Tumor necrosis factor-α signaling in macrophages

Tumor necrosis factor-α (TNFα) was cloned over 2 decades ago and its identification in part led to the discovery of a super family of tumor necrosis factors (TNFs) and their receptors. TNFα signals through two transmembrane receptors, TNFR1 and TNFR2, and regulates a number of critical cell functions including cell proliferation, survival, differentiation, and apoptosis. Macrophages are the major producers of TNFα and interestingly are also highly responsive to TNFα. Aberrant TNFα production and TNF receptor signaling have been associated with the pathogenesis of several diseases, including rheumatoid arthritis, Crohn's disease, atherosclerosis, psoriasis, sepsis, diabetes, and obesity. TNFα has been shown to play a pivotal role in orchestrating the cytokine cascade in many inflammatory diseases and because of this role as a "master-regulator" of inflammatory cytokine production, it has been proposed as a therapeutic target for a number of diseases. Indeed anti-TNFα drugs are now licensed for treating certain inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. In this review we discuss the discovery of TNFα and its actions especially in regulating macrophage biology. Given its importance in several human diseases, we also briefly discuss the role of anti-TNFα therapeutics in the treatment of inflammatory diseases.     

The role of tumour necrosis factor-α and tumour necrosis factor receptor signalling in inflammation-associated systemic genotoxicity

Chronic inflammatory diseases are characterised by systemically elevated levels of tumour necrosis factor (TNF)-α, a proinflammatory cytokine with pleiotropic downstream effects. We have previously demonstrated increased genotoxicity in peripheral leukocytes and various tissues in models of intestinal inflammation. In the present study, we asked whether TNF-α is sufficient to induce DNA damage systemically, as observed in intestinal inflammation, and whether tumour necrosis factor receptor (TNFR) signalling would be necessary for the resultant genotoxicity. In the wild-type mice, 500 ng per mouse of TNF-α was sufficient to induce DNA damage to multiple cell types and organs 1-h post-administration. Primary splenic T cells manifested TNF-α-induced DNA damage in the absence of other cell types. Furthermore, TNFR1(-/-)TNFR2(-/-) mice demonstrated decreased systemic DNA damage in a model of intestinal inflammation and after TNF-α injection versus wild-type mice, indicating the necessity of TNFR signalling. Nuclear factor (NF)-κB inhibitors were also able to decrease damage induced by TNF-α injection in wild-type mice. When TNF-α administration was combined with interleukin (IL)-1β, another proinflammatory cytokine, DNA damage persisted for up to 24 h. When combined with IL-10, an anti-inflammatory cytokine, decreased genotoxicity was observed in vivo and in vitro. TNF-α/TNFR-mediated signalling is therefore sufficient and plays a large role in mediating DNA damage to various cell types, subject to modulation by other cytokines and their mediators.     

Interleukin 8 and mast cell-generated tumor necrosis factor-α in neutrophil recruitment

In the reverse passive Arthus reaction in mouse skin and immune injury of mouse dermal basement membrane, neutrophil (PMN) infiltration in mast cell deficient WBB6F₁-W/W^v (W/W^v) mice was only 40% of that in WBB6F₁-+/+ (+/+) mice that had a normal mast cell repertoire. An anti-tumor necrosis factor- (TNF-) monoclonal antibody (mAb) decreased PMN infiltration by 35–80% in +/+ but not W/W^v mice. In addition, an anti-human interleukin-8 (IL-8) MAb, DM/C7, inhibited PMN infiltration of the skin induced by either intradermal administration of recombinant human IL-1 or immune complex deposition. In both models of immune complex injury, DM/C7 reduced PMN infiltration by 40–60% in +/+ mice but not W/W ^v mice. PMN infiltration and the sensitivity of this infiltration to anti-TNF- or DM/C7 MAb in W/W^v mice whose mast cell population had been restored was indistinguishable from the influx observed in +/+ mice. These data suggest that TNF-, IL-8, and mast cells play a fundamental role in PMN recruitment following immune complex injury.     

Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis.     

Pneumocystis carinii stimulates in vitro production of tumor necrosis factor-α by human macrophages

The ability of Pneumocystis carinii to induce tumor necrosis factor (TNF)- release by monocytes/macrophages from immunocompetent humans was investigated. Monocytes and monocyte-derived macrophages from healthy individuals produced an increased amount of TNF- when exposed to P. carinii cysts obtained from rats with steroid-induced pneumocystosis. The cysts induced increased TNF- production in a dose-dependent manner; baseline TNF- production was restored after addition of an anti-P. carinii hyperimmune serum. Kinetics experiments showed that the secretion of TNF- occurs early and reachs a maximal peak after 8 h. Since TNF- is directly lethal to P. carinii in vitro, it is suggested that the production of this cytokine in response to the cysts may be one of the mechanisms for the control of this parasitic infection.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

The effect of tumour necrosis factor-α and insulin on equine digital blood vessel function in vitro

Objective and design

Insulin and inflammatory cytokines may be involved in equine laminitis, which might be associated with digital vascular dysfunction. This study determined the effects of TNF-α and insulin on the endothelial-dependent relaxant responses of equine digital blood vessels and on equine digital vein endothelial cell (EDVEC) cGMP production.

Material

Isolated rings of equine digital arteries (EDAs) and veins (EDVs) were obtained and EDVECs were cultured from horses euthanized at an abattoir.

Methods

The effect of incubation with TNF-α (10 ng/ml) and/or insulin (1,000 μIU/ml) for 1.5 h or overnight under hyperoxic and hypoxic conditions on carbachol (endothelium-dependent) induced relaxation was assessed. The time course and concentration dependency of the effect of TNF-α and the effect of insulin (1,000 μIU/ml) on EDVEC cGMP production was determined.

Results

Incubation of EDAs overnight with TNF-α under hypoxic conditions resulted in endothelial-dependent vascular dysfunction. EDVs produced a more variable response. TNF-α increased EDVEC cGMP formation in a time- and concentration-dependent manner. Insulin had no significant effects.

Conclusions

There is a mismatch between the results obtained from isolated vessel rings and cultured endothelial cells suggesting TNF-α may reduce the biological effect of NO by reducing its bioavailability rather than its formation, leading to endothelial cell dysregulation.     

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1) and tumor necrosis factor-alpha (TNF-) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 and TNF- were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 and TNF- were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)₂-vitamin D₃, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF- stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 and TNF- failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 and l nM TNF-) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.     

The role of Gr-1+ cells and tumour necrosis factor-α signalling during Clostridium difficile colitis in mice

The host response to Clostridium difficile infection in antibiotic-treated mice is characterized by robust recruitment of Gr-1⁺ cells, increased expression of inflammatory cytokines including tumour necrosis factor-α (TNF-α), and the development of severe epithelial damage. To investigate the role of Gr-1⁺ cells and TNF-α during C. difficile colitis, we treated infected mice with monoclonal antibodies against Gr-1 or TNF-α. Mice were challenged with vegetative cells of C. difficile strain VPI 10463 following treatment with the third-generation cephalosporin ceftriaxone. Ceftriaxone treatment alone was associated with significant changes in cytokine expression within the colonic mucosa but not overt inflammatory histopathological changes. In comparison, C. difficile infection following ceftriaxone treatment was associated with increased expression of inflammatory cytokines and chemokines including Cxcl1, Cxcl2, Il1b, Il17f and Tnfa, as well as robust recruitment of Ly6C^Mid Gr-1^High neutrophils and Ly6C^High Gr-1^Mid monocytes and the development of severe colonic histopathology. Anti-Gr-1 antibody treatment resulted in effective depletion of both Ly6C^Mid Gr-1^High neutrophils and Ly6C^High Gr-1^Mid monocytes: however, we observed no protection from the development of severe pathology or reduction in expression of the pro-inflammatory cytokines Il1b, Il6, Il33 and Tnfa following anti-Gr-1 treatment. By contrast, anti-TNF-α treatment did not affect Gr-1⁺ cell recruitment, but was associated with increased expression of Il6 and Il1b. Additionally, Ffar2, Ffar3, Tslp, Tff and Ang4 expression was significantly reduced in anti-TNF-α-treated animals, in association with marked intestinal histopathology. These studies raise the possibility that TNF-α may play a role in restraining inflammation and protecting the epithelium during C. difficile infection.     

Tumour necrosis factor-α drives Alport glomerulosclerosis in mice by promoting podocyte apoptosis

Chronic renal failure involves the progressive loss of renal parenchymal cells. For example, Alport syndrome develops from mutated type IV collagen that fosters the digestion of glomerular basement membranes and podocyte loss, followed by progressive glomerulosclerosis, ie Alport nephropathy. Here we show that autosomal recessive Alport nephropathy in collagen 4a3-deficient mice is associated with increased intrarenal expression of the pro-apoptotic cytokine tumour necrosis factor-alpha (TNF-α) in glomerular cells including podocytes as well as in infiltrating leukocytes. We therefore hypothesized that TNF-α contributes to Alport glomerulosclerosis by inducing podocyte apoptosis. To address this issue, we treated 4-week-old collagen 4a3-deficient mice with either vehicle or the TNF-α antagonist etanercept for a period of 5 weeks. Etanercept treatment prolonged mean survival from 68 to 81 days as compared to vehicle-treated mice. The beneficial effect of etanercept on survival was associated with a significant improvement of the glomerulosclerosis score, proteinuria, and the glomerular filtration rate at 9 weeks of age. Etanercept treatment specifically reduced the numbers of apoptotic podocytes, increased total podocyte counts, and increased the renal mRNA expression of nephrin and podocin without affecting markers of renal inflammation. TNF-α-induced podocyte loss is a previously unrecognized pathological mechanism of Alport glomerulosclerosis, and TNF-α blockade might be a therapeutic option to delay the progression of Alport nephropathy and potentially of other forms of glomerulosclerosis.     

Suppressive effects of E3330, a novel quinone derivative,on tumor necrosis factor-α generation from monocytes and macrophages

E3330 {(2E)-3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonly-2-propenoic acid}, a novel synthesized hepatoprotective compound, has suppressive effects on tumor necrosis factor- (TNF-) generation from monocytes/macrophagesin vitro. E3330 (1–100 M) reduced lipopolysaccharide (LPS, 10 mg/ml or 1g/ml)-induced TNF- generation from rat resident andPropionibacterium acnes (P. acnes)-elicited peritoneal macrophages, rat and human monocytes, rat Kupffer cells, and splenic mononuclear cells in a concentration-dependent manner. E3330 also (1–100 M) suppressed TNF- generation stimulated with egg-albumin immune complex in ratP. acnes-elicited peritoneal macrophages. Northern blot analysis showed that LPS-induced expression of TNF- messenger RNA (mRNA) in human blood monocytes was suppressed by E3330. These findings indicate that E3330 has a suppressive effect on TNF- generation from monocytes/macrophages, regardless of origin or species, and this effect is based in part on the suppression of TNF- mRNA expression.     

Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit.     

MicroRNA-23a is involved in tumor necrosis factor-α induced apoptosis in mesenchymal stem cells and myocardial infarction

Cell therapy has emerged as an attractive therapeutic modality to treat myocardial infarction (MI) via repairing damaged myocardium, and mesenchymal stem cells (MSCs) are an appealing therapeutic approach for cardiac regeneration. However, the clinical application of MSC-based therapy is restricted because of the poor survival of implanted cells, and this poor survival remains poorly understood. Using a tumor necrosis factor (TNF)-α-induced bone marrow (BM)-MSC injury model in vitro and a rat MI model in vivo, we showed in the current study that miR-23a was involved in TNF-α-induced BM-MSC apoptosis through regulating caspase-7 and that the injection of BM-MSCs overexpressing miR-23a could improve left ventricular (LV) function and reduce infarct size in the rat MI model. Our findings elucidate the etiology of MI and provide an alternative treatment strategy for patients with heart failure caused by MI who are not optimal candidates for surgical treatment.     

Distribution pattern of matrix metalloproteinases 1, 2, 3, and 9, tissue inhibitors of matrix metalloproteinases 1 and 2, and α2-macroglobulin in cases of generalized AA- and AL amyloidosis

Matrix metalloproteinases (MMPs) degrade basement membranes and connective tissue and play an essential role in the homeostasis of the extracellular matrix which is disrupted by the deposition of amyloid. This immunohistochemical study investigated the distribution pattern of matrix metalloproteinases (MMP-1, -2, -3, and -9) and their inhibitors [alpha 2-macroglobulin (alpha 2-M), tissue inhibitors of MMPs (TIMP)-1, and TIMP-2] in human AA- and AL amyloid deposits. Specimens of liver, kidney, and spleen from 22 autopsy cases were investigated. Nine patients had suffered from generalized AA amyloidosis, eight from generalized AL amyloidosis, and five from rheumatoid arthritis or tuberculosis with no histological evidence of amyloid. In all amyloidotic and non-amyloidotic patients, each protease and protease inhibitor was detected in almost every organ investigated. In the amyloidotic cases, there was no indication that a specific protease or protease inhibitor was absent or expressed, but a difference was observed in their spatial distribution patterns. The most noticeable difference was found in immunostaining of amyloid. Only MMP-1, -2, and -3, and alpha 2-M were present in AA amyloid deposits, and only TIMP-1 and TIMP-2 were found in deposits of AL amyloid. This is the first study to show that MMP-1, -2, and -3 are present in AA amyloid deposits. They may be involved in tissue remodeling or in proteolysis of the precursor and fibril proteins.