IL-18 contributes to host resistance against infection with Pseudomonas aeruginosa through induction of IFN-gamma production

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible (B6), but not resistant (BALB/c) mice. To determine mechanisms mediating resistance, the role of IFN-gamma, IL-12, and IL-18 was tested in BALB/c mice. RT-PCR analysis detected IFN-gamma mRNA expression levels in cornea that were significantly increased at 1-7 days postinfection. IL-18 mRNA was detected constitutively in cornea and, at 1-7 days postinfection, levels were elevated significantly, while no IL-12 mRNA was similarly detected. To test whether IL-18 contributed to IFN-gamma production, mice were treated with anti-IL-18 mAb. Treatment decreased corneal IFN-gamma mRNA levels, and bacterial load and disease increased/worsened, compared with IgG-treated mice. To stringently examine the role of IFN-gamma in bacterial killing, knockout (-/-) vs wild-type (wt) mice also were tested. All corneas perforated, and bacterial load was increased significantly in -/- vs wt mice. Because disease severity was increased in IFN-gamma(-/-) vs IL-18-neutralized mice, and since IL-18 also induces production of TNF, we tested for TNF-alpha in both groups. ELISA analysis demonstrated significantly elevated corneal TNF-alpha protein levels in IFN-gamma(-/-) vs wt mice after infection. In contrast, RT-PCR analysis of IL-18-neutralized vs IgG-treated infected mice revealed decreased corneal TNF-alpha mRNA expression. Next, to resolve whether TNF was required for bacterial killing, TNF-alpha was neutralized in BALB/c mice. No difference in corneal bacterial load was detected in neutralized vs IgG-treated mice. These data provide evidence that IL-18 contributes to the resistance response by induction of IFN-gamma and that IFN-gamma is required for bacterial killing.     

The p55 TNF-alpha receptor plays a critical role in T cell alloreactivity

TNF-alpha is known to be an important mediator of tissue damage during allograft rejection and graft-vs-host disease (GVHD), but its role in supporting T cell responses to allogeneic Ags is unclear. We have studied this question by comparing normal mice with those lacking the p55 (p55 TNFR-/-) or p75 (p75 TNFR-/-) TNF-alpha receptors as donors in well-defined bone marrow transplant (BMT) models. Recipients of p55 TNFR-/- cells had significantly reduced mortality and morbidity from GVHD compared with the other two sources of T cells. In vitro, T cells lacking the p55 (but not the p75) TNF-alpha receptor exhibited decreased proliferation and production of Th1 cytokines in MLC. This defect was only partially restored by exogenous IL-2 and affected both CD4+ and CD8+ populations. CD8+ p55 TNFR-/- proliferation was impaired independently of IL-2 whereas CTL effector function was impaired in an IL-2-dependent fashion. Inhibition of TNF-alpha with TNFR:Fc in primary MLC also impaired the proliferation and Th1 differentiation of wild-type T cells. BMT mixing experiments demonstrated that the reduced ability of p55 TNFR-/- donor cells to induce GVHD was due to the absence of the p55 TNFR on T cells rather than bone marrow cells. These data highlight the importance of TNF-alpha in alloreactive T cell responses and suggest that inhibition of the T cell p55 TNF-alpha receptor may provide an additional useful therapeutic maneuver to inhibit alloreactive T cell responses following bone marrow and solid organ transplantation.     

Tumor necrosis factor p55 and p75 receptors are involved in chemical-induced apoptosis of dentate granule neurons

Localized tumor necrosis factor-alpha (TNFalpha) elevation has diverse effects in brain injury often attributed to signaling via TNFp55 or TNFp75 receptors. Both dentate granule cells and CA pyramidal cells express TNF receptors (TNFR) at low levels in a punctate pattern. Using a model to induce selective death of dentate granule cells (trimethyltin; 2 mg/kg, i.p.), neuronal apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ end labeling, active caspase 3 (AC3)] was accompanied by amoeboid microglia and elevated TNFalpha mRNA levels. TNFp55R (55 kDa type-1 TNFR) and TNFp75R (75 kDa type-2 TNFR) immunoreactivity in AC3(+) neurons displayed a pattern suggestive of receptor internalization and a temporal sequence of expression of TNFp55R followed by TNFp75R associated with the progression of apoptosis. A distinct ramified microglia response occurred around CA1 neurons and healthy dentate neurons that displayed an increase in the normal punctate pattern of TNFRs. Neuronal damage was decreased with i.c.v. injection of TNFalpha antibody and in TNFp55R-/-p75R-/- mice that showed higher constitutive mRNA levels for interleukin (IL-1alpha), macrophage inflammatory protein 1-alpha (MIP-1alpha), TNFalpha, transforming growth factor beta1, Fas, and TNFRSF6-assoicated via death domain (FADD). TNFp75R-/- mice showed exacerbated injury and elevated mRNA levels for IL-1alpha, MIP-1alpha, and TNFalpha. In TNFp55R-/- mice, constitutive mRNA levels for TNFalpha, IL-6, caspase 8, FADD, and Fas-associated phosphatase were higher; IL-1alpha, MIP-1alpha, and transforming growth factor beta1 lower. The mice displayed exacerbated neuronal death, delayed microglia response, increased FADD and TNFp75R mRNA levels, and co-expression of TNFp75R in AC3(+) neurons. The data demonstrate TNFR-mediated apoptotic death of dentate granule neurons utilizing both TNFRs and suggest a TNFp75R-mediated apoptosis in the absence of normal TNFp55R activity.     

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   

CXC chemokine receptor 2 but not C-C chemokine receptor 1 expression is essential for neutrophil recruitment to the cornea in helminth-mediated keratitis (river blindness)

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2(-/-) and CCR1(-/-) mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1 alpha was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2(-/-) mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2(-/-) mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2(-/-) mice, which was similar to BALB/c mice. Furthermore, although MIP-1 alpha production was lower in CXCR2(-/-) mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.     

B7/CD28 costimulation is critical in susceptibility to Pseudomonas aeruginosa corneal infection: a comparative study using monoclonal antibody blockade and CD28-deficient mice

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.     

Role of LPS and receptor subtypes in the uptake of TNF by the murine lung

Tumor necrosis factor alpha (TNF) is an important mediator in lung injury. The kinetics of TNF uptake by the lung are not completely understood. In this study, we evaluated the role that lipopolysaccharide (LPS) and the two types of TNF receptor (p55 and p75) play in the uptake of circulating murine TNF by the murine lung. TNF radioactively labeled with 125I (I-mTNF) was administered intravenously (2 x 10(6) cpm/mouse) to mice with both receptors (wild-type) or to mice missing one (p55-/- or p75-/-) or both (p55-/- and p75-/-) TNF receptors. Blood to lung non-reversible sequestration (Ki) and reversible uptake (Vi) were measured with multiple-time regression analysis. Uptake by lung of I-mTNF in wild-type mice had reversible and non-reversible components. This uptake was decreased by intratracheal, but not by intravenous, LPS, suggesting modulation by local, rather than systemic, inflammation. The p75-/- deficient mice retained the Ki (saturable, non-reversible) component of TNF uptake, whereas p55-/- deficient mice retained the Vi (saturable, reversible) component of TNF uptake. Both Ki and Vi components of TNF uptake were absent in the lungs of p55-/- p75-/- deficient mice. These studies show that local inflammation inhibits the uptake of circulating I-mTNF by lung and that uptake consists of two distinguishable compartments: reversible uptake mediated by the p75 receptor and non-reversible sequestration mediated by the p55 receptor.     

Increased hypothalamic expression of the p75 tumor necrosis factor receptor in New Zealand obese mice.

Previous studies have demonstrated that tumor necrosis factor-alpha (TNF-alpha) production from adipose tissue is elevated in obese animal models and in obese humans. It plays an important role in the induction of insulin resistance in experimental animals. In this study, we examined hypothalamic tissue expression of TNF-alpha and its receptors and TNF-alpha expression of adipose tissue in lean C57BLKSJ+/+ and obese polygenic New Zealand obese (NZO) mice. Obese animals exhibited hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and hypercholesterinemia. Using RT-PCR, we observed increased expression (2.4-fold) of TNF receptor 2 (p75) in the hypothalamus of obese mice. TNF-alpha expression in adipose tissue of obese mice was eight times higher than in controls. TNF-alpha and TNF receptor 1 (p55) expression in hypothalamic tissue was similar in obese and lean animals. These results suggest that the hypothalamic TNF receptor 2 (p75) might play a role in obesity by modulating the actions of TNF-alpha in conditions of leptin resistance.     

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.     

Critical role of corneal Langerhans cells in the CD4- but not CD8-mediated immunopathology in herpes simplex virus-1-infected mouse corneas.

Previous studies have revealed that the RE strain of HSV type 1 (HSV-1) induces a tissue-destructive inflammatory response in the mouse cornea that is mediated by CD4 T lymphocytes, whereas the KOS strain of HSV-1 preferentially activates CD8 T lymphocytes in the cornea. Langerhans cells (LC) normally reside only at the periphery of the cornea but can migrate centripetally after HSV-1 infection. We studied the relative contribution of LC to the corneal inflammation induced by the KOS and RE strains of HSV-1. Ten days after infection, the central one-third of RE HSV-1-infected corneas contained an average of 5.7 LC/high-power field compared with 0.6 LC/high-power field in KOS-infected corneas. We hypothesized that the increased density of LC in RE HSV-1-infected corneas at the time of T lymphocyte infiltration contributed to the preferential activation of CD4 T lymphocytes in these corneas. To test this hypothesis, we gave mice a low dose of UV-B corneal irradiation (150 mJ/cm2) 1 day before infection with HSV-1. UV-B irradiation effectively prevented the migration of LC into the central cornea when measured 10 or 21 days after corneal infection with either HSV-1 strain. UV-B corneal irradiation had no effect on the CTL response to HSV-1 Ag in the regional lymph nodes after corneal infection with KOS or RE HSV-1. The delayed-type hypersensitivity response induced by both strains of virus, when measured 8 and 14 days after corneal infection, was significantly reduced by UV-B irradiation. UV-B irradiation significantly reduced the incidence (p = 0.0023) and severity (p = 0.0008) of corneal stromal disease induced by RE HSV-1 but did not significantly affect the stromal disease induced by KOS HSV-1. To distinguish between the effect of UV-B treatment on the afferent and efferent arms of the Ir in mice, we administered UV-B treatment to one eye, followed 24 h later by RE HSV-1 infection of both eyes. These mice developed a normal delayed-type hypersensitivity response, and stromal inflammation developed normally in the untreated eye. However, stromal inflammation was significantly reduced in the treated eye. Our findings suggest that LC play a critical role in the activation of HSV-reactive CD4 T lymphocytes in the cornea. Moreover, the type of corneal inflammation induced by different strains of HSV-1 may reflect their differential capacity to induce LC migration into the central cornea.     

TNF-alpha receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-alpha and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. beta-Galactosidase expression in mice receiving intratracheal instillation of Adv/beta-gal (adenovirus construct expressing beta-galactosidase) was transient (less than 14 days), but a significant early increase of beta-galactosidase expression was seen in mice lacking either or both TNF-alpha receptors. Absence of TNF-alpha or the p55 receptor significantly attenuated the Ab response to both adenovirus and beta-galactosidase. Human IL-10 expression in the lung suppressed local TNF-alpha production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with beta-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-alpha signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with beta-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with beta-galactosidase, and is nonimmunogenic in the lung.     

Macrophages restrict Pseudomonas aeruginosa growth,regulate polymorphonuclear neutrophil influx,and balance pro- and anti-inflammatory cytokines in BALB/c mice

The role of macrophages in Pseudomonas aeruginosa corneal infection in susceptible (cornea perforates), C57BL/6 (B6) vs resistant (cornea heals), BALB/c mice was tested by depleting macrophages using subconjunctival injections of clodronate-containing liposomes before corneal infection. Both groups of inbred mice treated with clodronate-liposomes compared with PBS-liposomes (controls) exhibited more severe disease. In B6 mice, the cornea perforated and the eye became extremely shrunken, whereas in BALB/c mice, the cornea perforated rather than healed. The myeloperoxidase assay detected significantly more PMN in the cornea of both groups of mice treated with clodronate-liposomes vs PBS-liposomes. In independent experiments, ELISA analysis showed that protein levels for IL-1 beta, macrophage-inflammatory protein 2, and macrophage-inflammatory protein 1 alpha, all regulators of PMN chemotaxis, also were elevated in both groups of mice treated with clodronate-liposomes. Bacterial plate counts in B6 mice treated with clodronate-liposomes were unchanged at 3 days and were higher in control-treated mice at 5 days postinfection (p.i.), whereas in BALB/c mice, bacterial load was significantly elevated in the cornea of mice treated with clodronate-liposomes at both 3 and 5 days p.i. mRNA expression levels for pro (IFN-gamma and TNF-alpha)- and anti (IL-4 and IL-10)-inflammatory cytokines also were determined in BALB/c mice treated with clodronate-liposomes vs control-treated mice. Expression levels for IFN-gamma were significantly elevated in mice treated with clodronate-liposomes at 3 and 5 days p.i., while IL-10 levels (mRNA and protein) were reduced. These data provide evidence that macrophages control resistance to P. aeruginosa corneal infection through regulation of PMN number, bacterial killing and balancing pro- and anti-inflammatory cytokine levels.     

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.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Local expression of tumor necrosis factor alpha and interleukin-2 correlates with protection against corneal scarring after ocular challenge of vaccinated mice with herpes simplex virus type 1.

To correlate specific local immune responses with protection from corneal scarring, we examined immune cell infiltrates in the cornea after ocular challenge of vaccinated mice with herpes simplex virus type 1 (HSV-1). This is the first report to examine corneal infiltrates following ocular challenge of a vaccinated mouse rather than following infection of a naive mouse. Mice were vaccinated systemically with vaccines that following ocular challenge with HSV-1 resulted in (i) complete protection against corneal disease (KOS, an avirulent strain of HSV-1); (ii) partial protection, resulting in moderate corneal disease (baculovirus-expressed HSV-1 glycoprotein E [gE]); and (iii) no protection, resulting in severe corneal disease (mock vaccine). Infiltration into the cornea of CD4+ T cells, CD8+ T cells, macrophages, and cells containing various lymphokines was monitored on days 0, 1, 3, 7, and 10 postchallenge by immunocytochemistry of corneal sections. Prior to ocular challenge, no eye disease or corneal infiltrates were detected in any mice. KOS-vaccinated mice developed high HSV-1 neutralizing antibody titers (> 1:640) in serum. After ocular challenge, they were completely protected against death, developed no corneal disease, and had no detectable virus in their tear films at any time examined. In response to the ocular challenge, these mice developed high local levels of infiltrating CD4+ T cells and cells containing interleukin-2 (IL-2), IL-4, IL-6, or tumor necrosis factor alpha (TNF-alpha). In contrast, only low levels of infiltrating CD8+ T cells were found, and gamma interferon (IFN-gamma)-containing cells were not present until day 10. gE-vaccinated mice developed neutralizing antibody titers in serum almost as high as those of the KOS-vaccinated mice (> 1:320). After ocular challenge, they were also completely protected against death. However, the gE-vaccinated mice developed low levels of corneal disease and virus was detected in one-third of their eyes. Compared with KOS-vaccinated mice, the gE-vaccinated mice had a similar pattern of IFN-gamma, but a delay in the appearance of CD4+ T cells, CD8+ T cells, and IL-4-, IL-6-, and TNF-alpha-containing cells. In sharp contrast to those of the KOS-vaccinated mice, no cells containing IL-2 were detected in the eyes of gE-vaccinated mice at any time. Mock-vaccinated mice developed no detectable neutralizing antibody titer and were not protected from lethal HSV-1 challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

TNF regulates thymocyte production by apoptosis and proliferation of the triple negative (CD3-CD4-CD8-) subset

TNF is a proinflammatory cytokine with opposing death/no-death effects in vivo and in vitro. Our studies showed that TNF regulates mouse thymocyte production, inducing both apoptosis and proliferation of the most immature CD3(-)CD4(-)CD8(-) triple negative (TN) subset within a broad range of dosages (10(1)-10(5) pg/ml) in the presence of IL-7. TNF apoptosis affected only the TN3 (CD44(-)CD25(+)) and TN4 (CD44(-)CD25(-)) subsets that expressed both TNFR-p55 and -p75. Although each TNFR alone could mediate TNF apoptosis, maximal apoptosis was seen in C57BL/6J wild type, which expressed both TNFRs. TNF also induced proliferation of TN3 cells at higher doses (10(4)-10(5) pg/ml) mediated only by TNFR-p75. Both anti-TNFR-p55 and -TNFR-p75 mAb inhibited apoptosis but only anti-p75 inhibited proliferation. TNF also regulated TN proliferation to IL-7 because TNFR knockout (KO), TNF KO, and TNF/lymphotoxin alpha and beta triple KO mice showed 2- to 3-fold increased responses not seen in C57BL/6J wild type. In vivo, TNFR KO mice showed thymic hypertrophy with a 60% increase in total thymocytes, with no effect on the CD4/CD8 subsets. We conclude that TNF maintains homeostatic control of total thymocyte production by negative selection of TN3 and TN4 prothymocytes and down-regulation of their proliferation to endogenous IL-7.     

Bone marrow B cell apoptosis during in vivo influenza virus infection requires TNF-alpha and lymphotoxin-alpha

Suppression of bone marrow myeloid and erythroid progenitor cells occurs after infection with a variety of different viruses. In this study, we characterize the alterations in bone marrow (BM) lymphocytes after influenza virus infection in mice. We found a severe loss of BM B cells, particularly CD43(low/-)B220(+) pre-B and immature B cells, in influenza virus-infected mice. Depletion of BM B lineage cells resulted primarily from cell cycle arrest and most likely apoptosis within the BM environment, rather than from increased trafficking of BM emigrants to peripheral lymphoid tissues. Use of gene-knockout mice indicates that depletion of BM B cells is dependent on TNF-alpha, lymphotoxin-alpha, and both TNF receptors, TNFR1-p55 and TNFR2-p75. Thus, TNF-alpha and lymphotoxin-alpha are required for loss of BM B lineage cells during respiratory infection with influenza virus.