuric compound that is known to activate matrix metalloproteinases, up-regulated ICAM-1 shedding. TIMP-3 (but not TIMP-1 or -2) effectively blocked cleavage. This profile suggests the involvement of the ADAM family of proteases in the cleavage of ICAM-1. The introduction of enzymatically active tumor necrosis factor-alpha-converting enzyme (TACE) into ICAM-1-expressing cells up-regulated cleavage. Small interfering RNA directed against TACE blocked ICAM-1 cleavage. ICAM-1 transfected into TACE-/- fibroblasts did not show increased shedding over constitutive levels in the presence of PMA, whereas cleavage did occur in ICAM-1-transfected TACE+/+ cells. These results indicate that ICAM-1 shedding is mediated by TACE. Blocking the shedding of ICAM-1 altered the cell adhesive function, as ICAM-1-mediated cell adhesion was up-regulated in the presence of TACE small interfering RNA and TIMP-3, but not TIMP-1. However, cleavage was found to occur at multiple sites within the stalk domain of ICAM-1, and numerous point mutations within the region did not affect cleavage, indicating that TACE-mediated cleavage of ICAM-1 may not be sequence-specific.     

A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1

Homotypic adhesion by phorbol ester-stimulated lymphocytes requires LFA-1 and Mg+2 and does not involve like-like interactions between LFA-1 molecules on adjacent cells. The latter finding suggested that a second molecule, distinct from LFA-1, also participates in LFA-1-dependent adhesion. The identification of such a molecule was the object of this investigation. After immunization with LFA-1-deficient EBV-transformed lymphoblastoid cells, a MAb was obtained that inhibits phorbol ester-stimulated aggregation of LFA-1+ EBV lines. This MAb defines a novel cell surface molecule, which is designated intercellular adhesion molecule 1 (ICAM-1). ICAM-1 is distinct from LFA-1 in both cell distribution and structure. In SDS-PAGE, ICAM-1 isolated from JY cells is a single chain of Mr = 90,000. As shown by MAb inhibition, ICAM-1 participates in phorbol ester-stimulated adhesion reactions of B lymphocyte and myeloid cell lines and T lymphocyte blasts. However, aggregation of one T lymphocyte cell line (SKW-3) was inhibited by LFA-1 but not ICAM-1 MAb. It is proposed that ICAM-1 may be a ligand in many, but not all, LFA-1-dependent adhesion reactions.     

Tumor necrosis factor and interleukin 1 activate phospholipase in rat chondrocytes

Tumor necrosis factor (TNF) and interleukin 1 (IL-1) are both cytokines of macrophage origin with similar activity on several cell types. We investigated whether TNF can, analogously to IL-1, stimulate phospholipase activity of chondrocytes. Addition of each of these cytokines to cells, isolated from the xiphisternum of adult rats, resulted in a time- and dose-dependent increase in phospholipase activity in both secreted and membrane-associated form. Moreover, TNF and IL-1 both induce a transformation of chondrocyte morphology. In conclusion, TNF stimulates chondrocyte phospholipase activity and extends the long list of actions shared by IL-1 and TNF in a diversity of cellular systems.     

Tumor necrosis factor (TNF) receptor type 2 is an important mediator of TNF alpha function in the mouse ovary

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.