Tumor necrosis factor production by human T-cells stimulated with bacterial superantigens

Tumor necrosis factor (TNF) production from T-cells stimulated with superantigenic exotoxins, staphylococcal enterotoxin B and streptococcal pyrogenic exotoxin A was investigated in the presence of cells bearing distinct isotypes of HLA class II molecules. The main T-cell subset for TNF production was investigated in parallel. Similarly high levels of TNF production were induced upon stimulation with the toxins in the presence of DR⁺ or DQ⁺ cells, but only marginal levels of TNF production were induced in the presence of DP⁺ cells. Although both CD4⁺ T-cells and CD8⁺ T-cells produced TNF-α and TNF-β in response to toxin stimulation in the presence of HLA class II⁺ cells, the former T-cell subset was the major source of producers of TNF-α and TNF-β.     

Gene expression and production of tumour necrosis factor by a rat basophilic leukaemia cell line (RBL-2H3) with IgE receptor triggering

This study evaluated the gene expression of tumour necrosis factor (TNF) and the molecular weight of the cytotoxic factor in a subline of a rat basophilic leukaemia cell line, RBL-2H3. After IgE receptor triggering with a specific antigen that was associated with histamine release, cytotoxic activity in the cell lysates and supernatants increased for 2 hr during the culture of RBL-2H3 cells. Furthermore, calcium ionophore A23187 could induce release of histamine and cytotoxic activity from RBL-2H3 cells. However, compound 48/80, lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) were unable to induce the release of either histamine or cytotoxic activity from the cells. These data suggested that, at least in part, there was a common pathway in histamine release and production of cytotoxic activity. A protein synthesis inhibitor, cycloheximide, did not affect histamine release, but inhibited the induction of cytotoxic activity. This cytotoxic activity from RBL-2H3 cells was completely neutralized by anti-mouse TNF rabbit serum. With Northern blot analysis, mouse TNF cDNA probe could hybridize with RNA isolated from RBL-2H3 cells. TNF mRNA was induced as early as 1 hr after stimulation with specific antigen and decreased by 4 hr. Moreover, the molecular weight (MW) of the released cytotoxic factor from RBL-2H3 cells triggered with IgE receptors was approximately 17,000 by SDS-PAGE, which was compatible to that of TNF. Thus, it is concluded that the gene expression and production of TNF occurred in RBL-2H3 cells after IgE receptor triggering in association with histamine release, suggesting that TNF produced by basophils and mast cells may play an important role in allergic reaction through its wide range of biological activity.     

Tumor necrosis factor production by human macrophages stimulated in vitro by Plasmodium falciparum.

Production of tumor necrosis factor by human macrophages may be induced in vitro by cytoadherent and noncytoadherent strains of Plasmodium falciparum, with an optimal ratio of one to three parasitized erythrocytes per macrophage. Centrifuged and heated crude culture supernatants have the same effect, thus showing the existence of a thermostable soluble factor able to induce this expression. In vitro kinetic experiments have shown that the secretion of tumor necrosis factor appears early, with a maximal peak at 8 h.     

Glucocorticoids suppress the production of tumour necrosis factor by lipopolysaccharide-stimulated human monocytes.

We have investigated the modulating effect of steroids on the in vitro production of tumour necrosis factor (TNF) by lipopolysaccharide (LPS)-stimulated human monocytes. Dexamethasone, at concentrations ranging from 10(-8) to 10(-6) M, and cortisol, at concentrations 10(-7) and 10(-6) M, suppressed the TNF production in a dose-dependent manner. The highest concentrations of dexamethasone or cortisol reduced the TNF production to 21 +/- 2% and 48 +/- 8% of the control value, respectively. The effect of dexamethasone was time dependent, and an incubation time of 48 hr was required to reduce the TNF production to 21% of control. The effect of dexamethasone decreased when the incubation time became shorter, and the mean TNF production ranged from 49% to 72% of control when dexamethasone was added later than 8 hr before LPS addition, at the time of LPS addition, or within 1 hr after LPS addition. The magnitude of the TNF-suppressing effect of dexamethasone varied greatly from donor to donor. Only the glucocorticoids, and not the sex steroids or the mineralocorticoids, significantly reduced the TNF production.     

Salmonellae activate tumor necrosis factor alpha production in a human promonocytic cell line via a released polypeptide.

Invasive strains of Salmonella spp. cause both systemic and localized infections in humans. The ability to resist infection and some aspects of the tissue pathology associated with the presence of Salmonella in the gastrointestinal tract have been shown to be mediated in part by the induction of tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine produced by activated macrophages and lymphocytes. Recent reports indicate that TNF-alpha is involved in the induction of human immunodeficiency virus replication by Salmonella in the latently infected human promonocytic cell line U1. In the present study, we investigated the effects of Salmonella on TNF-alpha production in U1 cells and a related cell line, U38. Unlike Escherichia coli or Yersinia enterocolitica, salmonellae rapidly induce TNF-alpha expression in these cells through a released factor(s). Time course experiments show that the kinetics of TNF-alpha production by U38 cells stimulated with Salmonella conditioned medium closely resemble those observed in response to live Salmonella. The observation that TNF-alpha levels are elevated by 60 min after exposure to either bacteria or their conditioned medium suggests that the soluble inducer is continuously released or shed by the bacteria and that the signal acts rapidly to increase TNF-alpha production. Furthermore, the ability to produce the TNF-alpha inducer is shared by at least four Salmonella serotypes and does not correlate with the abilities to invade and to survive within phagocytes. Treatment of active conditioned medium with trypsin, but not low pH, high temperature, or urea, significantly inhibits its TNF-alpha-inducing effect on U38 cells, a finding which points to a polypeptide product of Salmonella as the mediator of TNF-alpha production. Gel filtration chromatography of Salmonella conditioned medium reveals two peaks of activity, consistent with molecular masses of approximately 150 and 110 kDa.     

Stimulators of tumour necrosis factor production released by damaged erythrocytes.

We sought to characterize factors released by sonicated human erythrocytes that stimulate peripheral blood mononuclear cells (PBMC) to release tumour necrosis factor-alpha (TNF). This response is not inhibited by polymyxin B, indicating that contaminating lipopolysaccharide (LPS) is not responsible. When erythrocyte lysates are fractionated by reverse-phase chromatography using a gradient of n-propanol on Sep-Pak C18 cartridges, the TNF-inducing activity elutes as a single peak. The erythrocyte-derived TNF-inducing activity is unaffected by digestion with proteases but is destroyed by mild base hydrolysis or digestion by lipases, indicating that compounds containing ester-linked acyl chains may be essential. These properties are similar to those of TNF stimulators that we have previously identified in erythrocytes infected with malaria parasites, except that the TNF-inducing activity per cell is about 200 times higher in parasitized erythrocytes than in uninfected erythrocytes. Lipase-digested erythrocyte lysates inhibit the TNF-inducing factors of both normal and malaria-infected erythrocytes, suggesting that lipase digestion creates partial structures which compete with active components for macrophage receptors. Such receptors may recognize a common structure that contains an inositol monophosphate (IMP)-like component, as IMP also inhibits the TNF response to erythrocyte-derived factors and to parasite lysates whereas it does not affect the response to LPS. We conclude that lysed erythrocytes release specific cytokine-inducing factors that may contribute to the fever response to non-infectious tissue injury.     

Tumor necrosis factor production by human granulocytes.

Human polymorphonuclear leukocytes kill WEHI 164 cells in an 18-hour 51Cr release assay. Antibody to human tumor necrosis factor (TNF) blocks the lysis of targets mediated by human granulocytes. Resting granulocytes produce an undetectable amount of TNF, if any. Granulocytes stimulated with Staphylococcus aureus release 250-500 U/ml TNF alpha. The specificity of the released TNF in the WEHI 164 cytotoxicity assay was confirmed by using neutralizing anti-TNF alpha monoclonal antibodies. The thymidine uptake of endothelial cells was inhibited by granulocyte-derived TNF. The identity of TNF alpha was further confirmed by molecular weight determination, by gel filtration on Sephacryl S-200, with a result of approximately 44,000. Besides their antimicrobial capacity, therefore, granulocytes may contribute to tumor rejection, inflammation and septic infections by releasing TNF.     

The production of tumour necrosis factor a (TNF-{alpha}) by human endometrial cells in culture

The production of tumour necrosis factor a (TNF-) by culturedhuman endometrial epithelial and stromal cells prepared fromendometrium obtained at different stages in the menstrual cyclehas been investigated. TNF- was not detectable in the supernatantsof stromal cell cultures prepared from endometrial tissue obtainedat any time in the menstrual cycle. TNF- production by endometrialepithelial cells in culture varied depending on the time inthe cycle at which the endometrial tissue was taken. Cells preparedfrom tissue obtained during the late proliferative phase ofthe cycle produced more TNF- than those prepared from tissueobtained at other times in the cycle. In addition, a small increasein TNF- production was seen by cells prepared from tissue obtainedduring the mid-secretory phase of the cycle. Interieukin 1 (IL-1)(1.4–140 pmol/1) caused a dose-dependent increase in TNF-production by cells prepared from both proliferative and secretoryendometrium. Maximum LL-1-stimulated increases in TNF- productionwere similar in cells from both proUferative and secretory endometriumand typically reached from four to 10 times basal values. Highdoses of progesterone, either alone or in the presence of oestradiol,also affected TNF-a production by epithelial cells. TNF- productionby cells prepared from proliferative endometrium was increasedby progesterone. In contrast, TNF- production by cells preparedfrom secretory endometrium was decreased in the presence ofprogesterone. The effects of steroids on TNF- production wereless marked than that of IL-1, with values increasing or decreasingto a maximum of three times the basal value. Placental protein14 (PP14) (0.18 and 1.8 nmol/1) also increased TNF- productionby cells prepared from proliferative tissue, but had no effecton its production by cells prepared from secretory endometrium.PP14-stimulated TNF- levels typically only reached a maximumof two times basal values.     

Increased tumour necrosis factor alpha production by neutrophils in patients with hepatitis B.

AIMS--To investigate the role of serum and neutrophil tumour necrosis factor alpha (TNF alpha) in patients with viral hepatitis. METHODS--The activities of serum and neutrophil TNF alpha were measured using a bioassay of in vitro cytotoxicity against L929 cells in 57 patients with viral hepatitis and 20 healthy blood donors. RESULTS--Both serum and neutrophil TNF alpha in patients with chronic active hepatitis (CAH) and subacute fulminant hepatitis (SAFH) increased compared with those in normal controls (p < 0.01). No such differences were seen in patients with acute hepatitis. Serum and neutrophil TNF alpha were obviously reduced in patients with CAH and SAFH during convalescence compared with the active period (p < 0.05; p < 0.01). Furthermore, serum TNF alpha was significantly increased in patients with SAFH and complications compared with those without (p < 0.01), and in patients with SAFH who died compared with those who survived (p < 0.01). Neutrophil TNF alpha was significantly higher in patients with SAFH and secondary bacterial infections (p < 0.05). CONCLUSIONS--Production of serum and neutrophil TNF alpha is increased in patients with CAH and SAFH, suggesting that neutrophil TNF alpha causes liver injury in these patients.     

Platelet-activating factor (PAF-acether) enhances the concomitant production of tumour necrosis factor-alpha and interleukin-1 by subsets of human monocytes.

The production of the cytokines tumour necrosis factor (TNF) and interleukin-1 (IL-1) by human monocytes was analysed following their stimulation with muramyl dipeptide (MDP; 1 microgram/ml), in the absence or presence of graded concentrations of platelet-activating factor (PAF). Significantly enhanced production of both TNF and IL-1 was observed at two concentration ranges of PAF: a major enhancement was observed at 10(-8)-10(-6) M and this was blocked by the PAF antagonist BN 52021 (10(-4) M). A second enhancement was observed at 10(-15)-10(-14) M PAF, which was not blocked by BN 52021. Monocytes isolated either by adherence or counterflow elutriation had similar responses to PAF. The biologically inactive precursor-metabolite, lyso-PAF, had no effect on cytokine production. PAF was shown to augment the production of both bioactive TNF and IL-1 and immunoreactive TNF-alpha and IL-1 alpha and beta. Fractionation of monocytes on a discontinuous Percoll gradient yielded a denser subpopulation, which responded preferentially to higher PAF concentrations, while the less dense subpopulation responded to both concentration ranges. These data indicate that PAF can modulate monocyte functions as related to cytokine production, and may thus contribute to amplification of inflammatory reactions and regulation of immune responses by interacting with subsets of human monocytes.     

Membrane-bound tumour necrosis factor alpha: immunocytochemical and ultrastructural studies of human monocytes and monocytic cell line and its induction by tumour cells in vitro.

Monoclonal antibody against recombinant human tumour necrosis factor alpha (rTNF) was used for the immunochemical detection of TNF in human blood monocytes and monocytic cell line U 937. Cells stimulated with phorbol myristate acetate (PMA) showed strong surface but not cytoplasmic staining. Unstimulated cells demonstrated weak or no staining. At early time after stimulation (1-2h) a spot reaction was seen in the Golgi area of the cytoplasm of stimulated cells. Coculture of tumour cells with monocytes also resulted in the induction of membrane TNF. Ultrastructural studies confirmed TNF localization within the cell membrane. These results indicate that TNF can be detected within the cells by immunocytochemistry which may make feasible studies on TNF appearance in cellular infiltrates in the tissues.     

Production of interleukin-1 but not tumor necrosis factor by human monocytes stimulated with pneumococcal cell surface components.

While there is considerable evidence that both interleukin-1 (IL-1) and tumor necrosis factor (TNF) are central mediators of inflammation caused by gram-negative bacteria and endotoxin, the roles of these two mediators in gram-positive infection are unknown. Pneumococcal infections are characterized by an intense inflammatory reaction in infected tissues. Current evidence suggests that the component of the pneumococcus which causes this inflammation in many body sites is the cell wall. We determined the ability of native pneumococcal cell wall, lipoteichoic acid, and cell wall subcomponents to stimulate secretion of IL-1 and TNF from human monocytes. Each pneumococcal cell surface component was found to have a different specific activity for induction of IL-1. Teichoication was an important determinant of this activity: teichoicated species were at least 10,000-fold more potent than endotoxin and 100-fold more potent than teichoic acid-free peptidoglycan. IL-1-inducing activity was greatly reduced by chemical alteration of the teichoic acid. In contrast to endotoxin, cell wall did not induce production of TNF. This dissociation of the production of IL-1 and TNF during the response of the human monocyte to pneumococcal surface components suggests that, in at least some circumstances, the mechanisms for generation of an inflammatory response to infection may be fundamentally different between gram-positive and gram-negative disease.     

Involvement of phospholipase A2 activation in tumour cell killing by tumour necrosis factor.

Earlier studies have indicated a possible role for arachidonate metabolism in the direct cytolysis of tumour cells by tumour necrosis factor (TNF) in vitro. In this study, the involvement of arachidonate metabolism has been investigated further with the following results: (i) Cytolysis of human U937 tumour cells by recombinant TNF was reduced by dexamethasone and quinacrine, agents which inhibit phospholipase A2. (ii) U937 and L929 cells, which are susceptible to TNF cytolysis, released arachidonic acid and its metabolites within 5 hr of TNF challenge, before cell death was apparent. In contrast, U937/R and L929/R, which are resistant to the cytolytic effects of TNF, did not release arachidonate products on TNF challenge. (iii) rTNF cytolysis of U937 cells was not reduced by inhibitors of the cyclo-oxygenase and lipo-oxygenase pathways of arachidonic acid metabolism. Cytolysis was reduced, however, by inhibitors of the arachidonate metabolic pathway involving cytochrome P450-dependent reductase, but only at reagent concentrations that also inhibited phospholipase A2 activity. Overall, these observations indicate a role for phospholipase A2 but not for arachidonic acid or its metabolites in the direct cytolysis of tumour cell lines by TNF.     

Inhibition of tumour necrosis factor and natural cytotoxic cell lytic activities by a spleen cell-elaborated factor.

Natural cytotoxic (NC) cell lytic activity is mediated by tumour necrosis factor (TNF), a protein with potent cytolytic activity on certain target cells. TNF also appears to mediate a wide range of other important biological activities (e.g. interferon-like anti-viral activity, induction of granulocyte-monocyte colony-stimulating factor, mediation of endotoxin-induced shock). Evidence is presented here that spleen cells from normal, untreated mice produce a factor(s) that inhibits both NC and TNF cytolytic activity. The factor(s) has a molecular weight greater than 10,000. Since indomethacin inhibits production by spleen cells of the NC/TNF inhibitory factor, it is suggested that prostaglandins are involved in the regulation of its production. Additionally, these studies indicate that the factor(s) does not function by inactivation of either NC effectors or TNF molecules, or by inhibition of the binding of NC cells or TNF to targets. Instead, the data suggest that the factor(s) acts on the targets rendering them refractory to TNF binding. Moreover, since the factor(s) acts slowly and requires protein synthesis in the target to function, it appears that the inhibitory activity is mediated via de novo-synthesized proteins from the target cells. At present, it is not known whether such a factor functions in vivo, although it is conceivable that its in vivo role is to modulate the pathological potential of TNF by protecting certain cells from NC or TNF lysis.     

Adrenomedullin suppresses tumour necrosis factor alpha-induced CXC chemokine ligand 10 production by human gingival fibroblasts

Periodontal disease is an inflammatory disorder characterized by the involvement of chemokines that are important for the recruitment of leucocytes. Several cytokines, including tumour necrosis factor alpha (TNF-alpha), are involved in regulating levels of chemokines in periodontal disease. CXC chemokine ligand 10 (CXCL10) is a chemokine related to the migration of T helper 1 cells. In this study, we examined CXCL10 expression in human gingival fibroblasts (HGFs). Moreover, we investigated the effects of adrenomedullin (AM), which is a multi-functional regulatory peptide, on the production of CXCL10 by HGFs. We revealed that TNF-alpha stimulation induced CXCL10 production by HGFs. HGFs expressed AM and AM receptors, calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein (RAMP) 2, mRNAs constitutively. AM treatment supressed CXCL10 production by TNF-alpha-stimulated HGFs. Moreover, we elucidated that AM produced by HGFs inhibited CXCL10 production by HGFs, because AM antagonist enhanced CXCL10 production by HGFs. TNF-alpha treatment enhanced CRLR and RAMP2 mRNA expression in HGFs. Furthermore, AM is expressed in human periodontal tissues, including both inflamed and clinically healthy tissues. These results suggest that the CXCL10 produced by HGFs may be involved in the migration of leucocytes into inflamed tissues and related to exacerbation of periodontal disease. AM might be a therapeutic target of periodontal disease, because AM can inhibit CXCL10 production by HGFs.     

Tumor necrosis factor production by human sarcoid alveolar macrophages.

Tumor necrosis factor (TNF) is an oncolytic peptide that may also exert many other biologic effects. Experimentally, immunologically activated mononuclear phagocytes stimulated with endotoxin (LPS) produce TNF, while resting mononuclear phagocytes stimulated with LPS produce little TNF. To date, the ability of human alveolar macrophages (AMs) to produce TNF has not been clearly delineated. As pulmonary sarcoidosis is a granulomatous inflammatory disorder characterized by immunologically activated AMs, we investigated the production of TNF by AMs obtained by bronchoalveolar lavage from 7 normal volunteers and 13 patients with pulmonary sarcoidosis. The AMs were cultured with and without LPS, and TNF production was assessed by an in vitro cytotoxicity assay. Unstimulated sarcoid and normal AMs produced little TNF, but LPS stimulation enhanced TNF production by both normal and sarcoid AMs. Furthermore, LPS-stimulated sarcoid AMs produced more TNF than normal AMs (84.9 +/- 16.7 versus 32.5 +/- 10.2 units/million cells, P less than 0.05). It is concluded that human AMs can produce TNF and that sarcoid AMs are primed and can produce significantly more TNF, compared with normal AMs.