Tumor Necrosis Factor Receptor I Research Articles

Unmasking of a protective tumor necrosis factor receptor I–mediated signal in the collagen‐induced arthritis model

To examine the relative importance of tumor necrosis factor receptor I (TNFRI) signaling in the hematopoietic tissue compartment in the progression of collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). DBA/1 mice were administered a lethal radiation dose and were then rescued with bone marrow derived from either DBA/1 or TNFRI(-/-) mice. CIA was then induced, and disease progression was characterized. Surprisingly, mice with CIA that received TNFRI(-/-) donor marrow developed increased disease severity as compared with control mice with CIA. This could not be attributed to an increased primary response to collagen or to the contribution of a non-DBA genetic background. In mice that received TNFRI(-/-) bone marrow, histologic markers of advanced disease were evident shortly after initiation of the immune response to collagen and long before clinical evidence of disease. Serum TNFalpha was undetectable, whereas serum interleukin-12 p40 levels were increased, at the end point of the study in mice that received TNFRI(-/-) bone marrow. These data raise the intriguing possibility of the existence of an antiinflammatory, TNFRI-mediated circuit in the hematopoietic compartment. This circuit bears a resemblance to the switch in TNFalpha function that has been observed during the resolution of bacterial infections. These data suggest that TNFRI-mediated signals in the radioresistant tissues contribute to disease progression, whereas TNFRI-mediated signals in the radiosensitive tissues can contribute to protection from disease. We thus put forward the hypothesis that the degree of response to TNFalpha blockade in RA is dependent in part on the relative genetic strengths of these 2 pathways.

Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro‐inflammatory and anti‐inflammatory agents: key role for TNF‐α

Apoptosis of inflammatory cells and their subsequent clearance (efferocytosis) by macrophages (Mphis) are key mechanisms orchestrating successful resolution of inflammation. Although the powerful proinflammatory agents lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha) influence rates of inflammatory cell apoptosis, little is known about their effects on efferocytosis. We have demonstrated that LPS and TNF-alpha potently inhibit efferocytosis of neutrophils by monocyte-derived Mphis. Inhibition was both concentration and time dependent, although the effect of TNF-alpha was more rapid. We have found that soluble TNF receptor-I attenuated LPS inhibition of phagocytosis, indicating that TNF-alpha production is critical. Inhibition of efferocytosis by LPS was found to be positively associated with Mphi production of TNF-alpha, but negatively with interleukin-10 (IL-10) release. A critical role for IL-10 in the regulation of phagocytosis was suggested by 2 important findings: LPS inhibition was observed more rapidly in the presence of an anti-human IL-10 receptor-alpha antibody, and efferocytosis by IL-10-deficient Mphis was markedly reduced compared to wild-type Mphis. Furthermore, exogenous IL-10 and glucocorticoids reversed inhibitory effects of LPS on efferocytosis via suppression of TNF-alpha production. We suggest that efferocytosis is regulated in an autocrine manner by pro- and anti-inflammatory mediators, and the inflammatory milieu determines whether inflammation successfully resolves.

TAK1 prevents TRAIL‐induced cell death through an NF‐κB‐independent mechanism

TAK1 functions downstream of several cytokines such as TNF, and activates both MAPK and NF‐κB pathways. We have previously determined that TAK1 is essential for preventing TNF‐induced cell death in epithelial tissues such as skin epidermis and intestinal epithelium. Deletion of TNF receptor I (TNFRI) largely rescued cell death caused by TAK1 deletion; however, dead cells in double (TNFRI and TAK1)‐deficient epithelial tissues were still increased compared to TNFRI knockout tissues. Here, we attempted to determine the TNF‐independent mechanism of cell death in TAK1‐deficient cells. TNF‐Related Apoptosis‐Inducing Ligand (TRAIL) belongs to TNF family, and is also known to induce cell death. We found that TAK1 deletion increased sensitivity to TRAIL‐killing. Although NF‐κB is one of the most important cell survival factors, we found that TRAIL did not induce NF‐κB activation even in TAK1 wild type cells, and that NEMO/IKKγ deletion did not increase sensitivity to TRAIL. In contrast, we found that expressions of several antioxidant genes were reduced in TAK1‐deficient cells, and that treatment of exogenous antioxidant butylated hydroxyanisole (BHA) rescued TRAIL‐killing. These results suggest that TAK1 regulates antioxidant gene expression and contributes to TRAIL‐resistance independently of NF‐κB activity. This work was supported by grants from Crohn's and Colitis Foundation of America and NIH (GM68812).

Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases

Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic–spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial–gut axis in human disease.

Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases

Tumor necrosis factor (TNF) is key to the pathogenesis of various arthritic diseases and inflammatory bowel disease (IBD). Anti-TNF therapies have proved successful in the clinical treatment of these diseases, but a mechanistic understanding of TNF function is still lacking. We have investigated early cellular mechanisms of TNF function in these diseases using an established TNF transgenic model, which develops a spondyloarthritis-like disease characterized by peripheral joint arthritis, sacroiliitis, enthesitis, and Crohn's-like IBD. Bone marrow grafting experiments demonstrated that development of arthritis requires TNF receptor I (TNFRI) expression in the radiation-resistant compartment, which is also known to be a sufficient target of TNF in the development of Crohn's-like IBD in the same model. Early activation of synovial fibroblasts and intestinal myofibroblasts could also be demonstrated by perturbed expression of matrix metalloproteases and their inhibitors. Notably, selective Cre/loxP-mediated TNFRI expression in mesenchymal cells resulted in a fully arthritic–spondyloarthritic and intestinal phenotype, indicating that mesenchymal cells are primary and sufficient targets of TNF in these pathologies. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate early common cellular pathways that may also explain the often observed synovial–gut axis in human disease.

Changes of cytokines in cirrhosis patients with advanced hepatocellular carcinoma treated by intra-arterial chemotherapy

Tumor necrosis factor (TNF) induces cancer cell-specific apoptosis by binding to a TNF-related apoptosis-inducing ligand. Binding of the Fas ligand on cytotoxic T lymphocytes to the Fas receptor on hepatocytes is also known to induce apoptosis. The aim of this study was to clarify changes of cytokines in patients with liver cirrhosis (LC) and advanced hepatocellular carcinoma (aHCC) receiving intra-arterial combination chemotherapy. Twenty-one adult Japanese LC patients with aHCC received intra-arterial combination chemotherapy. The serum levels of TNF-alpha, soluble TNF receptor-I (sTNFr-I), soluble Fas ligand (sFas L), and soluble Fas (sFas) were evaluated. Thirteen of the 21 patients (group R) showed an objective response, while the other eight patients (group N) showed no response. The serum level of TNF-alpha was lower after chemotherapy than before chemotherapy in group N, but there was no difference of serum sTNFr-I levels between before and after chemotherapy and there were also no differences between the two groups. The serum sFas levels were higher after chemotherapy than before chemotherapy in group N, while there was no difference among groups. These results indicate that a high serum TNF-alpha level and a low serum sFas level might be important for successful combined arterial chemotherapy in LC patients with aHCC.

Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin

Solamargine (SM), a major steroidal alkaloid glycoside, was purified from Solanum incanum plant. SM exhibited the most cytotoxic effect comparing with that of cisplatin (cDDP), methotrexate (MTX), 5-fluorouracil (5-FU), epirubicin (EPI) and cyclophosphamide (CP) against human breast cancer cells. In this study, SM induces apoptosis of the breast cancer cells and the mechanism was characterized. SM up-regulated the expressions of external death receptors, such as tumor necrosis factor receptor I (TNFR-I), Fas receptor (Fas), TNFR-I-associated death domain (TRADD), and Fas-associated death domain (FADD). SM also enhanced the intrinsic ratio of Bax to Bcl-2 by up-regulating Bax and down-regulating Bcl-2 and Bcl-xL expressions. These effects resulted in the release of mitochondrial cytochrome c and activation of caspase-8, -9 and -3 in the cells, indicating that SM triggered extrinsic and intrinsic apoptotic pathways of breast cancer cells. Similar to function way of SM, cDDP causes cancer cell apoptosis though caspase-8/caspase-3 and Bax/cytochrome c pathways, but the resistance to cDDP is correlated with Bcl-2 and Bcl-xL overexpression. However, the overexpression of Bcl-2 and Bcl-xL can be broken through by SM. The combined treatment of SM and cDDP significantly reduced Bcl-2 and Bcl-xL expressions, and enhanced Bax, cytochrome c, caspase-9 and -3 expressions in breast cancer cells. Thus, the combined use of SM and cDDP may be effective in cDDP-resistant breast cancer.

Progressive Biliary Destruction Is Independent of a Functional Tumor Necrosis Factor-α Pathway in A Rhesus Rotavirus-Induced Murine Model of Biliary Atresia

Rhesus rotavirus (RRV)-inoculated neonatal BALB/c mice develop an immune-mediated inflammation of extra- and intrahepatic bile ducts that progresses to biliary obstruction and death by 3 wk of age. Livers of diseased animals demonstrate increased numbers of T lymphocytes with elevated expression of helper T cell type 1 (Th1) cytokines at 1 wk, which transitions to increased numbers of macrophages and high expression of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha by 2 wk. We employed both pharmacologic and genetic approaches for attenuation of TNF-alpha to determine whether it plays a causal role in injury. First, RRV-inoculated BALB/c mice were subjected to multiple treatments with either the TNF receptor I (TNF-RI)-Fc fusion protein etanercept or neutralizing antibodies to mouse TNF-alpha. Also, TNF-RI(-/-) mice were injected with RRV in the same manner as wild-type mice. In all cases, TNF inhibition did not reduce the severity or incidence of disease. Survival curves of mice given blocking agents were similar to those of control RRV-inoculated mice, and survival of challenged TNF-RI(-/-) mice was worse than that of wild-type mice, likely because of the prolonged presence of infectious RRV. In all experimental groups, markers of disease were unchanged from those of control mice. In summary, although RRV-inoculated BALB/c mice have highly elevated expression of TNF-alpha, this cytokine does not play an obligate role in disease progression.

Excreted urinary mediators in an animal model of experimental immune nephritis with potential pathogenic significance

Currently, proteinuria is viewed as the earliest indicator of renal disease in immune-mediated nephritis. The objective of this study was to determine whether additional mediators may be excreted in the urine during immune-mediated nephritis, using an experimental model with a well-defined disease course. Urine samples from mice with anti-glomerular basement membrane (anti-GBM) antibody-induced experimental nephritis were screened using a focused immunoproteome array bearing 62 cytokines/chemokines/soluble receptors. Molecules identified through this screening assay were validated using an enzyme-linked immunosorbent assay. One of these molecules was further evaluated for its pathogenic role in disease, using antibody-blocking studies. Compared with B6 and BALB/c mice, in which moderately severe immune-mediated nephritis develops, the highly nephritis-susceptible 129/Sv and DBA/1 mice exhibited significantly increased urinary levels of vascular cell adhesion molecule 1 (VCAM-1), P-selectin, tumor necrosis factor receptor I (TNFRI), and CXCL16, particularly at the peak of disease. Whereas some of the mediators appeared to be serum derived early in the disease course, local production in the kidneys appeared to be an important source of these mediators later in the course of disease. Both intrinsic renal cells and infiltrating leukocytes appeared to be capable of producing these mediators. Finally, antibody-mediated blocking of CXCL16 ameliorated experimental immune nephritis. These studies identified VCAM-1, P-selectin, TNFRI, and CXCL16 as a quartet of molecules that have potential pathogenic significance; the levels of these molecules are significantly elevated during experimental immune nephritis. The relevance of these molecules in spontaneous immune nephritis warrants investigation.

Execution of Macrophage Apoptosis by PE_PGRS33 of Mycobacterium tuberculosis Is Mediated by Toll-like Receptor 2-dependent Release of Tumor Necrosis Factor-α

Combating tuberculosis requires a detailed understanding of how mycobacterial effectors modulate the host immune response. The role of the multigene PE family of proteins unique to mycobacteria in the pathogenesis of tuberculosis is still poorly understood, although certain PE_PGRS genes have been linked to virulence. Tumor necrosis factor-alpha (TNF-alpha) is essential for successfully combating tuberculosis. In this study we provide evidence that PE_PGRS33, a surface exposed protein, elicits TNF-alpha release from macrophages in a TLR2 (Toll-like receptor 2)-dependent manner. ASK1 (apoptosis signal-regulating kinase 1) is activated downstream of TLR2. ASK1 activates the MAPKs p38 and JNK. PE_PGRS33-induced signaling leads to enhanced expression of TNF-alpha and TNF receptor I (TNFRI) genes. Mycobacterium smegmatis expressing PE_ PGRS33 elicits the same effects as purified PE_PGRS33. TNF-alpha release occurs even when internalization of the bacteria is blocked by cytochalasin D, suggesting that interaction of PE_ PGRS33 with TLR2 is sufficient to trigger the effects described. Release of TNF-alpha plays the determining role in triggering apoptosis in macrophages challenged with PE_PGRS33. The death receptor-dependent signals are amplified through classical caspase 8-dependent mitochondrial release of cytochrome c, leading to the activation of caspases 9 and 3. An important aspect of our findings is that deletions within the PGRS domain (simulating those occurring in clinical strains) attenuate the TNF-alpha-inducing ability of PE_PGRS33. These results provide the first evidence that variations in the polymorphic repeats of the PGRS domain modulate the innate immune response.

Transglutaminase Participates in UVB-Induced Cell Death Pathways in Human Corneal Epithelial Cells

Ultraviolet light (UVB) is known to cause apoptosis in human corneal epithelial cells. This study evaluates the role of transglutaminase in regulating tumor necrosis factor (TNF) receptor clustering as well as caspase activation in UVB-induced apoptosis in human corneal epithelial cells. A human corneal epithelial cell line was used. A single dose of UVB (20 mJ/cm2) was used as a stimulus. Cell viability and cell death were investigated by MTT, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL), and caspase-3 assays. Immunofluorescent staining was used to investigate TNF receptor-I clustering at various time intervals after UVB. Short interfering RNA was used to knock down transglutaminase-2 expression. Fluorescein-cadaverine uptake was used to assess transglutaminase activity. A noncovalent peptide delivery system was used to transfect guinea pig liver transglutaminase into corneal epithelial cells. UVB increased transglutaminase activity, reduced cell viability, and increased TUNEL staining. UVB or TNF-alpha promoted TNF-receptor-I clustering, a process inhibited by the transglutaminase inhibitor, mono-dansyl cadaverine. UVB also increased activated caspase-3, in a manner suppressible by mono-dansyl cadaverine. Intracellular delivery of exogenous transglutaminase markedly increase caspase-3 activation compared with the vehicle control. Transglutaminase enzymatic activity is involved in corneal epithelial cell death after UVB and appears to participate in two steps regulating this process, clustering of TNF receptor-I and caspase-3 activation.

Metallophilic macrophages are lacking in the thymus of lymphotoxin-β receptor-deficient mice

Lymphotoxin-beta receptor (LTbetaR) axis plays a crucial role in development and compartmentalization of peripheral lymphatic organs. But, it is also required for the appropriate function and maintenance of structural integrity of the thymus: in LTbetaR-deficient animals the clonal deletion of autoreactive lymphocytes is impaired and differentiation of thymic medullary epithelial cells is disturbed. In this study, using several markers, we showed that thymic metallophilic macrophages were lacking in LTbetaR-deficient mice. In tumor necrosis factor receptor-I (p55)-deficient mice (which we used as positive control) thymic metallophilic cells were located, similarly as in normal mice, in the thymic cortico-medullary zone at the junction of cortex and medulla. These findings show that LTbetaR is necessary for maintenance of metallophilic macrophages in the thymus and provide further evidence that these cells may represent a factor involved in thymic negative selection.

Application of RNAi inducible TNFRI knockdown cells to the analysis of TNFα-induced cytotoxicity

RNA interference (RNAi) has become a popular tool for downregulating in many species including mammalian cells. Therefore, suppression of target genes in mammalian cultured cells using RNAi may represent an ideal alternative to knockout studies for understanding the molecular mechanisms of chemical toxicity. Here, we assessed the potential of RNAi mediated gene knockdown in HeLa and HepG2 cells to cytotoxicity studies. Tumor necrosis factor receptor I (TNFRI) was chosen as a target gene because its signaling has been implicated in xenobiotic-induced toxicity. We optimized the design and performance of a vector-based RNAi experiment and then investigated viability of both HeLa and HepG2 cells exposed to TNFα. In addition, we examined gene expression profile of TNFRI knockdown HeLa cells after TNFα treatment, and then protein expression levels for several apoptosis-related genes of the cells. In both HeLa and HepG2 cells, TNFα exposure resulted in significantly reduced susceptibility of the knockdown cells to the cytotoxicity as compared with those of mock-transfected cells. Furthermore, the gene expression profiling and western blotting revealed that several genes including apoptosis and/or NF-κB pathway were downregulated in the knockdown HeLa cells. These results suggest that downregulation of the TNFRI gene in both HeLa and HepG2 cells by RNAi participates in resistance to TNFα-induced cytotoxicity. Therefore, this study raises the possibility that RNAi-based gene silencing in mammalian cells may be a valuable tool for elucidating the relationships between phenotypic changes and target gene functions in response to xenobiotic-induced cytotoxicity. Further exposure study using xenobiotics needs to be done to validate the potential utility of RNAi technology.

Neuroendocrine Modulation Induced by Selective Blockade of TNF‐α in Rheumatoid Arthritis

Tumor necrosis factor-alpha (TNFalpha) is a main actor in the pathogenesis of rheumatoid arthritis (RA), interacting with other molecules in complex mechanisms. The neuroendocrine system is known to be involved and Chromogranin A (CHGA) serum levels are elevated in patients with RA. We evaluated the effect of the selective blockade of TNF-alpha, induced by treatment with anti-TNF-alpha monoclonal antibodies (mAbs), on the serum levels of CHGA and on its correlation with TNF-alpha and TNF-alpha receptors (TNFRs) serum levels. Seven patients with RA have been treated with the anti-TNF-alpha mAb, infliximab. We measured the serum levels of TNF-alpha, its receptors (tumor necrosis factor receptor-I [TNFR-I] and tumor necrosis factor receptor-II [TNFR-II]), and CHGA before and during the treatment. We also measured, as a control, the serum levels of CHGA, TNF-alpha, and soluble TNFRs in 14 patients who were being treated with infliximab, adalimumab, or etanercept and in 20 matching negative controls. The serum levels of TNFR-I and TNFR-II, which are a sensitive marker for the TNF-alpha pathway, correlated with those of CHGA before treatment (Pearson's coefficient, respectively, 0.59 and 0.53). Treatment with anti-TNF-alpha mAb provided a significant clinical response in all patients and the correlation between CHGA and TNFR-I and TNFR-II was no more evident during treatment (respectively, -0.09 and -0.07). TNF-alpha blockade allows a clinical effect in patients with RA and modifies the correlation between CHGA and TNFRs, suggesting that TNF-alpha and CHGA reciprocally interfere in the pathogenesis of RA, through intermediate adaptors, whose identification warrants further studies.

TNF-receptor I defective in internalization allows for cell death through activation of neutral sphingomyelinase

The cytoplasmic tail of the tumor necrosis factor receptor I (TNF-RI) contains several functionally distinct domains involved in apoptotic signaling. Mutants of TNF-RI carrying deletions of the death domain (DD), internalization domain (TRID), and neutral sphingomyelinase domain (NSD), respectively, retransfected in cells devoid of TNF-RI and TNF-RII, constituted distinct tools to evaluate the specific role of each domain in downstream apoptotic signaling events. Deletion of DD abolishes activation of caspase-3 and -9 and apoptosis following treatment with TNF because of blocked assembly of the DISC. Nevertheless, TNF-RI ΔTRID, though lacking a DISC, still allows for residual activation of caspase-3 followed by cell death, although caspase-9 activation was not detected. This activity of caspase-3 is probably due to activation of neutral sphingomyelinase (N-SMase). Increased activity of this enzyme was detected in cells expressing TNF-RI ΔTRID following treatment with TNF, but not in any other cell line investigated. N-SMase is activated by factor associated with N-SMase (FAN). Because TNF-RI ΔTRID is retained at the cell surface, FAN may interact with the mutated receptor for a prolonged amount of time, thereby overactivating N-SMase. Double deletion of TRID and NSD abolished caspase-3 activation and apoptosis, confirming this hypothesis.

Proinflammatory cytokines and cardiac abnormalities in uncomplicated obesity: Relationship with abdominal fat deposition

Background and aim Obesity can be considered a state of chronic, low-grade inflammation. Particularly, visceral adipose tissue (VAT) seems to be an active compartment in pro-inflammatory molecule secretion. The possible existence of a correlation between circulating cytokines, their soluble receptors, abdominal fat accumulation and echocardiographic abnormalities in uncomplicated obesity was investigated. Methods and results Echocardiographic parameters, C-reactive protein (CRP), interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6-R), tumor necrosis factor-α (TNF-α) and soluble TNF receptor I (TNFR-I) were assessed in 27 normotensive obese women (age 33.3 ± 8.3 years; BMI 43.5 ± 4.8 kg/m 2) and 15 normal-weight controls (age 36.8 ± 8.2 years; BMI 22.6 ± 1.7 kg/m 2). VAT was assessed by CT. The obese patients had higher serum IL-6 ( p < 0.01), sIL-6-R ( p < 0.0001), sIL-6-R/IL-6 complex ( p < 0.05), TNF-α ( p < 0.02), sTNF-α-RI ( p < 0.03) and CRP ( p < 0.0001) levels than normal women. Moreover, end-diastolic septum thickness (SW), end-diastolic posterior wall thickness (PW), absolute and indexed left ventricular mass, deceleration time (DT), myocardial performance index (MPI) and isovolumetric relaxation time (IVRT) were correlated with sIL-6-R, sIL-6-R/IL-6 complex and CRP levels. Interestingly, sIL-6-R, sIL-6-R/IL-6 complex, CRP, SW, PW, DT and MPI were higher in patients with a VAT area >130 cm 2 than those with <130 cm 2. Conclusion In normotensive obese women several pro-inflammatory molecules correlate with both echocardiographic abnormalities and the amount of intra-abdominal fat; these results may support a role for visceral fat in predisposing to cardiac dysfunction, possibly through a low-grade state of inflammation.

Synergistic Effect of Platelet-Activating Factor and Tumor Necrosis Factor-α on Corneal Myofibroblast Apoptosis

Elimination of myofibroblasts after repair of corneal injury is essential for the maintenance of corneal transparency. In the current study, the role of platelet-activating factor (PAF) in combination with tumor necrosis factor (TNF)-alpha in corneal myofibroblast apoptosis was explored. Porcine corneal myofibroblasts (PCMs) were obtained from subcultured fibroblasts plated at a low density (5 cells/mm2). Mouse anti-alpha-smooth muscle actin antibody was used to identify the cell phenotype. Immunofluorescence was performed to localize PAF and TNF-alpha receptors in those cells. The reactivity of the antibodies was characterized by Western blot analysis. To induce myofibroblast apoptosis, PCMs were treated for 24 to 72 hours with methylcarbamyl-PAF (cPAF, 300 nM), a nonhydrolyzable PAF analogue, TNF-alpha (20 ng/mL), and TNF-alpha+cPAF, with or without LAU-0901 (150 nM), a novel PAF antagonist. Apoptosis was assayed by Hoechst 33258 and TUNEL staining and DNA laddering. 6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Images were recorded by fluorescence microscope. Immunofluorescence with a PAF-receptor (N terminus) polyclonal antibody showed that the receptor was expressed in both plasma and nuclear membranes of myofibroblasts. TNF-alpha receptor II (TNF-RII) was localized in the cytoplasm, whereas TNF-receptor I (TNF-RI) was found in both cytoplasm and plasma membrane. Treatment with TNF-alpha for 24, 48, and 72 hours induced apoptosis in 18%, 24%, and 32%, respectively, of the myofibroblasts. Western blot analysis showed expression of single bands corresponding to the molecular weights of the receptors. Treatment with cPAF induced apoptosis in 10%, 18%, and 26% of the cells, respectively. However, treatment with both cytokines induced apoptosis in 42%, 78%, and 86%, respectively, of the cells, demonstrating a synergistic action between PAF and TNF-alpha. Blocking the PAF receptor with LAU-0901 inhibited the synergistic effect induced by PAF. Corneal myofibroblasts express a PAF receptor in the nuclear membrane, and they also express TNF-RI and RII. The synergistic effect on myofibroblast apoptosis by PAF and TNF-alpha suggests that during corneal stromal wound healing, PAF acting in conjunction with other cytokines could play an important role in eliminating these cells.

Interluekin-6, Interleukin-8, and Soluble Tumor Necrosis Factor Receptor-I in the Cord Blood as Predictors of Chronic Lung Disease in Premature Infants

Interluekin-6, Interleukin-8, and Soluble Tumor Necrosis Factor Receptor-I in the Cord Blood as Predictors of Chronic Lung Disease in Premature Infants

The MC160 Protein Expressed by the Dermatotropic Poxvirus Molluscum Contagiosum Virus Prevents Tumor Necrosis Factor Alpha-Induced NF-κB Activation via Inhibition of I Kappa Kinase Complex Formation

The pluripotent cytokine tumor necrosis factor alpha (TNF-alpha) binds to its cognate TNF receptor I (TNF-RI) to stimulate inflammation via activation of the NF-kappaB transcription factor. To prevent the detrimental effects of TNF-alpha in keratinocytes infected with the molluscum contagiosum virus (MCV), this poxvirus is expected to produce proteins that block at least one step of the TNF-RI signal transduction pathway. One such product, the MC160 protein, is predicted to interfere with this cellular response because of its homology to other proteins that regulate TNF-RI-mediated signaling. We report here that expression of MC160 molecules did significantly reduce TNF-alpha-mediated NF-kappaB activation in 293T cells, as measured by gene reporter and gel mobility shift assays. Since we observed that MC160 decreased other NF-kappaB activation pathways, namely those activated by receptor-interacting protein, TNF receptor-associated factor 2, NF-kappaB-inducing kinase, or MyD88, we hypothesized that the MC160 product interfered with I kappa kinase (IKK) activation, an event common to multiple signal transduction pathways. Indeed, MC160 protein expression was associated with a reduction in in vitro IKK kinase activity and IKK subunit phosphorylation. Further, IKK1-IKK2 interactions were not detected in MC160-expressing cells, under conditions demonstrated to induce IKK complex formation, but interactions between the MC160 protein and the major IKK subunits were undetectable. Surprisingly, MC160 expression correlated with a decrease in IKK1, but not IKK2 levels, suggesting a mechanism for MC160 disruption of IKK1-IKK2 interactions. MCV has probably retained its MC160 gene to inhibit NF-kappaB activation by interfering with signaling via multiple biological mediators. In the context of an MCV infection in vivo, MC160 protein expression may dampen the cellular production of proinflammatory molecules and enhance persistent infections in host keratinocytes.

Modulation of CD28 expression with anti–tumor necrosis factor α therapy in rheumatoid arthritis

The immune system of patients with rheumatoid arthritis (RA) is characterized by the accumulation of CD4+ T cells deficient in CD28 expression and the up-regulation of tumor necrosis factor alpha (TNFalpha). Previous in vitro studies have shown that TNFalpha induces transcriptional silencing of the CD28 gene. Because reduced expression of CD28 in T cells compromises immunocompetence, we examined whether CD28 expression is reduced in patients with RA in vivo and whether the reduction is related to TNFalpha. Patients with RA and age-matched individuals were recruited. Peripheral blood mononuclear cells were stained for CD3, CD4, CD8, CD28, TNF receptor I (TNFRI), and TNFRII, and analyzed by quantitative flow cytometry. The number of CD28 and TNFR molecules was monitored in a subgroup of patients with RA undergoing treatment with anti-TNFalpha. In addition to higher frequencies of CD28null T cells, patients with RA had significantly reduced numbers of CD28 and TNFRI molecules on CD4+,CD28+ T cells. Normal expression could be restored in vitro by overnight culture, suggesting that CD28 in patients was modulated by exogenous factors. In contrast, treatment with TNFalpha in vitro resulted in further down-regulation. CD28 expression was normalized in patients undergoing TNFalpha-neutralizing therapy. Overproduction of TNFalpha in RA induces a global down-regulation of CD28 in CD4+ T cells and may cause reduced sensitivity to costimulatory signals in T cell responses.