Sublytic complement protects prostate cancer cells from tumour necrosis factor-α-induced cell death

Inflammation is a critical component of tumour progression. Although complement and tumour necrosis factor (TNF)-α potentially exert significant anti-tumour effects, both mediators may also promote tumour progression. It has been demonstrated that sublytic complement confers resistance on tumour cells not only against lytic complement, but also other danger molecules such as perforin. In low concentrations, TNF promotes survival of malignant cells rather than exerting cytotoxic activity. In this study, we tested if sublytic complement is able to interfere with TNF-mediated tumour cell killing. Our results demonstrate that either subcytotoxic concentrations of TNF or sublytic complement rescue prostate carcinoma cells (DU145) from TNF-α-mediated cell death. Upon pretreatment with low-dose TNF-α, but not upon pre-exposure to sublytic complement, TNF resistance was associated with the down-regulation of TNF receptor 1 (TNF-R1) expression. Complement-induced protection against TNF-mediated apoptosis accompanied the induction of anti-apoptotic proteins [B cell leukaemia/lymphoma (Bcl)-2 and Bcl-xL] at an early stage followed by inhibition of the TNF-induced decrease in the amount of Bcl-2 and Bcl-xL. Cell protection also accompanied the inhibition of caspase-8 activation, poly (ADP-ribose) polymerase (PARP)-1 cleavage and the activation of nuclear factor (NF)-κB. Our data extend our current view on the induction of tumour cell resistance against cytotoxic mediators supporting the role of the tumour microenvironment in mediating protection against the anti-cancer immune response.     

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.     

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.     

Role of tumor necrosis factor-α in the pathogenesis of indomethacin-induced small intestinal injury in mice

The pathogenesis of small intestinal damage caused by non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is still unclear. For this reason, there is currently no therapeutic strategy for ameliorating such damage. On the other hand, molecular treatment strategies targeting tumor necrosis factor (TNF)-α exert beneficial effects on intestinal lesions in patients with inflammatory bowel disease (IBD). To clarify the participation of TNF-α in NSAID-induced small intestinal damage, we investigated the effects of indomethacin administration in mice with targeted deletion of the TNF-α gene. Indomethacin (10?mg/kg) was administered subcutaneously to male C57BL/6 (wild-type: WT) mice and TNF-α-deficient (TNF-α-/-) mice to induce small intestinal damage. The ulcer score, the tissue-associated myeloperoxidase (MPO) activity as an index of neutrophil infiltration, and the expression of keratinocyte chemoattractant (KC) mRNA in the small intestinal mucosa were measured. In addition, we performed a TUNEL assay to evaluate indomethacin-induced apoptosis of intestinal epithelial cells and measured the expression of caspase-3 protein and Bcl-2 mRNA. The ulcer score, MPO activity, and expression of KC mRNA were significantly increased after indomethacin administration. These increases were significantly inhibited in TNF-α-/- mice compared with WT mice. Apoptotic cells were observed by the TUNEL assay in the area of the ulcerative lesion, and they were significantly fewer in TNF-α-/- mice compared with WT mice. The expression of cleaved caspase-3 protein was induced by indomethacin administration, and significantly inhibited in TNF-α-/- mice compared with that of WT mice. The expression level of Bcl-2 mRNA in indomethacin-treated TNF-α-/- mice was significantly higher than that in WT mice. TNF-α plays an important role in the pathogenesis of indomethacin-induced small intestinal damage. These results suggest that TNF-α could become a new therapeutic target for NSAID-induced small intestinal damage.     

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.     

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.     

Neuropeptides stimulate production of interleukin-1β, interleukin-6, and tumor necrosis factor-α in human dental pulp cells

Objective:Orthodontic tooth movement causes inflammatory reactions in the periodontal membrane and dental pulp. It has been reported that substance P (SP) and calcitonin gene-related peptide (CGRP), both sensory neuropeptides, are manifested in the dental pulp of rats during experimental tooth movement, suggesting that they might be involved in the dental pulp inflammation during orthodontic tooth movement. However, the relationships between neuropeptides and pro-inflammatory cytokines have not been fully elucidated.Materials and methods:Human dental pulp (HDP) fibroblasts were prepared from 6 healthy young volunteers (3 males, 3 females; 15–25 years old) during the course of orthodontic treatment. HDP cells were incubated for 24 h in fresh medium containing 2% FCS in the presence of various concentrations of CGRP (10^–12 to 10^–4 M) and SP (10^–12 to 10^–4 M), and the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)- present in the media were determined using commercially available high-sensitivity enzyme-linked immunosorbent assay kit.Results:We examined the effects of stimulation by these neuropeptides on the production of inflammatory cytokines in HDP fibroblasts, and found that the levels of IL-1, IL-6, and TNF- increased in a time- and concentration-dependent manner. However, the neuropeptides did not act synergistically to increase cytokine secretion in HDP cells or significantly modify LPS-induced cytokine production by HDP cells.Conclusions:Our results suggest that human pulp fibroblasts may be involved in the progress of inflammation in pulp tissue during orthodontic tooth movement, as they produced large amounts of IL-1, IL-6, and TNF- following stimulation with neuropeptides.Received 2 March 2003; returned for revision 14 July 2003; accepted by M.J. Parnham 17 December 2003     

Induction of latency-associated peptide (transforming growth factor-β(1)) expression on CD4+ T cells reduces Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a transforming growth factor-β-dependent manner

CD4(+) T cells expressing the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β(1))] play an important role in the modulation of immune responses. Here, we identified a novel peptide ligand (GPC(81-95) ) with an intrinsic ability to induce membrane-bound LAP (TGF-β(1)) expression on a subpopulation of human CD4(+) T cells (using flow cytometry; ranging from 0·8% to 2·6%) and stimulate peripheral blood mononuclear cells to release LAP (TGF-β(1) ) (using ELISPOT assay; ranging from 0·03% to 0·16%). In spite of this low percentage of responding cells, GPC(81-95) significantly reduced Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a TGF-β(1) - and CD4(+) T-cell-dependent manner. The results demonstrate that GPC(81-95) is a useful tool to study the functional properties of a subpopulation of LAP (TGF-β(1))(+) CD4(+) T cells and suggest a pathway that can be exploited to suppress inflammatory response.     

Macrophages from IBD patients exhibit defective tumour necrosis factor-α secretion but otherwise normal or augmented pro-inflammatory responses to infection

Defects in macrophage function have been implicated in the establishment of Crohn's disease (CD). However, the response of macrophages from CD patients to live bacteria, particularly Mycobacterium avium subsp. paratuberculosis (MAP), has not been addressed. Considering MAP has long been associated to CD, our objective was to assess whether macrophages from CD patients showed impaired inflammatory response to infection by MAP comparing to M. avium subsp. avium (MA) and other live intestinal commensal bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, ulcerative colitis (UC) patients and controls. Following in vitro infection with MAP, MA, Escherichia coli or Enterococcus faecalis, cytokine levels and cell surface receptor expression were evaluated at different time points. Macrophages from CD patients showed impaired TNF-α secretion in response to bacterial challenge, but augmented IL-23 secretion and preserved IL-12 secretion and CD-40 expression. In addition, CD macrophages showed low IL-10 secretion. Macrophages from IBD patients showed increased expression of TLR-2 and -4, unaffected by infection. Differences in cytokine secretion observed after bacterial challenge were not MAP-specific, as other bacteria (E. coli and MA) showed similar effects. Macrophages from UC patients showed a less compromised TNF-α synthesis in response to mycobacterial infection than CD macrophages, with increased constitutive IL-12 secretion, and preserved IL-10 secretion. The increased IL-23 levels in response to infection and decreased IL-10 production observed in macrophages from CD patients may contribute to the inflammatory exacerbation observed in those patients.     

Tumor necrosis factor-α antibodies (infliximab,adalimumab and certolizumab) in Crohn's disease: systematic review and meta-analysis

Introduction

This meta-analysis compares the effectiveness and safety of tumor necrosis factor α (TNF-α) antibodies (infliximab, adalimumab and certolizumab) with either a placebo or each of them in the treatment of Crohn''s disease (CD).

Material and methods

A systematic review of literature published up to November 2012 was performed and a meta-analysis of identified studies was carried out. We searched the following databases: PubMed, EMBASE, The Cochrane Library and others. Only randomized or clinical controlled trials were included.

Results

Nineteen clinical trials fulfilled the established criteria (5 studies for infliximab vs. placebo, 6 for each adalimumab or certolizumab vs. placebo and 2 comparing infliximab with adalimumab). The results of meta-analysis showed that anti-TNF therapy in patients with CD is safe and statistically significantly more effective when compared with the placebo for induction of remission at week 4 (RB = 1.90, 95% CI: 1.55–2.33, p < 0.00001), maintenance of remission at weeks 20–30 (RB = 1.86, 95% CI: 1.61–2.15, p < 0.00001) and at weeks 48–56 (RB = 2.75, 95% CI: 2.13–3.54, p < 0.00001) in patients who responded to the induction therapy and patients randomized before the induction. Anti-TNF agents were also superior to the placebo in fistula healing (during short-term induction, as well as long-term maintenance) and inducing CR-70 but not CR-100 at week 4. Moreover, the anti-TNF therapy had a significant effect on achieving both CR-70 and CR-100 during long-term maintenance.

Conclusions

Infliximab, adalimumab and certolizumab are effective as both induction and maintenance therapy in moderate to severe Crohn''s disease in adults, including patients with fistulas. The safety profile was acceptable.     

Seladin-1 is a novel lipopolysaccharide (LPS)-responsive gene and inhibits the tumour necrosis factor-�� production and osteoclast formation in response to LPS

Selective Alzheimer disease indicator‐1 (seladin‐1) is a broadly expressed oxidoreductase and is related to Alzheimer disease, cholesterol metabolism and carcinogenesis. The effect of lipopolysaccharide (LPS) on the expression of seladin‐1 was examined using RAW 264.7 macrophage‐like cells and murine peritoneal macrophages. Lipopolysaccharide induced the expression of seladin‐1 protein and messenger RNA in those macrophages. The seladin‐1 expression was also augmented by a series of Toll‐like receptor ligands. The LPS augmented the expression of seladin‐1 via reactive oxygen species generation and p38 activation. Seladin‐1 inhibited LPS‐induced activation of p38 but not nuclear factor‐κB and inhibited the production of tumour necrosis factor‐α in response to LPS. Moreover, seladin‐1 inhibited LPS‐induced osteoclast formation and enhanced LPS‐induced alkaline phosphatase activity. Therefore, it was suggested that seladin‐1 might be an LPS‐responsible gene product and regulate the LPS‐induced inflammatory response negatively.     

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.     

Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice

One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms - bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.     

The functional tumor necrosis factor-α (308A/G) polymorphism modulates attentional selection in elderly individuals

There has been increasing interest in understanding the role of inflammatory processes for cognitive functions in aging using molecular genetic approaches. Though this has mostly been evaluated in pathological aging, little is known about the relevance for cognitive functions in healthy aging in humans. On the basis of behavioral data and neurophysiological data (event-related potentials and time–frequency decomposition) we show that the A-allele of the functional tumor necrosis factor (TNF)-α -308 A/G polymorphism confers dysfunction in a number of cognitive processes: prolonged attentional selection indexed by a delayed P1/N1 complex, an increased P3a, which is interpreted as an enhanced distractibility by nonrelevant stimuli and compromised response selection mechanisms, as indexed by a reduced frontocentral N2. Time–frequency analyses show that allelic variations further exert their effects by modulating alpha and beta frequency oscillations. On a neurobiological level, these effects might be because of the interaction of TNF-α with glutamatergic neural transmission by which TNF-α is known to boost apoptotic mechanisms in elderly individuals.     

NLRP12 negatively modulates inducible nitric oxide synthase (iNOS) expression and tumor necrosis factor-α production in Porphyromonas gingivalis LPS-treated mouse macrophage cell line (RAW264.7)

Objective

The aim of the present study is to investigate the participation of NLRP12 in Porphyromonas gingivalis LPS-activated mouse macrophages.

Methods

NLRP12-depleted mouse macrophages were stimulated with P. gingivalis LPS (1 μg/ml.). At indicated time points, the treated cells were lysed and the supernatant from treated cells was collected. Gene and protein expression of NLRP12 and iNOS were determined by RT-PCR and immunoblotting, respectively. The level of TNF-α production in the supernatant of the activated cells was determined by ELISA.

Results and conclusion

NLRP12 was upregulated in response to stimulation with P. gingivalis LPS. In addition, when NLRP12 was depleted in P. gingivalis LPS-treated macrophages, an increase in TNF-α production and iNOS expression were observed when compared to those of the control cells, indicating that NLRP12 downregulates the inflammatory cytokine and antimicrobial molecule production in the macrophages.