Differential roles of p55 and p75 tumor necrosis factor receptors on stretch-induced pulmonary edema in mice

Ventilator-induced lung injury plays a crucial role in the outcome of patients with acute lung injury. Previous studies have shown a role for the cytokine tumor necrosis factor-alpha (TNF) in stretch-induced alveolar neutrophil recruitment, but the involvement of TNF in stretch-induced pulmonary edema is unclear. We investigated the effects of TNF through its individual p55 and p75 receptors on early pulmonary edema formation during high stretch ventilation, before neutrophil infiltration. Anesthetized wild-type or TNF receptor single/double knockout mice were ventilated with high tidal volume ( approximately 38 ml/kg) for 2 h or until they developed arterial hypotension. Pulmonary edema was assessed by physiological parameters including respiratory mechanics and blood gases, and by lavage fluid protein, lung wet:dry weight ratio, and lung permeability measurements using fluorescence-labeled albumin. High stretch ventilation in wild-type and TNF receptor double knockout animals induced similar pulmonary edema, and only 25-30% of mice completed the protocol. In contrast, the p55 receptor knockout mice were strongly protected from edema formation, with all animals completing the protocol. Myeloperoxidase assay indicated that this protective effect was not associated with decreased pulmonary neutrophil sequestration. The p75 receptor knockout mice, however, displayed increased susceptibility to edema formation, and no animals survived the full 2 h. These results demonstrate a novel role for TNF signaling (independent from its effects on neutrophil recruitment) specifically through the p55 receptor, in promoting high stretch-induced pulmonary edema, whereas p75 signaling may play an opposing role.     

Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.     

Tumor necrosis factor (TNF) protects resistant C57BL/6 mice against herpes simplex virus-induced encephalitis independently of signaling via TNF receptor 1 or 2

Tumor necrosis factor (TNF) is a multifunctional cytokine that has a role in induction and regulation of host innate and adaptive immune responses. The importance of TNF antiviral mechanisms is reflected by the diverse strategies adopted by different viruses, particularly members of the herpesvirus family, to block TNF responses. TNF binds and signals through two receptors, Tnfrsf1a (TNF receptor 1 [TNFR1], or p55) and Tnfrsf1b (TNFR2, or p75). We report here that herpes simplex virus 1 (HSV-1) infection of TNF-/- mice on the resistant C57BL/6 genetic background results in significantly increased susceptibility (P < 0.0001, log rank test) to fatal HSV encephalitis (HSE) and prolonged persistence of elevated levels of virus in neural tissues. In contrast, although virus titers in neural tissues of p55-/- N13 mice were elevated to levels comparable to what was found for the TNF-/- mice, the p55-/- N13 mice were as resistant as control C57BL/6 mice (P > 0.05). The incidence of fatal HSE was significantly increased by in vivo neutralization of TNF using soluble TNFR1 (sTNFR1) or depletion of macrophages in C57BL/6 mice (P = 0.0038 and P = 0.0071, respectively). Strikingly, in vivo neutralization of TNF in HSV-1-infected p55-/- p75-/- mice by use of three independent approaches (treatment with soluble p55 receptor, anti-TNF monoclonal antibody, or in vivo small interfering RNA against TNF) resulted in significantly increased mortality rates (P = 0.005), comparable in magnitude to those for C57BL/6 mice treated with sTNFR1 (P = 0.0018). Overall, these results indicate that while TNF is required for resistance to fatal HSE, both p55 and p75 receptors are dispensable. Precisely how TNF mediates protection against HSV-1 mortality in p55-/- p75-/- mice remains to be determined.     

Tissue inhibitor of metalloproteinase 3 regulates TNF-dependent systemic inflammation

Host response to infectious agents must be rapid and powerful. One mechanism is the release of presynthesized membrane-bound TNF. TNF shedding is mediated by TNF-alpha converting enzyme, which is selectively inhibited by the tissue inhibitor of metalloproteinase 3 (TIMP3). We show that loss of TIMP3 impacts innate immunity by dysregulating cleavage of TNF and its receptors. Cultured timp3-/- macrophages release more TNF in response to LPS than wild-type macrophages. In timp3-/- mice, LPS causes serum levels of TNF and its receptors to rise more rapidly and remain higher compared with wild-type mice. The altered kinetics of ligand and receptor shedding enhances TNF signaling in timp3-/- mice, indicated by elevated serum IL-6. Physiologically, timp3-/- mice are more susceptible to LPS-induced mortality. Ablation of the TNF receptor gene p55 (Tnfrsf1a) or treatment with a synthetic metalloproteinase inhibitor rescues timp3-/- mice. Thus, TIMP3 is essential for normal innate immune function.     

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.     

TNF-alpha regulates corneal Langerhans cell migration

Langerhans cells (LC) belong to the dendritic cell family and mediate Ag presentation in the cornea and ocular surface. Under normal physiological conditions, the central cornea is devoid of LC. Centripetal migration of LC plays a critical role in promoting immunoinflammatory responses in the eye including allograft rejection and herpetic keratitis. The molecular mechanisms responsible for ocular LC migration are poorly understood. To examine whether TNF-alpha mediates corneal LC migration and to establish the interaction of IL-1 and TNF-alpha in regulating LC migratory capacity, we utilized gene-targeted knockout mice lacking IL-1 receptor I (IL-1RI-/-), TNF receptor I (p55-/-), TNF receptor II (p75-/-), or both (p55-/-p75-/-). LC migration was induced by thermal cautery or cytokine injection and enumerated by an immunofluorescence assay. Migration of LC after cauterization and TNF-alpha injection was significantly depressed in both p55-/- and p75-/- mice. Similarly, in the first 72 h after intracorneal injection of IL-1alpha, LC migration was reduced in p55-/-, p75-/-, and p55-/-p75-/- mice. In contrast, injection of TNF-alpha in IL-1RI-/- mice led to normal migration of corneal LC indistinguishable from wild-type controls. These results suggest that the IL-1 induction of corneal LC migration is largely mediated by TNFR function, whereas TNF-alpha induction of LC migration is independent of IL-1RI activity. Moreover, the data suggest that both p55 and p75 signaling pathways are important in mediating LC migration in the cornea.     

Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-alpha receptors

This study was designed to investigate the mechanisms through which tumor necrosis factor (Tnf) modulates ozone (O(3))-induced pulmonary injury in susceptible C57BL/6J (B6) mice. B6 [wild-type (wt)] mice and B6 mice with targeted disruption (knockout) of the genes for the p55 TNF receptor [TNFR1(-/-)], the p75 TNF receptor [TNFR2(-/-)], or both receptors [TNFR1/TNFR2(-/-)] were exposed to 0.3 parts/million O(3) for 48 h (subacute), and lung responses were determined by bronchoalveolar lavage. All TNFR(-/-) mice had significantly less O(3)-induced inflammation and epithelial damage but not lung hyperpermeability than wt mice. Compared with air-exposed control mice, O(3) elicited upregulation of lung TNFR1 and TNFR2 mRNAs in wt mice and downregulated TNFR1 and TNFR2 mRNAs in TNFR2(-/-) and TNFR1(-/-) mice, respectively. Airway hyperreactivity induced by acute O(3) exposure (2 parts/million for 3 h) was diminished in knockout mice compared with that in wt mice, although lung inflammation and permeability remained elevated. Results suggested a critical role for TNFR signaling in subacute O(3)-induced pulmonary epithelial injury and inflammation and in acute O(3)-induced airway hyperreactivity.     

Dual effects of p38 MAPK on TNF-dependent bronchoconstriction and TNF-independent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome

The administration of endotoxins from Gram-negative bacteria induces manifestations reminding of acute respiratory distress syndrome. p38 MAPKs have been implicated in this pathology. In this study, we show that the specific p38 alpha,beta MAPK inhibitor, compound 37, prevents LPS-induced bronchoconstriction and neutrophil recruitment into the lungs and bronchoalveolar space in a dose-dependent manner in C57BL/6 mice. Furthermore, TNF induction and TNF signals were blocked. In TNF-deficient mice, bronchoconstriction, but not neutrophil sequestration, in the lung was abrogated after LPS administration. Therefore, TNF inhibition does not explain all of the effects of the p38 MAPK inhibitor. The p38 alpha,beta MAPK inhibitor also prevented LPS-induced neutrophilia in TNF-deficient mice. In conclusion, LPS provokes acute bronchoconstriction that is TNF dependent and p38 MAPK mediated, whereas the neutrophil recruitment is independent of TNF but depends on LPS/TLR4-induced signals mediated by p38 MAPK.     

Genetic deletion of the tumor necrosis factor receptor p60 or p80 sensitizes macrophages to lipopolysaccharide-induced nuclear factor-kappa B,mitogen-activated protein kinases,and apoptosis

Whether deletion of tumor necrosis factor (TNF) receptor 1 or 2 affects lipopolysaccharide (LPS)-mediated signaling is not understood. In this report, we used macrophages derived from wild type (wt) mice and from mice null for the type 1 receptor (p60-/-), the type 2 receptor (p80-/-), or both (p60-/- p80-/-) to investigate the effect of these receptors on LPS-mediated activation of NF-kappaB, mitogen-activated protein kinases, and apoptosis. LPS activated NF-kappaB by 3-4-fold in wt cells but by 9-10-fold in p60-/-, p80-/-, and p60-/- p80-/- macrophages. These results correlated with the IkappaBalpha kinase activation, which is needed for NF-kappaB activation. LPS-induced cyclooxygenase-2 and inducible NO synthase proteins and NO production were maximum in p60-/- p80-/- macrophages and minimum in wt cells. LPS activated C-Jun N-terminal kinase, p38MAPK, and extracellular signal-regulated kinase in wt cells, but the levels were much higher in p60-/-, p80-/-, and p60-/- p80-/- cells. LPS-induced cytotoxicity, poly(ADP-ribose) polymerase cleavage, and annexin V staining were also highest in p60-/- p80-/- cells and lowest in wt cells. The difference in LPS signaling was unrelated to the expression of LPS receptors, CD14, or toll-like receptor 4. Overall, our studies indicate that deletion of either of the TNF receptors sensitizes the macrophages to LPS and provide evidence for cross-talk between TNF and LPS signaling.     

Divergent roles for p55 and p75 tumor necrosis factor receptors in the pathogenesis of MOG(35-55)-induced experimental autoimmune encephalomyelitis

To clarify the role of tumor necrosis factor (TNF) in the inflammatory aspects of autoimmunity vs its potential role in the apoptotic elimination of autoreactive effector cells, we assessed the roles of the p55 (TNFR1/Tnfrsf1a/CD120a) and p75 (TNFR2/Tnfrsf1b/CD120b) TNF receptors in the pathogenesis of MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE). TNFR p55/p75(-/-) double knockout mice were completely resistant to clinical disease. TNFR p55(-/-) single knockout mice were also totally resistant to EAE, exhibiting reduced MOG(35-55)- specific proliferative responses and Th1 cytokine production, despite displaying equivalent DTH responses. Importantly, IL-5 was significantly increased in p55(-/-) mice. In contrast, p75(-/-) knockout mice exhibited exacerbated EAE, enhanced Th1 cytokine production, and enhanced CD4(+) and F4/80(+) CNS infiltration. Thus, p55/TNFR1 is required for the initiation of pathologic disease, whereas p75/TNFR2 may be important in regulating the immune response. These results have important implications for therapies targeting p55 and p75 receptors for treating autoimmune diseases.     

A selective role for the TNF p55 receptor in autocrine signaling following IFN-gamma stimulation in experimental autoimmune uveoretinitis

IFN-gamma stimulates macrophage activation and NO production, which leads to destruction of the retina in experimental autoimmune uveoretinitis. In this study, we investigate the mechanism of disease resistance in TNF p55 receptor-deficient animals. We show that although T cell priming is relatively unaffected, macrophages lacking the TNF p55 receptor fail to produce NO following IFN-gamma stimulation because of a requirement for autocrine TNF-alpha signaling through the TNF p55 receptor. In contrast to the impaired activation of NO synthesis, MHC class II up-regulation was indistinguishable in wild-type and TNFRp55-/- mice stimulated with IFN-gamma. These defects could be overcome by stimulating macrophages with LPS. Together, these results show that selected aspects of IFN-gamma activation are controlled by autocrine secretion of TNF-alpha, but that this control is lost in the presence of signals generated by pathogen-associated molecular patterns recognizing receptors.     

Development of allergic contact dermatitis requires activation of both tumor necrosis factor-receptors

We investigated the role of the TNF receptors, type I (p55TNFR) and type II (p75TNFR), in a mouse model of contact hypersensitivity, i.e., a model of a delayed type hypersensitivity (DTH) allergic reaction. Mice deficient for p55TNFR or p75TNFR were used to investigate the functions of these receptors in development of the DTH reaction. We show that both TNF receptors have a strong influence on the overall outcome of the DTH reaction, with the two TNF receptors exerting distinct functions. Dendritic cells of mice lacking p55TNFR had a defect in allergen uptake but showed normal migration into regional lymph nodes. In contrast, dendritic cells of p75TNFR-deficient mice showed diminished migration into regional lymph nodes after allergen contact, whereas the allergen uptake was independent of the p75TNFR. Thus, both TNF receptors are required for the development of a complete DTH reaction.     

Dissection of antiviral and immune regulatory functions of tumor necrosis factor receptors in a chronic lymphocytic choriomeningitis virus infection

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). While the roles of perforin-dependent cytotoxicity, IFN-gamma, and TNF-alpha in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75(-/-)) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55(-/-)) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55(-/-) and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55(-/-), and p75(-/-) mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.     

A low level of TNF-alpha mediates hemorrhage-induced acute lung injury via p55 TNF receptor

Acute lung injury after hemorrhagic shock (HS) is associated with the expression of tumor necrosis factor (TNF)-alpha in the lung. However, the role of TNF-alpha and its receptors in this pulmonary disorder remains obscure. This study examined the temporal relationship of pulmonary TNF-alpha production to neutrophil accumulation during HS and determined the role of TNF-alpha in neutrophil accumulation and lung leak. HS was induced in mice by removal of 30% of total blood volume. Lung TNF-alpha was measured by ELISA. Neutrophil accumulation was detected by immunofluorescent staining, and microvascular permeability was assessed using Evans blue dye. Although HS induced a slight and transient increase in lung TNF-alpha, neutrophil accumulation preceded the increase in TNF-alpha. However, lung neutrophil accumulation and lung leak were abrogated in TNF-alpha knockout mice, and both were restored by administration of recombinant TNF-alpha to TNF-alpha knockout mice before HS. Neutrophil accumulation and lung leak were abrogated in mice lacking the p55 TNF-alpha receptor, but neither was influenced by p75 TNF-alpha receptor knockout. This study demonstrates that a low level of pulmonary TNF-alpha is sufficient to mediate HS-induced acute lung injury during HS and that the p55 TNF-alpha receptor plays a dominant role in regulating the pulmonary inflammatory response to HS.     

LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor

Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent on endogenously produced tumor necrosis factor (TNF)-alpha. The present study was undertaken to determine whether membrane-associated or secreted TNF-alpha signaling through the p55 or p75 receptor was responsible for survival and hepatic injury after lipopolysaccharide administration in D-galactosamine-sensitized mice. Transgenic mice expressing null forms of TNF-alpha, the p55 and p75 receptor, and mice expressing only a cell-associated form of TNF-alpha were challenged with 8 mg D-galactosamine and 100 ng lipopolysaccharide. Mortality and apoptotic liver injury were only seen in wild-type and p75 knockout mice. p75 Knockout mice had significantly higher concentrations of plasma TNF-alpha than any other experimental group (P 相似文献   

p75 neurotrophin receptor regulates agonist-induced pulmonary vasoconstriction

A member of the TNF receptor family, the p75 neurotrophin receptor (p75(NTR)) has been previously shown to play a role in the regulation of fibrin deposition in the lung. However, the role of p75(NTR) in the regulation of pulmonary vascular tone in the lung is unknown. In the present study, we evaluated the expression of p75(NTR) in mouse pulmonary arteries and the putative role of p75(NTR) in modulating pulmonary vascular tone and agonist responsiveness using wild-type (WT) and p75(NTR) knockout (p75(-/-)) mice. Our data indicated that p75(NTR) is expressed in both smooth muscle and endothelial cells within the pulmonary vascular wall in WT mice. Pulmonary artery rings from p75(-/-) mice exhibited significantly elevated active tension due to endothelin-1-mediated Ca(2+) influx. Furthermore, the contraction due to capacitative Ca(2+) entry (CCE) in response to phenylephrine-mediated active depletion of intracellular Ca(2+) stores was significantly enhanced compared with WT rings. The contraction due to CCE induced by passive store depletion, however, was comparable between WT and p75(-/-) rings. Active tension induced by serotonin, U-46619 (a thromboxane A(2) analog), thrombin, 4-aminopyridine (a K(+) channel blocker), and high extracellular K(+) in p75(-/-) rings was similar to that in WT rings. Deletion of p75(NTR) did not alter pulmonary vasodilation to sodium nitroprusside (a nitric oxide donor). These data suggest that intact p75(NTR) signaling may play a role in modulating pulmonary vasoconstriction induced by endothelin-1 and by active store depletion.     

Death receptors mediate the adverse effects of febrile-range hyperthermia on the outcome of lipopolysaccharide-induced lung injury

We have shown that febrile-range hyperthermia enhances lung injury and mortality in mice exposed to inhaled LPS and is associated with increased TNF-α receptor activity, suppression of NF-κB activity in vitro, and increased apoptosis of alveolar epithelial cells in vivo. We hypothesized that hyperthermia enhances lung injury and mortality in vivo by a mechanism dependent on TNF receptor signaling. To test this, we exposed mice lacking the TNF-receptor family members TNFR1/R2 or Fas (TNFR1/R2(-/-) and lpr) to inhaled LPS with or without febrile-range hyperthermia. For comparison, we studied mice lacking IL-1 receptor activity (IL-1R(-/-)) to determine the role of inflammation on the effect of hyperthermia in vivo. TNFR1/R2(-/-) and lpr mice were protected from augmented alveolar permeability and mortality associated with hyperthermia, whereas IL-1R(-/-) mice were susceptible to augmented alveolar permeability but protected from mortality associated with hyperthermia. Hyperthermia decreased pulmonary concentrations of TNF-α and keratinocyte-derived chemokine after LPS in C57BL/6 mice and did not affect pulmonary inflammation but enhanced circulating markers of oxidative injury and nitric oxide metabolites. The data suggest that hyperthermia enhances lung injury by a mechanism that requires death receptor activity and is not directly associated with changes in inflammation mediated by hyperthermia. In addition, hyperthermia appears to enhance mortality by generating a systemic inflammatory response and not by a mechanism directly associated with respiratory failure. Finally, we observed that exposure to febrile-range hyperthermia converts a modest, survivable model of lung injury into a fatal syndrome associated with oxidative and nitrosative stress, similar to the systemic inflammatory response syndrome.