Tumor Necrosis Factor Binding Research Articles

Apoptosis mediated by the TNF-related cytokine and receptor families.

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin beta-receptor (LT beta R), among others. The MHC-encoded cytokines, TNF and LT alpha, form homomeric trimers, whereas LT beta assembles into heterotrimers with LT alpha, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LT beta R are involved in induction NF kappa B and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the "death domain" homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways.

TNF Binding Protein Prevents Hyperlactatemia and Inactivation of PDH Complex in Skeletal Muscle during Sepsis

Background.Hyperlactatemia is a metabolic complication of hypermetabolic, hyperdynamic sepsis. An important mechanism responsible for elevating plasma lactate concentrations in sepsis is altered regulation of the pyruvate dehydrogenase complex (PDH) in skeletal muscle. We investigated the ability of a specific tumor necrosis factor binding protein, TNFbp, to modulate lactate concentrations and skeletal muscle PDH activity in a rodent model of chronic abdominal sepsis.Materials and methods.We examined the regulation of lactate metabolism in four groups of animals: Control, Control + TNFbp, Septic, and Septic + TNFbp. Chronic (5 days) sepsis was induced by the creation of a stable intraabdominal abscess using a sterile fecal-agar pellet inoculated withE. coliplusB. Fragilisas the foreign body nidus. TNFbp (1 mg/kg/day) was injected subcutaneously daily.Results.Sepsis increased plasma and skeletal muscle lactate concentrations 2-fold compared with control. In septic rats treated with TNFbp, plasma and skeletal muscle lactate concentrations were significantly decreased compared with untreated septic rats. In skeletal muscle, sepsis resulted in a 70% decrease in the proportion of the PDH in the active form compared with controls. The sepsis-induced inhibition in the PDH complex activity was prevented by TNFbp. PDH kinase was enhanced 1.8-fold in sepsis, and the increase in PDH kinase activity was prevented by treatment with TNFbp. TNFbp treatment did not have any effects on plasma lactate or the proportion of active skeletal muscle PDH activity in control animals.Conclusions.TNFbp prevents the sepsis-induced hyperlactatemia and derangements in skeletal muscle lactate concentrations and PDH activity. These observations suggest that TNF is an important mediator responsible for lactate dyshomeostasis during sepsis.

Tumour necrosis factor is trafficked to a mitochondrial tumour necrosis factor binding protein.

Tumour necrosis factor is trafficked to a mitochondrial tumour necrosis factor binding protein.

The effect of tumor necrosis factor binding protein and interleukin-1 receptor antagonist on the development of abdominal aortic aneurysms in a rat model

Purpose: Tumor necrosis factor (TNF), interleukin 1 (IL-1), and matrix metalloproteases have been noted to be elevated in human abdominal aortic aneurysms (AAAs) as compared with normal and occlusive aortic disease. Because TNF and IL-1 have been shown to cause release of proteases that weaken the aortic matrix, it has been suggested that these cytokines may play a central role in the aortic dilatation process. To substantiate this hypothesis, we investigated the effects of TNF and IL-1 antagonists, tumor necrosis factor binding protein (TNF-BP) and interleukin-1 receptor antagonist (IL-1RA), on the development of AAAs in a well-described rat model. Methods: Isolated segments of infrarenal aorta of 16 rats were perfused with porcine elastase. In the treated group, eight rats were given intravenous TNF-BP prior to elastase perfusion, at 48 hours and at 96 hours. In the control group, eight rats were given only intravenous vehicle at the same time intervals. Isolated segments of infrarenal aorta of an additional 16 rats were perfused with porcine elastase in a similar fashion. In the treated group, eight rats were given intraperitoneal IL-1RA prior to celiotomy and every eight hours. In the control group, eight rats were given only intraperitoneal vehicle at the same time intervals. On the sixth postoperative day, all rats underwent celiotomy and measurement of the infrarenal aortic diameter with a micrometer while the animal was alive. Aortic specimens were collected on day six for hematoxylin and eosin staining, trichrome staining, and gel polyacrylamide gel electophoresis (PAGE) zymography. Results: TNF-BP was completely able to block post elastase dilation, whereas IL-1Ra seemed to have no effect. Hematoxylin and eosin staining and trichrome staining revealed that animals treated with TNF-BP had less of an inflammatory response and preservation of the elastin and smooth muscles in the media of the aortic wall as compared with animals treated with IL-1RA or vehicle. Zymography was not able to detect significant protease activity in the aortic wall of any of the rats at six days. Conclusion: TNF-BP, but not IL-1RA, may inhibit the development of AAAs in this model. (J Vasc Surg 1998;28:522-6.)

DIFFERENT ROLES OF TUMOUR NECROSIS FACTOR α AND INTERLEUKIN 1 IN MURINE STREPTOCOCCAL CELL WALL ARTHRITIS

In this study two different aspects of tumour necrosis factor α (TNF-α) and interleukin 1 (IL-1) in locally induced murine streptococcal cell wall arthritis (SCW) were investigated. First, the kinetics and interdependence of TNF-α and IL-1 release; and second; their involvement in inflammation and cartilage destruction. Kinetic studies showed that the TNF-α peak level preceded the IL-1 peak level. However, in vivo neutralization of TNF-α did not result in decreased IL-1 bioactivity or immunoreactivity, suggesting that there is no dominant TNF-α-dependent IL-1 release in this model. Inflammation was studied by measuring knee joint swelling and inflammatory cell influx. Impact on cartilage was studied by measuring chondrocyte proteoglycan synthesis and cartilage proteoglycan depletion. The role of TNF-α in these phenomena was investigated using anti-TNF-α antibodies and tumour necrosis factor binding protein (TNFbp). Similarly, the role of IL-1 was studied using anti-IL-1 antibodies or IL-1 receptor antagonist (IL-1Ra). Anti-TNF-α treatment significantly reduced joint swelling, whereas this effect was not found by using anti-IL-1 or IL-1Ra. In contrast, neutralization of IL-1, but not TNF-α, resulted in a significant decrease of chondrocyte proteoglycan synthesis inhibition. Moreover, histology revealed that anti-IL-1 treatment reduced cartilage proteoglycan depletion and inflammatory cell influx. Combined anti-TNF-α/anti-IL-1 treatment significantly suppressed both inflammation and cartilage damage. However, the impact on these separate parameters did not exceed the effects of either anti-TNF-α or anti-TNF-1. It can be concluded that both TNF-α and IL-1 exert specific activities in SCW arthritis. The involvement of TNF-α in this model is limited to joint swelling, whereas IL-1 plays a dominant role in cartilage destruction and inflammatory cell influx.

Resistance to etoposide-induced apoptosis in a Burkitt's lymphoma cell line.

Burkitt's lymphoma cells that vary in their phenotypic characteristics show significantly different degrees of susceptibility to radiation-induced apoptosis. Propensity to undergo apoptosis is reflected in the degradation of substrates such as DNA-dependent protein kinase but the status of bcl-2, c-myc and p53 has been uninformative. In this study, we have focused on 2 Epstein-Barr virus (EBV)-associated Burkitt's cell lines, one (WW2) susceptible and the other (BL29) resistant to etoposide-induced apoptosis. Differences in expression of BHRF1, an EBV gene that is homologous to the Bcl-2 proto-oncogene and known to inhibit apoptosis, or changes in apoptosis inhibitory proteins (IAPs), did not appear to account for the difference in susceptibility in the 2 cell lines. Cytoplasmic extracts from etoposide-treated WW2 cells caused apoptotic changes in nuclei isolated from either BL29 or WW2 cells, whereas extracts from BL29 cells failed to do so. In addition, extracts from etoposide-treated WW2 cells degraded the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an important indicator of apoptosis, but this protein was resistant to degradation by BL29 extracts. It appears likely that caspase 3 (CPP32) is involved in this degradation since it was activated only in the apoptosis susceptible cells and the pattern of cleavage of DNA-PKcs was similar to that reported previously with recombinant caspase 3. As observed previously, addition of caspase 3 to nuclei failed to induce morphological changes indicative of apoptosis, but addition of caspase 3 to nuclei in the presence of extract from the resistant cells led to apoptotic changes. We conclude that resistance to apoptosis in BL29 cells is due to a failure of etoposide to activate upstream effectors of caspase activity.

Hydrogen peroxide induces tumor necrosis factor α–mediated cardiac injury by a P38 mitogen-activated protein kinase–dependent mechanism

Background: Oxidant stress caused by ischemia or endotoxemia induces myocardial dysfunction and cardiomyocyte death; however, mechanisms responsible remain unknown. We hypothesized that hydrogen peroxide (H 2O 2) induces myocardial dysfunction and cardiomyocyte death via P38 mitogen-activated protein kinase (MAPK)–mediated myocardial tumor necrosis factor (TNF) production. Methods: Langendorff perfused rat hearts (6/group) were subjected to oxidant stress (H 2O 2 infusion; 300 mmol/L × 80 minutes), with and without prior infusion of a specific P38 kinase MAPK inhibitor (P38i = 1 mmol/L/min × 5 minutes) or TNF neutralization (20 mg TNF binding protein (BP)/min × 80 minutes). Developed pressure (DP), coronary flow, and end-diastolic pressure were continuously recorded. Myocardial creatine kinase (CK) loss was measured in the coronary effluent, and tissue TNF was measured in myocardial homogenates. Results: Eighty minutes of H 2O 2 infusion induced a 6.5-fold increase in myocardial TNF production, which was associated with a 70% decrease in DP and increase in CK loss. P38 MAPK inhibition or TNF-BP decreased myocardial TNF production, cardiomyocyte death, and myocardial dysfunction. Conclusions: These results demonstrate that H 2O 2 alone induces myocardial TNF production. P38 MAPK is an oxidant-sensitive enzyme that mediates oxidant-induced myocardial TNF production, cardiac dysfunction, and cardiomyocyte death. (Surgery 1998;124:291-7.)

Hydrogen peroxide induces tumor necrosis factor alpha-mediated cardiac injury by a P38 mitogen-activated protein kinase-dependent mechanism.

Oxidant stress caused by ischemia or endotoxemia induces myocardial dysfunction and cardiomyocyte death; however, mechanisms responsible remain unknown. We hypothesized that hydrogen peroxide (H2O2) induces myocardial dysfunction and cardiomyocyte death via P38 mitogen-activated protein kinase (MAPK)-mediated myocardial tumor necrosis factor (TNF) production. Langendorff perfused rat hearts (6/group) were subjected to oxidant stress (H2O2 infusion; 300 mmol/L x 80 minutes), with and without prior infusion of a specific P38 kinase MAPK inhibitor (P38i = 1 mmol/L/min x 5 minutes) or TNF neutralization (20 mg TNF binding protein (BP)/min x 80 minutes). Developed pressure (DP), coronary flow, and end-diastolic pressure were continuously recorded. Myocardial creatine kinase (CK) loss was measured in the coronary effluent, and tissue TNF was measured in myocardial homogenates. Eighty minutes of H2O2 infusion induced a 6.5-fold increase in myocardial TNF production, which was associated with a 70% decrease in DP and increase in CK loss. P38 MAPK inhibition or TNF-BP decreased myocardial TNF production, cardiomyocyte death, and myocardial dysfunction. These results demonstrate that H2O2 alone induces myocardial TNF production. P38 MPAK is an oxidant-sensitive enzyme that mediates oxidant-induced myocardial TNF production, cardiac dysfunction, and cardiomyocyte death.

Tumor Necrosis Factor Binding Protein Improves Incisional Wound Healing in Sepsis

Background.Sepsis is associated with poor wound healing; however, the exact role of tumor necrosis factor (TNF) as a mediator of sepsis-induced alterations in different types of tissue repair is unknown. This study examines the effects of a specific TNF antagonist (TNFbp) on the healing of intestinal anastomoses, incisional wounds, and polyvinyl (PVA) sponge implants in chronic abdominal sepsis.Methods.Three groups of male Sprague–Dawley rats were studied: control, sepsis, and sepsis + TNFbp. Jejunal resection and anastomosis were performed through a 4-cm upper midline incision on day 1. On day 3, sepsis was induced by creation of a chronic abdominal abscess. Saline (0.1 ml) or TNFbp (1.0 mg/kg, 0.1 ml) was injected subcutaneously every day starting 4 h prior to sepsis. On day 7, the wound-breaking strength (WBS) of the skin incision and intestinal anastomoses was determined using a tensiometer. Wound histology and collagen deposition were evaluated by comparison of Sirius red-stained sections. The hydroxyproline content of PVA sponges was used to quantitate collagen content under the different experimental conditions.Results.Septic mortality (20% vs 26%) was not significantly altered by TNFbp. Septic animals demonstrated a reduction in food consumption on days 3 to 5 that was not affected by TNFbp administration. Neither sepsis nor TNFbp altered the breaking strength or histologic appearance of intestinal anastomoses. However, the breaking strength of incisional wounds was decreased by 40% in septic rats (P< 0.001 vs controls). Administration of TNFbp to septic rats significantly improved incisional WBS (P< 0.01 vs sepsis), but not to control levels. Serius red staining of incisional wounds and PVA sponges demonstrated a decrease in collagen organization and deposition in septic rats that was ameliorated by TNFbp. Similarly, the reduction in hydroxyproline content of PVA sponges from septic animals was prevented by TNFbp.Conclusions.The process of tissue repair in intestine and skin wounds appears to be significantly different following the septic insult. The healing of jejunal anastomoses was refractory to the catabolic effects of sepsis. In contrast, collagen deposition and organization are significantly decreased in cutaneous wounds during chronic sepsis. TNFbp significantly ameliorated the inhibitory effects of sepsis on cutaneous wound healing. These results suggest that TNF is an important mediator of the decrease in collagen deposition observed in cutaneous wounds during the septic state.

PLASMA ENDOTOXIN, PLASMA AND STOOL PRO AND AND INFLAMMATORY CYTOKINES AND STOOL SHIGA TOXIN IN PATIENTS WITH SHIGELLOSIS

F13 We measured on admission, day 3 and 5, and 3 weeks after discharge plasma concentrations of endotoxin (ETX), granulocyte colony stimulating factor(GCSF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a), interleukin 6 (IL 6), interleukin I-receptor antagonist (IL-I RA), tumor necrosis factor binding protein (TNF-BP): stool concentrations of G-CSF, GM CSF, TNFa, IL-6, IL I RA; and stool Shiga toxin (STX) in 157 patients (pts) with shigellosis and 103 pts with watery diarrhoea. Twenty six (17%) of the shigellosis pts were infected with S. dysenteriae type I (SDI) and developed hemolytic uraemic syndrome (HUS), 36(23%) pts (33 & 3 pts had SDI and S. flexneri infection respectively) with leukemoid reaction (LR) but did not have HUS, 65 (41 %) pts had SDI infection without HUS or LR, and 30 (19%) pts had Shigella infections other than SDI and did not have HUS or LR. Mean peak ETX concentrations were higher in pts with Shigella infection compared to watery diarrhoea (5.00 vs 0.51 unit/ml; P<0.001), pts with HUS had higher mean peak ETX (9.46 vs 4.02 unit/ml; P=0.078) compared to other pts infected with Shigella without HUS. Similarly mean peak plasma concentration of G-CSF (1.83 vs 0.02 pg/ml), GM-CSF (0.38 vs 0 pg/ml), TNF-a (4.32 vs 0.31 pg/ml), IL 6 (0.08 vs 0 pg/ml), IL I RA (4.50 vs 1.71 pg/ml), TNF-BP (1.48 vs 0 pg/ml) were significantly higher in pts infected with Shigella compared to pts with watery diarrhoea. Pts with HUS had significantly higher mean peak plasma GM-CSF (0.74 vs 0.31 pg/ml). TNF-A (7.07 vs 3.74 pg/ml), IL-6 (0.19 vs 0.05 pg/ml), IL-I RA (7.59 vs 3.75 pg/ml), TNF BP (6.48 vs 0.43 pg/ml) compared to pts infected with Shigella but did not developed HUS. Pts infected with SDI infection without HUS or LR had higher mean peak stool G-CSF (2741.17 vs 1551.73 pg/ml; P=0.048), GM-CSF (99.07 vs 64.55 pg/ml; P=0.303), TNF -a(515.77 vs 284.67 pg/ml; P= 0.1 13), IL-6 (420.08 vs 290.43 pg/ml; P= 0.101), IL-I RA (4146.04 vs 3531.96 pg/ml; P=0.299) compared to other Shigella infected pts. Pts with SDI had more frequent SXT detectable in stool. In summary, the plasma concentrations of endotoxin, "pro" and anti inflammatory cytokines were markedly higher in pts with shigellosis who develop HUS compared to those who did not; and stool concentrations of "pro" and anti-inflammatory cytokines were higher in SDI infected pts who did not have HUS or LR compared to other Shigella infected pts.

Induction of stress-activated protein kinases/c-Jun N-terminal kinases by the p55 tumour necrosis factor receptor does not require sphingomyelinases.

Ceramide has been implicated in the activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK). Binding of tumour necrosis factor (TNF) to its 55 kDa receptor (TR55) leads to the generation of ceramide through activation of either acid or neutral sphingomyelinase (A/N-SMase) as well as to potent activation of SAPK/JNK. We have examined a putative role of both N- and A-SMase in the TR55-dependent activation of SAPK/JNK. The analysis of TR55 deletion mutants expressed in 70Z/3 pre-B cells revealed that activation of SAPK/JNK occurs independently of N-SMase. Although both SAPK/JNK and A-SMase are activated by the death domain of TR55, pharmacological prevention of the TR55-dependent activation of A-SMase, or proteolytic degradation of A-SMase in 70Z/3 cells, did not impair SAPK/JNK activation, indicating that SAPK/JNK are not secondary to A-SMase. In addition, proteolytic degradation of A-SMase also did not affect SAPK/JNK activation by ultraviolet (UV-C) irradiation, arguing against a general role of A-SMase in stress-mediated responses. Furthermore, fibroblasts from Niemann-Pick A patients deficient in A-SMase did not show altered activation of SAPK/JNK in response to either TNF or UV-C. These results suggest that TR55 can activate SAPK/JNK without direct participation of sphingomyelinases or ceramide.

Neutralization of pyrogen-induced tumour necrosis factor by its type 1 soluble receptor in guinea-pigs: effects on fever and interleukin-6 release.

1. A soluble form of the tumour necrosis factor (TNF) type 1 receptor (referred to as TNF binding protein, TNF-bp) at a dose of 1 mg per animal, or an equivalent volume of solvent, was injected together with 10 microg kg-1 lipopolysaccharide (LPS) or 50 microg kg-1 muramyl-dipeptide (MDP) directly into the arterial circulation of guinea-pigs and the effects on circulating TNF or interleukin-6 (IL-6) and on abdominal temperature were studied. 2. At 15 or 60 min after injection, LPS-induced and MDP-induced circulating TNF was below the detection limit of the assay and thus completely neutralized in animals treated with TNF-bp. In the control group, TNF was still below the limit of detection in most animals 15 min after LPS was injected; in some animals small traces of TNF could already be detected at that time. However, 60 min after administration of LPS, large amounts of TNF (19508 +/- 4682 pg ml-1) were measured in the control group. MDP-induced TNF in plasma was below the limit of detection 15 min after MDP was injected, and rose to 10862 +/- 3029 pg ml-1 60 min after injection. 3. Low levels of circulating IL-6 (20-40 international units (IU) ml-1) were measured in all groups of animals 15 min after injection of LPS or MDP. This value corresponds to the baseline activity of IL-6 in plasma of guinea-pigs. One hour after administration of LPS, IL-6 rose to 5442 +/- 1662 IU ml-1 in the control group and to a significantly lower value of 1485 +/- 179 IU ml-1 in guinea-pigs treated with TNF-bp. One hour after injection of MDP, circulating IL-6 was 2614 +/- 506 IU ml-1 in the control group, while the corresponding value in animals treated with TNF-bp again was significantly lower (873 +/- 312 IU ml-1). 4. The second phase of the characteristic biphasic LPS fever in guinea-pigs was significantly attenuated in animals treated with TNF-bp. The shorter first phase of the febrile response to LPS was identical in both groups of animals. 5. The late phase of MDP-induced fever (7-22 h after injection) was depressed by treatment with TNF-bp, while the first phase of MDP-induced fever (0-7 h after injection) was significantly enhanced by the neutralization of TNF by TNF-bp.

Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins.

During toxicologic evaluation of a dimeric PEG-linked protein, tumor necrosis factor binding protein (TNF-bp), vacuolation of renal cortical tubular epithelium was seen in male and female Sprague-Dawley rats (200-300 g) given i.v. doses of 40, 20, or 10 mg/kg every other day for 3 months. Tubular lesions in rats treated with 20 or 40 mg/kg for 3 months were only partially reversible after a 2-month recovery period. Despite the presence of marked vacuolation, there were no changes in BUN, creatinine, urinalysis parameters, urinary NAG, urinary B2-microglobulin, or fractional sodium excretion. Single i.v. doses > or = 20 mg/kg TNF-bp caused similar but milder changes. However, equivalent doses of PEG alone or the non-PEG-linked TNF-bp did not cause light microscopic evidence of vacuolation. Treatment of rats with another PEG-linked protein of similar molecular weight resulted in similar changes. Immunostaining for TNF-bp revealed positivity in the apical cytoplasm of renal tubular epithelium within 1 h of i.v. dosing. Immunostaining of kidneys from chronically dosed rats indicated that protein was present in some vacuoles as long as dosing continued; however, kidneys from animals on a reversibility study had vacuoles but no immunostaining for TNF-bp. These results, along with a study that showed more severe lesions with PEG-linked proteins of lower molecular weight and minimal if any lesions with PEG-linked proteins > 70 kDa, suggest that TNF-bp is filtered through the glomerulus and that the protein with attached PEG is reabsorbed by the proximal tubules. Vacuolation may be a result of fluid distension of lysosomes due to the hygroscopic nature of PEG. These studies demonstrated that PEG-linked proteins have the capacity to induce renal tubular vacuolation at high doses. However, the change was not associated with alteration of clinical pathology or functional markers.

Epstein-Barr virus-mediated B-cell proliferation is dependent upon latent membrane protein 1, which simulates an activated CD40 receptor.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for the immortalization of human B cells and is linked etiologically to several human tumors. LMP1 is an integral membrane protein which acts like a constitutively active receptor. It binds tumor necrosis factor (TNF)-receptor-associated factors (TRAFs), activates NF-kappaB and triggers the transcription factor AP-1 via the c-Jun N-terminal kinase (JNK) cascade, but its specific contribution to B-cell immortalization has not been elucidated fully. To address the function of LMP1, we established B cell lines with a novel mini-EBV plasmid in which the LMP1 gene can be regulated at will without affecting the expression of other latent EBV genes. We demonstrate here that continuous expression of LMP1 is essential for the proliferation of EBV-immortalized B cells in vitro. Re-induction of LMP1 expression or activation of the cellular CD40 receptor both induce the JNK signaling cascade, activate the transcription factor NF-kappaB and stimulate proliferation of these B cells. Our findings strongly suggest that LMP1 mimics B-cell activation processes which are physiologically triggered by CD40-CD40 ligand signals. Since LMP1 acts in a ligand-independent manner, it replaces the T cell-derived activation signal to sustain indefinite B-cell proliferation.

Differential effects of interleukin-1 receptor antagonist and tumor necrosis factor binding protein on fatty-streak formation in apolipoprotein E-deficient mice.

The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17beta was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility.

The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor.

Tumor necrosis factor (TNF) can induce a variety of cellular responses at low picomolar concentrations. This is in apparent conflict with the published dissociation constants for TNF binding to TNF receptors in the order of 100-500 pM. To elucidate the mechanisms underlying the outstanding cellular sensitivity to TNF, we determined the binding characteristics of TNF to both human TNF receptors at 37 degrees C. Calculation of the dissociation constant (Kd) from the association and dissociation rate constants determined at 37 degrees C revealed a remarkable high affinity for TNF binding to the 60-kDa TNF type 1 receptor (TNF-R1; Kd = 1.9 x 10(-11) M) and a significantly lower affinity for the 80-kDa TNF type 2 receptor (TNF-R2; Kd = 4.2 x 10(-10) M). The high affinity determined for TNF-R1 is mainly caused by the marked stability of ligand-receptor complexes in contrast to the transient interaction of soluble TNF with TNF-R2. These data can readily explain the predominant role of TNF-R1 in induction of cellular responses by soluble TNF and suggest the stability of the TNF-TNF receptor complexes as a rationale for their differential signaling capability. In accordance with this reasoning, the lower signaling capability of homotrimeric lymphotoxin, compared with TNF, correlates with a lower stability of the lymphotoxin-TNF-R1 complex at 37 degrees C.

In vitro inhibition of binding of tumor necrosis factor (TNF)-alpha by monoclonal antibody to TNF receptor on glioma cell and monocyte.

The use of monoclonal antibodies to the tumor necrosis factor (TNF) receptors, the TNF-p55 receptor (TNF-p55R) and the TNF-p75 receptor (TNF-p75R), was evaluated to reduce the effects of TNF caused by binding to TNF-p75R. Competitive binding of anti-TNF-p55R (mAbp55R) and anti-TNF-p75R monoclonal antibodies (mAbp75R) with iodine-125-labeled TNF-alpha to GL-9 glioma cells and U937 histiocytic lymphoma cells was evaluated. The effects of mAbp55R and mAbp75R on the growth suppression by TNF-alpha of GL-9 cells and TNF-alpha production in U937 cells were also examined. mAbp75R bound to U937 cells competitively with TNF-alpha and suppressed TNF-alpha production by U937, but had no effect on the growth inhibition of GL-9 human glioma cell by TNF-alpha in vitro. These findings suggest that co-administration of TNF-p75R antagonist with TNF-alpha may decrease the toxicity of TNF-alpha administration resulting in a better therapeutic result.

Determination of disulfide bonds in highly bridged disulfide-linked peptides by matrix-assisted laser desorption/ionization mass spectrometry with postsource decay.

Matrix-assisted laser desorption/ionization mass spectrometry with postsource decay was used to generate fragment ions from peptide fragments containing heteropeptides linked together by two disulfide bonds. Postsource decay analysis of these peptide samples generates a series of singly charged fragment ions that, in addition to the peptide sequence ions, provide useful information for assigning disulfide arrangement in highly bridged disulfide-linked peptides. The assignment was made possible by fragmentation at peptide bonds between two Cys residues in a peptide that constitutes the highly bridged fragment, while retaining the disulfide linkage to the other peptide. Fragmentation using other types of instruments, such as quadrupole ion-trap mass spectrometry with collision-induced dissociation, usually did not generate such fragment ions. The data obtained from postsource decay also provide fragment ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds. The present method is a highly sensitive technique which requires no further sample handling and should be complementary to other classical chemical methods. The method proved useful in facilitating the assignment of disulfide structure in tumor necrosis factor binding protein (TNFbp), which contains 162 amino acids and 13 disulfide bonds (Jones, M.; et al. Biochemistry, in press). Postsource decay analysis of large disulfide-containing peptides usually produces no fragmentation but generates a series of high-intensity ions derived from both symmetric and nonsymmetric cleavages of disulfide bonds.

Neuroprotective effects of TNF binding protein in focal cerebral ischemia

The effect of tumor necrosis factor binding protein (TNFbp) was studied in mice subjected to a permanent middle cerebral artery occlusion (MCAO). TNFbp is a dimeric form of the type I soluble TNF receptor linked to polyethylene glycol (TNFbp), and binds and inhibits TNF- α. TNFbp produced a significant reduction in the cortical infarct volume (22.6±3.5 mm 3 immediately after MCAO; 25.2±2.4 mm 3 1 h after MCAO) compared with vehicle-treated animals (30.3±3.7 mm 3 immediately post MCAO; 31±3.7 mm 3 1 h after MCAO (mean±S.D.) when administered intracranially up to 60 min post-occlusion. The neuroprotective effect of TNFbp was sustained in mice for 2 weeks after MCAO. DNA fragmentation at the margin of the cortical infarcts was dramatically reduced in mice treated with TNFbp whereas all control animals showed consistent and obvious DNA fragmentation 2 weeks after MCAO. TNFbp could have therapeutic value for the treatment of ischemic stroke if the problem of delivery to brain can be overcome.

LPS induces late cardiac functional protection against ischemia independent of cardiac and circulating TNF-alpha.

Lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-alpha independently induce cardioprotection against ischemia in the rat at 24 h after administration, suggesting that endogenously synthesized TNF-alpha may play a role in LPS-induced protection. The purposes of this study were 1) to delineate the time course of LPS-induced cardiac functional protection against ischemia and its relation with myocardial and circulating TNF-alpha profile, 2) to examine whether prior protein synthesis inhibition abrogates the protection, and 3) to assess the effects of TNF-alpha inhibition and neutralization on the protection. Rats were treated with LPS (0.5 mg/kg i.p.). Cardiac functional resistance to normothermic global ischemia-reperfusion was examined at sequential time points after LPS treatment in isolated hearts by the Langendorff technique. Myocardial and circulating TNF-alpha was determined by enzyme-linked immunosorbent assay at 1-24 h after LPS treatment. Protection was apparent at 24 h, 3 days, and 7 days but not at 2 or 12 h. Maximal protection at 3 days was abolished by cycloheximide pretreatment (0.5 mg/kg i.p. 3 h before LPS treatment). Increases in myocardial and circulating TNF-alpha preceded the acquisition of protection. Dexamethasone pretreatment (4.0 or 8.0 mg/kg i.p. 30 min before LPS treatment) abolished peak increase in myocardial TNF-alpha and substantially suppressed circulating TNF-alpha (54.3 and 85.9% inhibition, respectively) without an influence on the maximal protection. Similarly, maximal protection was not affected by TNF binding protein (40 or 80 microg/kg i.v. immediately after LPS treatment). The results suggest that LPS-induced cardiac functional protection against ischemia is a delayed and long-lasting protective response that may involve de novo protein synthesis. Although LPS-induced increase in myocardial and circulating TNF-alpha precedes the delayed protection, it may not be required for the delayed protection.