We have identified a putative signalling feature of the cytoplasmic domains of the tumour necrosis factor (TNF) family members based on available amino acid sequence data. A casein kinase I (CKI) consensus sequence is conserved in the cytoplasmic domain of six of 15 members of the type II integral membrane TNF ligand family. We examined the phosphorylation state of transmembrane tumour necrosis factor-alpha (mTNF) with [32P]orthophosphate labelling and in vitro kinase assays, in lipopolysaccharide-stimulated RAW264.7 cells. A dimeric form of the type I soluble TNF receptor (sTNFR) was found to dephosphorylate mTNF. This effect could be prevented by treatment with phosphatase inhibitors. Recombinant CKI was able to phosphorylate mTNF that had been dephosphorylated by sTNFR ligation in vivo, and this was less effective if phosphatase inhibitors had been used to prevent mTNF dephosphorylation. A mutated form of mTNF, lacking the CKI recognition site, cannot be phosphorylated by the enzyme. Binding of sTNFR to mTNF induced an increase in intracellular calcium levels in RAW264.7 cells, implying the presence of an associated signalling pathway. We predict that this CKI motif is phosphorylated in other TNF ligand members, and that it represents a new insight into the mechanism of 'reverse signalling' in this cytokine family.

BackgroundHidradenitis Suppurativa (HS) is a painful, chronic inflammatory skin disease. Global estimates of prevalence vary between 0.03% and 4% of the population.Our main aim was to determine HS prevalence in the Australian adult population focussing on the demographics, management pathways and diagnosis rate of individuals living with HS.MethodsIn this population-based cross-sectional study, 17,050 individuals representative of the Australian adult population were asked through face-to-face household interviews to answer a previously validated HS screening questionnaire with high diagnostic power. Individuals who screened positive were asked additional questions, including previous diagnosis of HS and number/type of physicians consulted regarding their condition.Results11,433 Australian residents answered the HS questionnaire, 88 screening positive for HS (0.77%; 95% CI 0.62–0.95). Considering the previously reported sensitivity (0.97) and positive predictive value (0.85) of the screening questionnaire, HS prevalence was estimated to be 0.67% (95% CI 0.53%-0.84%). 6 of 88 suspected HS individuals reported a pre-existing HS diagnosis (6.8%; 95% CI 3.2%-14.1%). 25.6% of the undiagnosed individuals suspected of having HS had not seen any clinicians regarding their boils; the remaining ones had consulted General Practitioners (96.7%), and clinicians from different specialties. Comparisons of individuals who screened positive for HS versus those who screened negative demonstrated statistically significant differences in gender (p = 0.0046), age (p<0.0001), BMI (p = 0.0307), smoking status (p<0.0001), employment status (p<0.0001) and income (p = 0.0321).ConclusionsThe prevalence of HS in Australia was estimated to be 0.67% (95% CI 0.53%-0.84%). The diagnosis rate amongst the suspected HS cases was low, which appeared to be due to a combination of patients not seeking help and decentralization of care. Individuals suspected of having HS were more likely to be females, young, obese, smokers, unemployed or at home duties and having lower annual personal income in comparison with individuals not suspected of having HS.

Tumor necrosis factor alpha (TNF-alpha) is initially synthesized as a type II integral membrane protein (transmembrane TNF-alpha) after macrophage activation. In this study we have investigated some aspects of the regulation of expression and biological activity of transmembrane TNF-alpha by both soluble TNF-alpha receptors (sTNF-alphaR) and inhibitors of TNF-alpha processing. We show, using the technique of receptor-mediated ligand precipitation (RMLP), that a dimeric construct of the type I sTNF-alphaR binds to transmembrane TNF-alpha, expressed on the mouse macrophage cell line RAW264.7, under cell culture conditions. This interaction between sTNF-alphaR and transmembrane TNF-alpha does not prevent processing and release of soluble TNF-alpha. A specific hydroxamic acid-based inhibitor of processing, BB1101 (British Biotech), was found to increase the total cellular levels of whole-cell, 26-kDa, precursor TNF-alpha by 2.2-fold. However, the inhibitor increased the levels of precursor TNF-alpha present solely on the cell surface (i.e., transmembrane TNF-alpha) by 5.1- to 7.5-fold. This increase in the levels of transmembrane TNF-alpha on the activated human monocytoid cell line mono mac 6 was associated with a similar (6.7-fold) increase in TNF-alpha-mediated cytotoxicity toward the human adenocarcinoma cell line Colo 205, which is sensitive only to the transmembrane form of TNF-alpha. Mono mac 6 cells, expressing transmembrane TNF-alpha, were found to be killing the Colo 205 target cells through apoptosis. This cytotoxicity could be neutralized by pre-incubating the mono mac 6 cells with either sTNF-alphaR or polyclonal anti-TNF-alpha serum.

The mouse macrophage cell-line RAW264.7, stimulated with lipopolysaccharide, was used as a model for the study of the production of tumor necrosis factor (TNF) isoforms. TNF is synthesised initially as a 26 kDa transmembrane precursor, which is then processed enzymatically by a protease to release a mature molecule of 17 kDa. Dose-dependent production of transmembrane TNF was assessed by fractionation of cell membranes and Western blot analysis followed by autoradiography and densitometry. Isoforms of both the precursor and mature molecules were separated using two-dimensional (2-D) electrophoresis with immobilised pH gradient 3-10 linear gels as the first dimension. After radiolabelling of cells with 35S, both cell-associated and supernate-associated TNF isoforms were immunoprecipitated. A large number of protein spots were visualised on the 2-D gel map, for both the transmembrane and mature TNF species, more than have been detected previously using one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The likelihood that these putative isoforms were the result of differential glycosylation was tested by preincubating the cells with tunicamycin. This had the effect of reducing the number of protein spots, notably the higher molecular weight species. There were a number of precursor TNF isoforms that were unchanged upon tunicamycin treatment and these presumably reflect protein modifications other than glycosylation.

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