TNF-induced enterocyte apoptosis and detachment in mice: induction of caspases and prevention by a caspase inhibitor, ZVAD-fmk

Injection of mouse recombinant TNF to mice induced apoptosis and detachment of the enterocytes of the tip of the villi, evident after 30 to 90 minutes, which resulted in a shrinkage of the villi. Injection of TNF increased the expression of caspase 1, 2, 3, and 6 as well as of cathepsin D in the mucosal wall, which was maximal 30 minutes after TNF injection. Caspase 1 and 3 were not induced in TNFR1-deficient mice in which TNF does not induce apoptosis and detachment. The administration of a caspase inhibitor (ZVAD-fmk, 300 microg) decreased enterocyte detachment and apoptosis, as well as villus atrophy, whereas a caspase 3 inhibitor (Z-DEVD-cmk) had no effect. The results indicate that the induction of caspases by TNF is the cause of their detachment in the lumen and of the resulting villus atrophy.     

The Role of p53 in Bleomycin-Induced DNA Damage in the Lung : A Comparative Study with the Small Intestine

To elucidate the role of p53 and apoptosis in the pathogenesis of lung injury, we examined histological changes, expressions of p53 and p21waf1/cip1 (p21), apoptosis, DNA double strand breaks, cell kinetics, and DNA synthesis in C57/BL6 mice (p53+/+) and mice deficient for p53 (p53-/-) at 2 hours to 7 days after a single intravenous administration of bleomycin. We also compared these parameters between the lung cells and small intestinal epithelial cells to explore potential differences in their response to DNA damage. Bleomycin induced p21 expression in a p53-dependent manner in p53+/+ mice but neither p53 nor p21 expression in p53-/- mice. In the lung of both groups of mice, focal inflammation followed by fibrosis was observed, but there was no evidence of apoptosis. Cells with DNA breaks and those undergoing DNA synthesis were unequivocally increased, but the cycling cell fraction remained unchanged, suggesting that the DNA synthesis detected in the lung reflected unscheduled DNA synthesis for repair of damaged DNA. DNA breaks and unscheduled DNA synthesis were prolonged in p53-/- mice compared to p53+/+ mice. By contrast, in the small intestine, marked cell cycle arrest and extensive apoptosis were evoked in the cycling crypt cells of both groups of mice, but these changes were milder and DNA breaks remained detectable for a longer time in p53-/- mice than in p53+/+ mice. Among the resting enterocytes in the villi, apoptosis was observed almost equally in both groups, but repair of DNA breaks was significantly delayed in the p53-/- mice. These observations imply that apoptosis is mediated largely by the p53-dependent pathway in the crypts but exclusively by the p53-independent pathway in the villi, that this pathway is particularly important in DNA repair in the villi, and that despite this difference in the significance of apoptosis, p53 plays an important role in DNA repair in both the crypts and villi. Our results suggest that the lung cells and small intestinal cells respond to the bleomycin treatment in different ways in terms of the induction of apoptosis and that p53 carries out an essential role in the early response to and repair of DNA damage by a non-apoptotic mechanism which appears to be crucial in the noncycling lung cells and enterocytes. Importantly, the p53-p21 pathway and apoptosis are unlikely to be essential for bleomycin-induced tissue injury in the lung.     

Induction of CD14 expression in Lpsn,Lpsd and tumor necrosis factor receptor-deficient mice

The involvement of CD14 in lipopolysaccharide (LPS) recognition and signaling has been demonstrated in several studies. For this reason, we investigated whether the resistance of Lps^d mice to LPS might be related to an impaired CD14 expression. We compared the in vivo and in vitro expression of CD14 in Lpsⁿ (LPS sensitive) and Lps^d mice, and its modulation by LPS, killed gramnegative and gram-positive bacteria and double-stranded (ds)RNA. Untreated Lpsⁿ and Lps^d cultured macrophages (MΦ), expressed similar amounts of CD14 mRNA and membrane-bound (m)CD14. LPS enhanced CD14 expression only in Lpsⁿ MΦ, while all bacteria, or dsRNA, enhanced CD14 in Lpsⁿ and Lps^d MΦ. Similarly, in vivo administration of LPS induced or enhanced CD 14 mRNA in different organs of Lpsⁿ mice only, while bacteria or dsRNA in both types of mouse. Furthermore, exogenous recombinant tumor necrosis factor (TNF) induced in vivo and in vitro enhanced CD 14 expression in Lpsⁿ, Lps^d and also in TNF receptor 2-deficient (TNFR2−/−) mice, but failed to do so in TNFR1−/− mice, showing that TNFR1 mediates the effect of TNF on CD14. However, LPS, bacteria and dsRNA induced CD14 in both TNFR2−/− and TNFR1−/− mice to a similar extent, revealing that this induction does not require TNF signaling.     

Crucial role of tumor necrosis factor (TNF) receptor 2 and membrane-bound TNF in experimental cerebral malaria

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of experimental cerebral malaria (CM), but the respective role of its two types of receptors has not been established. A significant increase in the expression of TNF-receptor 2 (TNFR2, p75), but not of TNFR1 (p55), was found on brain microvessels at the time of CM in susceptible animals. Moreover, mice genetically deficient for TNFR2 (Tnfr2°) were significantly protected from experimental CM, in contrast to TNFR1-deficient (Tnfr1°) mice, which were as susceptible as wild-type mice. To identify the factors involved in the protection from CM conferred by the lack of TNFR2, we assessed in both knockout and control mice the serum concentrations of mediators that are critical for the development of CM, as well as the up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the brain microvessels. No significant difference in serum levels of TNF and interferon-γ was found between infected wild-type and Tnfr1° or Tnfr2° mice. Interestingly, the pronounced ICAM-1 up-regulation and leukocyte sequestration, typically occurring in brain microvessels of CM-susceptible animals, was detected in infected control and Tnfr1° mice – both of which developed CM – whereas no such ICAM-1 up-regulation or leukocyte sequestration was observed in Tnfr2° mice, which were protected from CM. Making use of microvascular endothelium cells (MVEC) isolated from wild-type, Tnfr1° or Tnfr2° mice, we show that soluble TNF requires the presence of both TNF receptors, whereas membrane-bound TNF only needs TNFR2 for TNF-mediated ICAM-1 up-regulation in brain MVEC. Thus, only in MVEC lacking TNFR2, neither membrane-bound nor soluble TNF cause the up-regulation of ICAM-1 in vitro. In conclusion, these results indicate that the interaction between membrane TNF and TNFR2 is crucial in the development of this neurological syndrome.     

In vivo evidence for a functional role of both tumor necrosis factor (TNF) receptors and transmembrane TNF in experimental hepatitis

The significance of tumor necrosis factor receptor 1 (TNFR1) for TNF function in vivo is well documented, whereas the role of TNFR2 so far remains obscure. In a model of concanavalin A (Con A)-induced, CD4⁺ T cell-dependent experimental hepatitis in mice, in which TNF is a central mediator of apoptotic and necrotic liver damage, we now provide evidence for an essential in vivo function of TNFR2 in this pathophysiological process. We demonstrate that a cooperation of TNFR1 and TNFR2 is required for hepatotoxicity as mice deficient of either receptor were resistant against Con A. A significant role of TNFR2 for Con A-induced hepatitis is also shown by the enhanced sensitivity of transgenic mice overexpressing the human TNFR2. The ligand for cytotoxic signaling via both TNF receptors is the precursor of soluble TNF, i.e. transmembrane TNF. Indeed, transmembrane TNF is sufficient to mediate hepatic damage, as transgenic mice deficient in wild-type soluble TNF but expressing a mutated nonsecretable form of TNF developed inflammatory liver disease.     

A murine transmembrane tumor necrosis factor (TNF) transgene induces arthritis by cooperative p55/p75 TNF receptor signaling

The arthritogenic activities of tumor necrosis factor (TNF) and its p55TNF-receptor (R) have been well documented in experimental animal models of arthritis, and in transgenic mice expressing wild-type or mutant transmembrane human TNF proteins in their joints. In this study we show that chronic inflammatory arthritis also develops in transgenic mice made to overexpress a mutant transmembrane from of the murine TNF protein (muTNF_Δ1–12) which is known to utilize efficiently both the p55 and the p75TNFR. Cross-breeding of the transgene into a TNF knockout background did not alter development of disease. Analysis of TNF bioactivity in sera from lipopolysaccharide-stimulated mice or ex vivo macrophage cultures demonstrated that the muTNF_Δ1–12 protein accumulates on the cell surface and is not processed to bioactive soluble TNF, indicating that transmembrane TNF is by itself sufficient to mediate pathogenesis of arthritis. Furthermore, using TNFR knockout mice, it is shown that development of transmembrane TNF-mediated arthritis requires the presence of the p55TNFR but is significantly delayed in the absence of the p75TNFR, suggesting a positive cooperation between the two TNFR in the arthritogenic process. These results indicate that blocking the activities of both soluble and transmembrane TNF may be required to effectively neutralize the pathogenic potential of this cytokine in arthritis.     

Tumor necrosis factor (TNF) receptor type 1 (p55) is a main mediator for TNF-α-induced skin inflammation

Tumor necrosis factor α (TNF-α) is a pleiotropic proinflammatory cytokine that elicits a large number of biological effects, including inflammatory and immuno-regulatory responses. Biological activities of TNF-α are mediated by two distinct TNF receptors, p55 type 1 receptor (TNFR1) and p75 type 2 receptor (TNFR2). To determine the role of TNF-α in the induction of inflammatory responses in the skin, gene-targeted mutant mice lacking either TNFR1 or TNFR2 were painted with irritant chemicals. Both phenol and croton oil painting onto the ears induced less inflammation in TNFR1(?) mice than normal and TNFR2(?) mice. Intradermal injection of TNF-α (0.2–200 ng for 3 days) into the ear induced less inflammation in TNFR1(?) mice than in normal mice. TNFR2(?) mice developed a normal inflammatory reaction to high doses of TNF-α (20–200 ng for 3 days), while they showed minimal reactivity to low doses of TNF-α (0.2–2 ng for 3 days). TNF-α is known to trigger the release of a series of other cytokines and to induce the expression of cell adhesion molecules, thus contributing to the development of inflammation. The levels of protein and mRNA for interleukin (IL)-6 were elevated in keratinocytes from normal as well as TNFR2(?) mice after treatment with TNF-α, while keratinocytes from TNFR1(?) mice did not show any up-regulation of IL-6. TNF-α induced intercellular adhesion molecule (ICAM)-1 expression in the keratinocytes from normal and TNFR2(?) mice, but not in those from TNFR1(?) mice. These results indicate that TNFR1 is critical for induction of skin inflammation by TNF-α.     

Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

Lack of TNFR2 expression by CD4+ T cells exacerbates experimental colitis

TNF plays fundamental roles in the induction and perpetuation of inflammation. The effects of TNF are mediated through TNF receptor (TNFR) 1 or 2. As these two receptors mediate different functions, selective targeting of one receptor may represent a more specific treatment for inflammatory disorders than the complete blocking of TNF. TNFR2 expression is up‐regulated in inflammatory bowel disease. Hence, we directly assessed the role of TNFR2 signaling in the CD4⁺ T‐cell transfer model of colitis using TNFR2^?/? or WT mice as donors of colitogenic CD4⁺CD45RB^hi T cells for transfer into syngeneic RAG2^?/? or RAG2^?/?TNFR2^?/? recipient mice. Although the absence of TNFR2 expression by non‐lymphoid cells of the recipient mice does not influence the course of colitis, transfer of TNFR2^?/? CD4⁺ T cells leads to an accelerated onset of disease and to more severe signs of inflammation. The enhanced colitogenic potential of TNFR2^?/? CD4⁺ T cells is associated with reduced activation‐induced cell death, resulting in an increased accumulation of TNFR2^?/? CD4⁺ T cells. Hence, TNFR2 signaling is crucial for the TNF‐dependent contraction of the disease‐inducing T cells. Therefore, a selective blocking of TNFR2 may lead to exacerbation rather than attenuation of T‐cell‐mediated inflammatory disorders.     

Tumour necrosis factor receptors and apoptosis of alveolar macrophages during early infection with attenuated and virulent Mycobacterium bovis

Apoptosis of macrophages has been reported as an effective host strategy to control the growth of intracellular pathogens, including pathogenic mycobacteria. Tumour necrosis factor-α (TNF-α) plays an important role in the modulation of apoptosis of infected macrophages. It exerts its biological activities via two distinct cell surface receptors, TNFR1 and TNFR2, whose extracellular domain can be released by proteolysis forming soluble TNF receptors (sTNFR1 and sTNFR2). The signalling through TNFR1 initiates the majority of the biological functions of TNF-α, leading to either cell death or survival whereas TNFR2 mediates primarily survival signals. Here, the expression of TNF-α receptors and the apoptosis of alveolar macrophages were investigated during the early phase of infection with attenuated and virulent mycobacteria in mice. A significant increase of apoptosis and high expression of TNFR1 were observed in alveolar macrophages at 3 and 7 days after infection with attenuated Mycobacterium bovis but only on day 7 in infection with the virulent M. bovis. Low surface expression of TNFR1 and increased levels of sTNFR1 on day 3 after infection by the virulent strain were associated with reduced rates of apoptotic macrophages. In addition, a significant reduction in apoptosis of alveolar macrophages was observed in TNFR1^−/− mice at day 3 after bacillus Calmette–Guérin infection. These results suggest a potential role for TNFR1 in mycobacteria-induced alveolar macrophage apoptosis in vivo. In this scenario, shedding of TNFR1 seems to contribute to the modulation of macrophage apoptosis in a strain-dependent manner.     

Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice

IL-1 has a number of effects on T cell growth but a specific role for IL-1 in T cell responses in vivo has not been elucidated. In this study the role of IL-1 in Th1/Th2 responses was examined in mice deficient for the IL-1 type 1 receptor (IL-1RI −/−) during cutaneous Leishmania major infection or following immunization with keyhole limpet hemocyanin (KLH). After inoculation of L. major stationary phase promastigotes into the hind footpad, both IL-1RI−/− and wild-type (WT) mice developed small lesions which resolved spontaneously. Lymphnode cells from infected IL-1RI−/− mice produced significantly more IL-4 and IL-10 than those from WT mice following antigenic stimulation in vitro. Splenocytes from IL-1RI−/− and WT mice showed similar levels of antigen-induced proliferation. In contrast, splenocyte cultures from the IL-1RI−/− mice contained significantly more IL-4 than those from WT mice. Similar results were also obtained after immunization with KLH. While lymph node cells from both IL-1RI−/− and WT mice displayed similar levels of KLH-specific proliferation, those from IL-1RI −/− mice produced significantly more IL-4 than those from WT mice. Conversely, antigen-stimulated lymph node cells from WT mice secreted significantly greater amounts of IFN-γ as compared with those from IL-1RI −/− mice. These data indicate that while IL-1 is not required for mounting an immune response or antigen-dependent proliferation, it appears to be required for normal regulation of Th1/Th2 responses and may function to negatively regulate IL-4 expression.     

Radiation-Induced p53 and p21WAF-1/CIP1 Expression in the Murine Intestinal Epithelium : Apoptosis and Cell Cycle Arrest

p53-dependent expression of p21^WAF-1/CIP1 has been studied in murine intestinal epithelium after exposure to ionizing radiation. In un-irradiated small intestine, neither p53 nor p21^WAF-1/CIP1 could be detected by immunohistochemistry. After irradiation (8 Gy), there was a time- and dose-dependent increase in the expression of both proteins. In the small bowel, the positional expression of p53 and p21^WAF-1/CIP1 was similar but not coincident. Both proteins could be observed throughout the crypts with greatest frequency of expression over the first 15 cell positions, which includes the stem cell population (approximately positions 3 to 5) and the proliferating, transit cell population (approximately positions 5 to 15). p53-positive cells were primarily distributed toward the base of the crypt relative to p21^WAF-1/CIP1. Subdivision of the p53-positive cell population revealed that the cells with strongest p53 immunoreactivity were positioned farther toward the base of the crypt, and their distribution was approximately coincident with the frequency distribution of apoptotic cells. Cells that were either weakly or moderately immunoreactive for p53 were located toward the middle of the crypt and were approximately coincident with the distribution of p21^WAF-1/CIP1. The numbers of both p53- and p21^WAF-1/CIP1-positive cells declined steadily with time, and by 6 days after irradiation there were very few immunoreactive cells to observe. Radiation-induced increase in p53 and p21^WAF-1/CIP1 expression was not detected in mice homozygously null for p53. Expression of p21^WAF-1/CIP1 and incorporation of tritiated thymidine were found to be mutually exclusive. In the large bowel, p21^WAF-1/CIP1 and p53 expression were observed along the entire length of the colonic crypts after irradiation (8 Gy), and, unlike in the small intestine, this expression was not only maintained but increased over 72 hours. p21^WAF-1/CIP1 immunoreactivity was detected in large intestine epithelium up to 6 days after irradiation. The differential expression of p21^WAF-1/CIP1, observed between the large and small bowel and within the small intestinal crypts, is discussed.     

Expulsion ofEchinostoma trivolvis (Cort, 1914) Kanev, 1985 and retention ofE. caproni Richard, 1964 (Trematoda: Echinostomatidae) in C3H mice: pathological,ultrasturctural, and cytochemical effects on the host intestine

C3H mice were infected with 30 metacercarial cysts of either echinostome to study the pathological, ultrastructural, and cytochemical effects of the infection on the mouse small intestine. In mice infected withEchinostoma caproni, the intestine showed villous atrophy with fused or eroded villi. The microvilli of the enterocytes were sparse and distorted and showed reduced alkaline phosphatase activity. The crypts of Lieberkuhn were hyperplastic and showed a marked reduction in goblet and Paneth cells. As compared with uninfected controls, there was a marked reduction in glucose-6-phosphatase activity in the enterocytes of the infected gut. Collagen fibers and the number of fibroblasts were increased under the epithelium. In mice infected withE. trivolvis, the tips of the intestinal villi were bent and blunted. The microvilli of the enterocytes were less tightly packed than those of uninfected controls. The mitochondria in the enterocytes were irregularly shaped, contained intracristal bodies, and showed increased cytochrome oxidase activity as compared with those of uninfected controls. The crypts were hyperplastic but showed an increase in the numbers of goblet and Paneth cells. The fibroblasts and collagen fibers showed abnormal development. The ultrastructural and cytochemical differences seen in this study reflect the uniqueness of the host-parasite relationship of each of these echinostome species in the gut of the C3H mouse.     

p53 Mediates TNF-Induced Epithelial Cell Apoptosis in IBD

Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS^−/− mice. IEC p53 levels and apoptosis were reduced in IL-10^−/− colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF–treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF–treated CUC patients.Human inflammatory bowel diseases (IBDs) are characterized by excessive crypt epithelial apoptosis, surface ulceration, distorted crypt architecture, diarrhea, and bleeding. Barrier disruption is linked to epithelial apoptosis caused by aberrant activation of innate and adaptive immune responses.^1–3 A hallmark of severe IBD is the overproduction of tumor necrosis factor (TNF) in mucosal tissue.^2,4 The importance of TNF in disease pathogenesis is underlined by the pronounced clinical improvement induced when anti-TNF antibodies reduce diarrhea, weight loss, and bleeding.^4,5 At the mucosal level, anti-TNF antibody treatment enhances mucosal healing with rapid re-epithelialization of ulcerated surfaces. Studies indicate some (eg, infliximab and adalimumab), but not all (eg, certolizumab), anti-TNF agents induce apoptosis of lamina propria cells, despite all three being able to enhance mucosal healing.^6,7 However, it remains unclear whether the effect of anti-TNF on mucosal healing is related to reduced epithelial apoptosis and, if so, through what mechanism.Overproduction of TNF in IBD has potent effects on mucosal adaptive and innate immune responses.^8,9 TNF participates in macrophage activation by enhancing antimicrobial functions.¹⁰ In response to TNF, macrophages increase production of reactive nitrosative species, such as nitric oxide (NO⁻) and its metabolite, peroxynitrite (ONOO⁻).¹¹ Inducible NO synthase (iNOS) blockade inhibits disease severity and epithelial apoptosis in animal models of IBD.^12,13 Data from human IBD studies suggest that NO⁻ and ONOO⁻ stabilize p53 and activate response pathways.^14,15 During tumorigenesis, NO⁻-induced mutations of p53 inactivate tumor suppressor function, with loss of protective effects.¹⁶ Thus, TNF-mediated activation of iNOS may be an important pathway for regulating epithelial cell apoptosis during colitis and colitis-induced dysplasia.Understanding TNF receptor signaling is complex and difficult to apply to in vivo systems. TNF receptor 1 (TNFR1) associates with the TNF receptor–associated death domain, which activates the extrinsic, caspase 8–linked pathway of apoptosis.^17,18 However, in some systems examined, TNF receptor–associated death domain is dispensable for TNF-induced apoptosis, and cross activation of TNFRl and TNFR2 converges unto common downstream signaling events, resulting in apoptosis mediated by intrinsic (mitochondrial) pathways.^17,19 The proliferative zone for intestinal epithelial cells (IECs) resides in lower crypt regions. Cellular proliferation requires enhanced mitochondrial function. Given that epithelial apoptosis in IBD occurs in proliferative crypt epithelial cells, we suspected that pathways involving induction of mitochondrial pathways were used. In addition, a comprehensive understanding of the role of TNF receptor signaling within the mucosal microenvironment requires that receptor deficiency be restricted to distinct populations participating in mucosal immune responses.Increased epithelial crypt cell apoptosis commonly occurs in ulcerative colitis (UC) and Crohn''s disease.^20,21 Numerous in vitro and in vivo model systems have studied this phenomenon, suggesting that TNF-mediated pathways play key roles in inducing programmed cell death in epithelial crypts. To model these pathways, a well-characterized model of T-cell activation was used that induces transient stem/progenitor cell activation, crypt IEC proliferation, and TNF-mediated diarrhea reminiscent of human IBD.^22,23 The reproducible kinetics of the model permitted identification of the events in immune-mediated apoptosis and allowed application to relevant gene knockout models. We recently reported that p53 is the major mediator of colonic crypt IEC apoptosis in colitis.²⁴ This article examines the upstream events leading up to p53 activation and IEC apoptosis. Results suggest a mechanism by which TNF signals, through both TNFR1 and TNFR2, stimulate iNOS-mediated p53-dependent apoptosis of crypt IECs. Studies in the IL-10^−/− murine model of colitis confirmed that TNF-induced iNOS led to activation of p53 and induced IEC apoptosis. Finally, we confirm that TNF-induced p53-mediated apoptosis also occurs in vivo during human UC. Overall, the findings suggest that T-cell activation causes TNF and iNOS-mediated stabilization of p53, followed by p53-mediated crypt cell apoptosis in IBD. These data have direct relevance to mechanisms of barrier disruption, ulceration, and initiation of dysplasia seen in p53 mutant crypts.     

Type 1 innate lymphoid cell aggravation of atherosclerosis is mediated through TLR4

ILC populations elaborate a similar cytokine expression pattern with helper T cell subsets Th1, Th2 and Th17. Recent studies indicate that CD 25+ILC 2 could alleviate atherosclerosis by altering lipid metabolism, whereas the depletion of CD 90‐expressing ILC s had no influence on atherosclerosis. Thus, these findings raise the question of whether ILC 1 cells react on atherosclerosis. Hence, our group attempted to explore the role of ILC 1 cells in atherosclerosis. We found that ILC 1 cells have a high Th1‐like gene expression of T‐bet and IFN ‐γ, which is distinct from ILC 2, ILC 3 or conventional NK (cNK ) cells. Moreover, atherosclerotic lesions were greatly reduced in ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs for depleting ILC 1 cells (ILC 1+cNK cells), compared to ApoE−/−Rag1−/− mice treated with anti‐IL ‐15R mA bs for depleting cNK cells, and these effects could be fully rescued through the adoptive transfer of ILC 1 cells sorted from the spleen of ApoE−/−TLR 4+/+ mice into ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs. However, the adoptive transfer of ILC 1 cells sorted from the spleen of ApoE−/−TLR 4−/− mice into ApoE−/−Rag1−/− mice treated with anti‐NK 1.1 mA bs blocked the progression of atherosclerosis, indicating that the pro‐atherosclerotic role of ILC 1 cells is dependent on TLR 4. Furthermore, oxLDL ‐induced increase in IFN ‐γ expression from ApoE−/− ILC 1 cells was correlated with the decrease in BACH 2 expression. Taken together, ILC 1 cells exist in atherosclerosis and aggravate atherosclerosis via increasing pro‐inflammatory cytokine expression in a TLR 4/BACH 2‐dependent manner.     

TNF alpha promotes proliferation of oligodendrocyte progenitors and remyelination

Here we used mice lacking tumor necrosis factor-alpha (TNF alpha) and its associated receptors to study a model of demyelination and remyelination in which these events could be carefully controlled using a toxin, cuprizone. Unexpectedly, the lack of TNF alpha led to a significant delay in remyelination as assessed by histology, immunohistochemistry for myelin proteins and electron microscopy coupled with morphometric analysis. Failure of repair correlated with a reduction in the pool of proliferating oligodendrocyte progenitors (bromodeoxyuridine-labeled NG2(+) cells) followed by a reduction in the number of mature oligodendrocytes. Analysis of mice lacking TNF receptor 1 (TNFR1) or TNFR2 indicated that TNFR2, not TNFR1, is critical to oligodendrocyte regeneration. This unexpected reparative role for TNF alpha in the CNS is important for understanding oligodendrocyte regeneration/proliferation, nerve remyelination and the design of new therapeutics for demyelinating diseases.     

Immune down-regulation and peripheral deletion of CD8 T cells does not require TNF receptor-ligand interactions nor CD95 (Fas, APO-1)

TNF receptor-ligand interactions and CD95 (Fas / APO-1) have been demonstrated to be involved in activation-induced death of mature T cells. Here, we examined the role of these molecules in the murine model of lymphocytic choriomeningitis virus (LCMV) infection using LCMV TCR transgenic (tg) mice lacking TNF, TNF receptor I (TNFR1), CD95 or both TNFR1 and CD95. This report demonstrates that neither TNF receptor-ligand interactions nor CD95 was required for down-regulation of LCMV-specific CD8 T cells following acute LCMV infection in vivo. Even LCMV-specific CD8 T cells lacking both TNFR1 and CD95 molecules declined after the acute phase of the infection with normal kinetics. Furthermore, peripheral deletion of LCMV-specific CD8 T cells induced by LCMV peptide injection or by adoptive transfer of tg spleen cells expressing the corresponding LCMV epitope was not impaired in mice lacking TNF, TNFR1 and / or CD95. Our data speak against an indispensable role of these molecules in antigen-induced apoptosis of CD8 T cells in vivo and suggest that T cell homeostasis after antigen challenge is controlled by additional mechanisms.