Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells

OBJECTIVE: Low serum levels of mannan binding lectin (MBL) are associated with increased risk of recurrent infections. We determined whether there was an association between serum MBL levels and the course and prognosis of rheumatoid arthritis (RA). METHODS: MBL was analyzed in sera from 99 patients with RA who were included in a longterm prospective study. RESULTS: Compared with controls, a high fraction of patients lacked detectable MBL in serum (11 vs 3%; p = 0.025). Comparing patients with MBL serum levels above and below the median revealed that those with levels below the median were younger at onset of RA (p = 0.043) and had higher erythrocyte sedimentation rate (p = 0.006), joint swelling score (p = 0.019), limitation of joint motion score (p = 0.027), and annual increase in radiographic destruction score (p = 0.053). CONCLUSION: MBL insufficiency may be a contributing pathogenetic factor in RA.     

Tumor necrosis factor-alpha-induced apoptosis in a human keratinocyte cell line (HaCaT) is counteracted by transforming growth factor-alpha

Pulmonary surfactant is a complex mixture of lipids and proteins that functions to keep alveoli from collapsing at the end of expiration. Dipalmitoylphosphatidylcholine has been identified as the most important component for lowering surface tension at the air-liquid interface. Hydrophobic surfactant apoproteins, SP-B and SP-C, play essential roles in the biophysical functions of the surfactant phospholipids. Hydrophilic surfactant apoproteins (SP-A and SP-D) that are members of C-type lectin superfamily, interact with phospholipids and glycolipids and modulate host defense functions in the lung. SP-A also plays an important role in regulating phospholipid homeostasis in the alveolar spaces. Recent advances in genetics and molecular biology have clarified the structure-function relationship of surfactant apoproteins.     

Cytotoxicity of lindane and paraquat to human hepatoma cell lines

A serosurvey was conducted in a random sample of 259 women and 231 men in 12 rural communities in Mwanza Region, Tanzania, using a type-specific ELISA for Herpes simplex virus type 2 (HSV-2) infection. Seroprevalence rose steeply with age to approximately 75% in women >=25 years old and 60% in men >=30. After adjusting for age and residence, HSV-2 prevalence was higher in women who were married, in a polygamous marriage, Treponema pallidum hemagglutination assay (TPHA)-positive, had more lifetime sex partners, or who had not traveled. Prevalence was higher in men who were married, had lived elsewhere, had more lifetime partners, had used condoms, or were TPHA-positive. HSV-2 infection was significantly associated with recent history of genital ulcer. The association between HSV-2 infection and lifetime sex partners was strongest in those <25 years old in both sexes. This association supports the use of HSV-2 serology as a marker of risk behavior in this population, particularly among young people.     

Expression and cleavage of tumor necrosis factor-alpha and tumor necrosis factor receptors by human monocytic cell lines upon direct contact with stimulated T cells

Tumor necrosis factor-alpha (TNF-alpha) is a potent cytokine in inflammatory processes. A variety of mechanisms that modulate its activity have been described, one being its binding to soluble receptors (sTNFR). In this study, we demonstrate that human monocytic cells such as THP-1 respond to direct contact with a membrane preparation of stimulated HUT-78 cells by producing TNF-alpha and by releasing sTNFR-p75, but not sTNFR-p55, with different kinetics. TNF-alpha concentration peaked after 12 h of contact and then decreased, whereas sTNFR-p75 production increased progressively upon cell/cell contact. The decrease in TNF-alpha concentration is not due to trapping of TNF-alpha by its soluble receptors or other soluble or cell-associated molecules, but rather to a proteolytic activity associated to THP-1 cells. On the other hand, the increase in sTNFR-p75 release does not result from an increase in the cleavage of pre-existing cell-associated sTNFR-p75 but from an increase in TNFR-p75 expression, immediately followed by the cleavage of its extracellular domain. Phenylmethylsulfonylfluoride, a serine protease inhibitor, has a negative effect on both TNF-alpha degradation and sTNFR-p75 release by THP-1 cells. Thus, there may be an enzymatic activity associated to THP-1 cells that plays an important role in the neutralization of TNF-alpha activity both by degrading the molecule and by cleaving its receptors at the cell surface.     

The specific type IV phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-alpha production by human mononuclear cells

Compounds suppressing the production of tumor necrosis factor-alpha are protective in animal models of septic shock. Recent studies demonstrated a beneficial effect of xanthine derivatives, which suppress tumor necrosis factor-alpha production by acting as non-specific cAMP phosphodiesterase inhibitors. In this experiment we tested the effect of (+/-)-rolipram (racemate) and its enantiomers on human mononuclear cells stimulated with lipopolysaccharide (LPS). Rolipram has a phenyl-pyrrolidinone structure, unrelated to the methylxanthines, and acts as a specific inhibitor of the type IV phosphodiesterase. Our results identify rolipram as a remarkably potent suppressor of the LPS-induced synthesis of tumor necrosis factor-alpha. When compared to the non-specific inhibitor pentoxifylline, the IC50 of (+/-)-rolipram (130 nM) is more than 500 times lower. The influence of rolipram on tumor necrosis factor-alpha production depended on the steric configuration of the molecule, since the (-)-enantiomer exhibited a five times lower IC50 than the (+)-enantiomer. The inhibitory effect of all substances tested is selective for tumor necrosis factor-alpha rather than interleukin-1 beta, since interleukin-1 beta production is only slightly influenced.     

Inhibition of tumor necrosis factor-alpha production by SK&F 98625, a CoA-independent transacylase inhibitor, in cultured rat peritoneal macrophages

When rat peritoneal macrophages were incubated in medium containing thapsigargin, tumor necrosis factor-alpha (TNF-alpha) production was increased time-dependently. In the presence of SK&F 98625, a CoA-independent transacylase inhibitor, the thapsigargin-induced TNF-alpha production was inhibited dose-dependently. Platelet-activating factor (PAF) and prostaglandin E2 (PGE2) production were also inhibited by SK&F 98625. The SK&F 98625-induced inhibition of TNF-alpha production was not prevented by addition of PGE2. PAF antagonists such as E6123, L-652,731 and CV-6209 partially inhibited the thapsigargin-induced TNF-alpha production, suggesting that concurrently produced PAF in thapsigargin-stimulated macrophages up-regulates TNF-alpha production. The inhibition by SK&F 98625 of thapsigargin-induced TNF-alpha production might be partly due to the inhibition of PAF production.     

Two chicken B cell lines resistant to ouabain for the production of chicken monoclonal antibodies

Two chicken B cell lines, termed MuH1 and MuH4, which are resistant to ouabain, were established as fusion partners for production of chicken monoclonal antibody from the parental cell lines R27H1 and R27H4 which were deficient in thymidine kinase activity. The MuH1 synthesized, but did not secrete mu chain, whereas the MuH4 secreted non-specific IgM. Fusions of the MuH1 and MuH4 with spleen cells from chickens immunized with human IgG resulted in successful preparations of hybridomas secreting antibodies specific to human IgG. Transformed cells insensitive to HAT appeared in many culture wells after cell hybridizations with R27H4, but on hybridization with MuH1 or MuH4 the appearance of such unfavorable cells could be avoided by the inclusion of ouabain in HAT medium. Thus, in comparison with the R27H4 line, the MuH1 and MuH4 lines did not provide higher fusion efficiencies, but gave increased opportunities to obtain hybridomas producing specific antibodies. Among a total of eight hybridomas, four derived from HuM1 and four derived from MuH4, seven secreted both IgG and IgM, and the remaining one derived from MuH1 secreted only IgG. In all eight hybridoma lines IgG was specifically reactive to human IgG.     

Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression

Recent data indicate a major role for IL-10 in suppressing immune and inflammatory reactions. To date, expression of human IL-10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL-6 expression. In this study we sought to determine whether non-hematopoietic human tumor cell lines produce IL-10 and, if so, what is the relationship between IL-10 and IL-6. Using ELISA, we determined IL-10 and IL-6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL-6 protein was secreted by 28 of the tumor cell lines; IL-10 was measurable in 15 cell lines. IL-6 secretion was maximal and most frequent in renal-cancer cell lines, while IL-10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL-10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio-active, as demonstrated in the mouse MC/9 mast-cell-line assay and in human mixed-lymphocyte reactions. In both assays, IL-10 bio-activity was neutralized by an anti-IL-10 monoclonal antibody. Expression of IL-6 and IL-10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL-1 alpha, and TNF-alpha strongly enhanced the release of IL-6 by RCC cells, but only marginally affected IL-10 production in colon-carcinoma cells. IL-10 secretion by colon-carcinoma cells was moderately stimulated by IFN-gamma and IL-4. Dexamethasone suppressed the release of IL-6, but had no inhibitory effect on IL-10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL-6 and/or IL-10, suggesting a role of these cytokines in solid-tumor development and anti-tumor immunity.     

The growth response to tumour necrosis factor alpha of human gynaecological cancer cell lines

Academic computing initiatives rank high on the list of priorities of many dental schools. However, outcomes of academic computing initiatives have not been presented. The objectives of this program evaluation were to: 1) document a strategic initiative for academic computing over a five-year period; 2) assess outcomes; and 3) demonstrate how outcomes assessment changed strategic goals for the future. In 1992, Temple University School of Dentistry developed an academic computing plan. The plan proposed to develop the computer literacy of faculty, teach students the computer skills they need to be successful in their careers, and introduce computer-aided instruction as a new teaching tool. Before a new five-year plan was developed in 1997, the original plan's outcomes were summarily assessed. Assessment instruments included faculty and student surveys, budgets, inventory records, and utilization statistics. The school has reached two of three goals of the 1992 plan. Eighty percent of all full-time faculty have computers, are computer literate, and use computers for a variety of purposes. The school has implemented a comprehensive predoctoral dental informatics curriculum. However, the implementation of computer-aided instruction has not met expectations. Goals of the 1998-2003 plan include establishing an online learning infrastructure, improving student access, implementing computer-based oral health records, and further improving the computer literacy of faculty and students. Planning and supporting academic computing initiatives is a substantial challenge. Factors such as institutional culture, capital investment, ongoing support, and technological change influence plans and their success. While process and structure can be assessed relatively easily, measures for changed educational outcomes are still lacking.     

Tumor necrosis factor-alpha and interleukin-6 release from white blood cells induced by different graft materials in vitro are affected by pentoxifylline and iloprost

Inflammatory mediators such as cytokines produced by white blood cells (WBCs) at the site of implantation are important for the biocompatibility of vascular grafts. The aim of the present study was to demonstrate the tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) release from WBCs incubated with expanded polytetrafluoroethylene (ePTFE) or woven Dacron grafts. In a second series the effects of pentoxifylline (PTX) and iloprost (ILO), both known to inhibit white blood cell function, on this release were determined. Woven Dacron grafts induced significantly higher release of both TNF-alpha and IL-6 compared to ePTFE. TNF-alpha was detectable first after 2 h, whereas IL-6 was seen after 4 h. Maximum values were reached at 6 and 12 h, respectively. The addition of an endotoxin gave more pronounced patterns of cytokine release not influenced by time. Preincubation with both PTX and ILO at final concentrations of 100 and 10 micrograms/mL, respectively, reduced significantly the TNF-alpha release without differences between the two graft materials, whereas the effect on the IL-6 release varied and was graft material-dependent. In conclusion, graft material-dependent induction of TNF-alpha and IL-6 from WBCs was demonstrated. PTX and ILO influenced the cytokine release. It might be suggested that graft material-induced cytokine production could contribute to intimal hyperplasia in vivo. The present findings encourage further studies regarding graft material-induced WBC alterations and the role of pharmacologic agents influencing this function.     

Responses of human glioblastoma cells to human natural tumor necrosis factor-alpha: susceptibility, mechanism of resistance and cytokine production studies

Responses and susceptibility of 14 human glioblastoma cell lines to human natural tumor necrosis factor-alpha (TNF) were studied in vitro. Susceptibility of glioblastoma cells to TNF varied in experimental conditions applied. Most of glioblastoma cell lines were resistant to cytotoxic activity of TNF in a MTT assay at concentrations below 16 U/ml for 72 h exposure. However, TNF at higher dose, in prolonged exposure and against low density of target cells was antiproliferative for certain glioblastoma cultures. TNF exposure at 10 U/ml for 48 h suppressed DNA synthesis in 9 of 14 glioblastoma cultures, but increased in 3 cultures. In addition, colony forming assay showed anti-clonogenic activity of TNF in 5 of 6 glioblastoma cell lines tested. In spite of their low susceptibility to TNF, glioblastoma cells well responded to TNF stimulation at low dose (10 U/ml) for a short period in the absence of cell damage. Productions of Interleukin-6 (IL-6), IL-8-like activity, granulocyte-macrophage colony stimulating factor (GM-CSF), prostaglandin E2 (PGE2) and manganous superoxide dismutase (Mn-SOD) were enhanced or induced by the low-dose TNF stimulation. Mn-SOD, a protein protective against oxidative cell damage, was well induced in time- and dose-dependent manner, however did not correlate with TNF resistance. Whereas the levels of PGE2 in TNF-susceptible cell lines, H-4 and SF-188, were higher than those of other lines. In conclusion, most of glioblastoma cells are resistant to TNF cytotoxic effects, but highly responsive to TNF stimulation. Its effect on glioblastoma cells appears to modulate cell differentiation rather than to kill the cells.     

alpha-Melanocyte stimulating hormone inhibits tumour necrosis factor-alpha stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines

alpha-Melanocyte stimulating hormone (alpha-MSH) was found significantly to reduce tumour necrosis factor-alpha (TNF-alpha) stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in normal adult cutaneous melanocytes. The maximum inhibitory response to alpha-MSH was obtained at around 10(-10) mol/L alpha-MSH when cells were coincubated with alpha-MSH and TNF-alpha for 24 h. alpha-MSH had little or no effect on basal ICAM-1 expression in melanocytes and the effects of alpha-MSH could be mimicked with 3-isobutyl-1-methylxanthine (IBMX). Preliminary data in three human melanoma cell lines also showed alpha-MSH and forskolin to be effective in significantly reducing TNF-alpha stimulated ICAM-1 expression over 24 h. The extent of the inhibition varied from cell line to cell line and was greatest in those cells with the highest number of alpha-MSH receptors. These data suggest that alpha-MSH has the ability to oppose the action of the pro-inflammatory cytokine TNF-alpha on melanocytes and melanoma cells.     

Interleukin-6 downregulates factor XII production by human hepatoma cell line (HepG2)

Involvement of the contact system of coagulation in the pathogenesis of various inflammatory diseases is suggested by reduced plasma levels of factor XII (Hageman factor) and prekallikrein generally considered to result from activation of the contact system. However, in many of these diseases patients develop an acute-phase response and, therefore, an alternative explanation for the decreased levels of factor XII could be the downregulation of factor XII gene expression in the liver as described for negative acute-phase proteins. We report here that interleukin-6 (IL-6), the principal cytokine mediating the synthesis of most acute-phase proteins in the liver, downregulates the production of factor XII by the human hepatoma cell line HepG2 by up to 75%. The decrease in protein secretion correlated with an equivalent decrease of factor XII mRNA likely indicating a pretranslational control of factor XII gene expression by IL-6. Downregulation of factor XII production by IL-6 in vitro parallelled that of transthyretin, a known negative acute-phase protein. Moreover, we show that, in patients developing an acute-phase response after immunotherapy with IL-2, plasma levels of factor XII correlate (r = .76, P < .0001) with those of transthyretin. Taken together, these results suggest that factor XII behaves as a negative acute-phase protein.     

Growth inhibitory response to activin A and B by human prostate tumour cell lines, LNCaP and DU145

Activins are growth and differentiation factors which have been shown to have proliferative and antiproliferative actions in many tissues. In addition, they have been implicated in tumourigenesis in reproductive tissues. Although activin and inhibin are present in rat ventral prostate, inhibin beta, but not alpha, subunit proteins have been detected in the human prostate epithelial tumour cell lines LNCaP, DU145 and PC3. With this absence of capacity to produce inhibins, the aims of this study were to determine the effect of activin A and B and follistatin on DNA synthesis by these human prostate tumour cell lines. The results demonstrate a differential response to exogenously added activin A and B on DNA synthesis in vitro by the tumour cell lines. The inhibitory effects were observed on LNCaP cells in the absence or presence of stimulation with 1 nM 5 alpha-dihydrotestosterone and on the androgen-independent DU145 cells, but not the PC3 cells. Activin A caused a dose-dependent inhibition of DNA synthesis and proliferation by LNCaP and androgen-independent DU145 cells which was maximal at 8 ng/ml. The effect of exogenously added activin A was completely reversed by follistatin, but not by inhibin A. The addition of human recombinant FS 288 alone (400 ng/ml) did not have any effect on DNA synthesis, whereas inhibin A alone (400 ng/ml) caused a significant inhibition of DNA synthesis. The capacity of all three cell lines to produce activins and follistatins was demonstrated by the expression of the mRNAs and confirmed by the localisation of immunoreactivity for these ligands to the cytoplasm of the tumour cells. The growth inhibitory response to activins A and B by LNCaP and DU145 cells, and the ability of follistatin to block these effects, suggest that the autocrine interactions between activins and follistatins have a role in the regulation of LNCaP and DU145 prostate tumour cell growth.