Differential effects of tumour necrosis factor-α and interleukin-12 on isopentenyl pyrophosphate-stimulated interferon-γ production by cord blood Vγ9 T cells

Lower numbers of Vγ9Vδ2 T cells in cord blood (CB) than in adult peripheral blood (PB), as well as their impaired ability to produce interferon-γ (IFN-γ) in response to stimulation, are associated with functional deficiency in the immune system in newborns. In this study, we stimulated CB Vγ9 T cells with their T-cell receptor-specific ligand, isopentenyl pyrophosphate (IPP), plus exogenous costimulatory cytokines such as interleukin-2 (IL-2), IL-12 and tumour necrosis factor-α (TNF-α), which are known to play important roles in the activation of PB γδ T cells. Our data show that CB Vγ9 T cells are able to produce IFN-γ at levels comparable to PB Vγ9 T cells by the addition of TNF-α in the presence of IPP and IL-2; however, under the same culture conditions, IL-12 does not efficiently activate CB Vγ9 T cells to produce IFN-γ. The frequency of TNF-α receptor II-positive Vγ9T cells and the expression levels of TNF-α receptor II are similar in CB and PB; in contrast, the frequency of IL-12 receptor βI (IL-12RβI)-positive Vγ9T cells and expression levels of IL-12RβI are significantly lower in CB than PB. TNF-α but not IL-12 increases the expression of IL-2Rβ on CB Vγ9 T cells. These results provide new insights into the role of TNF-α in the activation of CB Vγ9 T cells.     

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.     

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.     

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.     

Molecular mechanisms of the biphasic effects of interferon-γ on osteoclastogenesis

Although interferon-γ (IFN-γ) potently inhibits osteoclastogenesis, the suppressive effect is significantly reduced when osteoclast precursors are pre-exposed to the receptor activator of NF-κB (RANK) ligand (RANKL). However, the molecular mechanism underlying the biphasic effects of IFN-γ on osteoclastogenesis remains elusive. Here, we recapitulate the biphasic functions of IFN-γ in osteoclastogenesis in both tissue culture dishes and on bone slices. We further demonstrate that IFN-γ markedly suppresses the RANKL-induced expression of nuclear factor of activated T-cells c1 (NFATc1) in normal, but not RANKL-pretreated bone marrow macrophages (BMMs). Similarly, IFN-γ impairs the activation of the nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in normal, but not RANKL-pretreated, BMMs. These findings indicate that IFN-γ inhibits osteoclastogenesis partially by suppressing the expression of NFATc1 and the activation of the NF-κB and JNK pathways. Moreover, IFN-γ inhibits the RANKL-induced expression of osteoclast genes, but RANKL pretreatment reprograms osteoclast genes into a state in which they can no longer be suppressed by IFN-γ, indicating that IFN-γ inhibits osteoclastogenesis by blocking the expression of osteoclast genes. Finally, the IVVY(535-538) motif in the cytoplasmic domain of RANK is responsible for rendering BMMs refractory to the inhibitory effect of IFN-γ. Taken together, these findings provide important mechanistic insights into the biphasic effects of IFN-γ on osteoclastogenesis.     

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.     

Continuous recruitment, co-expression of tumour necrosis factor-α and matrix metalloproteinases, and apoptosis of macrophages in gout tophi

Gout tophi are characterised by foreign body granulomas consisting of mono- and multinucleated macrophages surrounding deposits of monosodium urate microcrystals. After primary formation, granulomas grow associated with degradation of the extracellular matrix. Based on this background, we have sought (1) to investigate whether during granuloma's growth new macrophages are recruited into the tophi, (2) to find in situ evidence for macrophages' active role in matrix degradation and (3) to examine whether shrunk cells seen within gout tophi are apoptotic. Immunohistochemistry showed that perivascular localised mononuclear cells are CD68+, S100A8+, S100A9+, 25F9-, representing freshly migrated monocytes/macrophages. In contrast, almost all CD68+ mono- and multinucleated cells arranged within granulomas were S100A8-, S100A9-, 25F9+, representing mature (non-migrating) macrophages. Serial sections revealed that macrophages co-express tumour necrosis factor (TNF)-alpha and matrix metalloproteinases (MMPs) 2 and 9. In situ end-labelling of fragmented DNA demonstrated that CD68+ macrophages undergo apoptosis within gout tophi. Our data show that macrophages are continuously recruited into the gout tophi. These macrophages co-produce the proinflammatory cytokine TNF-alpha and two TNF-alpha inducible lytic enzymes, MMP-2 and MMP-9, suggesting that TNF-alpha may induce MMP production followed by matrix degradation within foreign body granulomas. In parallel, macrophages undergo apoptosis, a phenomenon that may restrict the destructive potential of inflammatory macrophages.     

Synergistic actions of pemphigus vulgaris IgG,Fas-ligand and tumor necrosis factor-α during induction of basal cell shrinkage and acantholysis

This study tested a recently proposed “Basal Cell Shrinkage” hypothesis of pemphigus acantholysis through a quantitative analysis of individual and cooperative effects of pemphigus vulgaris (PV) IgG, Fas-ligand (Fas-L) and tumor necrosis factor-α (TNFα) on keratinocyte (KC) volume (i.e. cell size) and adhesive properties. Exposure of KC monolayers and MatTek EpiDermFT? tissues cultures to the physiologic concentrations of Fas-L, TNFα or IgGs from two PV patients resulted in various degrees of reversible changes, which were not observed in control cultures either exposed to normal IgG or left intact. Within 12–24 h of exposure, basal cells in experimental cultures lost their ability to form stress fibers, retracted cytoplasmic aprons and formed keratin aggregates, indicating that their cytoskeleton collapsed. The cell volume decreased significantly (p < 0.05) as the polygonal cell shape changed to a round one. The shrunk cells detached from their neighbors and the substrate, resulting in a reciprocal increase of both the areas of acantholysis and the number of detached KCs, respectively. Since in the skin of PV patients, KCs are targeted by autoantibodies concomitantly with being exposed to autocrine and paracrine pro-apoptotic and pro-inflammatory cytokines, we combined PV IgG with Fas-L and/or TNFα in the cell culture experiments. This amplified several fold an ability of PV IgG to cause basal cell shrinkage and detachment. The obtained results demonstrated for the first time that PV IgG works together with Fas-L and TNFα to induce acantholysis via basal cell shrinkage, which provides a novel mechanism explaining successful treatment of PV patients with TNFα inhibitors.     

Alterations of serum tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and sialic acids in malignant ovine theileriosis

The study was designed to evaluate the effect of the severity of Theileria lestoquardi (hirci) infection on tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and sialic acid concentration in blood sera in a naturally infected sheep. Infected animals (diseased group) comprised 50 Iranian fat-tailed sheep, about 1–2 years old, naturally infected with Theileria lestoquardi (hirci), were divided into three subgroups according to parasitemia rates (<2%, 2–4%, and >4%). As a control group, ten noninfected sheep were also selected. Hematological parameters and levels of TNF-α, IFN-γ, and sialic acid (TSA, LBSA, and PBSA) concentrations were measured in both groups. The results showed significant increases in the level of TNF-α, IFN-γ, and sialic acid concentrations in infected sheep compared with noninfected ones (P?<?0.05). The increased level of TNF-α and IFN-γ in infected sheep may indicate the role of these cytokines to inhibit the growth and development of the parasite in infected host cells. Also, it is speculated that preventing the establishment of parasites by inhibiting the differentiation of trophozoites to schizonts is another aspect of the enhanced activity of these substances. In addition, increased levels of sialic acid concentration during parasitemia presumably stimulate the host immune response and influence the parasite–host cell adhesion. Clearly, further molecular and biochemical investigations are needed to elucidate the exact mechanisms involved in immunopathogenesis of malignant ovine theileriosis.     

Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes

Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.     

Modulation of the endothelial procoagulant response to lipopoly-saccharide and tumour necrosis factor-α in-vitro: The effects of dexamethasone,pentoxifylline, iloprost and a polyclonal anti-human IL-1α antibody

Endothelial expression of tissue factor (TF), a potent procoagulant molecule, is increased in response to inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF) and interleukin-1 (IL-1). We have examined the effects of three antiinflammatory agents and a polyclonal anti-human IL-1 antibody on the human endothelial TF response toE. coli 0111:B4 LPS and recombinant TNF (rTNF) in vitro. In contrast to the expected inhibitory effect, dexamethasone, pentoxyfilline and iloprost failed to block TF expression when administered simultaneously or 30 minutes prior to stimulation with either LPS or rTNF. Inhibition of procoagulant activity was demonstrated with the anti-IL-1 antibody, suggesting that endothelial derived IL-1 is partially responsible for the TF response to the agonists employed. The failure of the antiinflammatory agents to inhibit endothelial TF expression highlights the possibility that therapeutic agents that modulate the circulating monocyte response to LPS and TNF may not ameliorate the endothelial dysfunction that is also induced by these inflammatory mediators.     

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.     

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.     

Associations between cytotoxic T lymphocyte-associated antigen-4 polymorphisms and serum tumor necrosis factor-α and interferon-γ levels in patients with chronic hepatitis B virus infection

Background  

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) polymorphism, which may affect host immune response including cytokines production, is thought to be associated with hepatitis B virus (HBV) infection. This study investigated the associations between CTLA4 polymorphism and serum tumor necrosis factor (TNF)-α and interferon (IFN)-γ levels in patients with chronic HBV infection.     

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.