Induction of tumour necrosis factor-alpha during haemodialysis. Influence of the membrane type.

Some of the secondary clinical effects induced by long-term haemodialysis in patients with end-stage renal failure have been related to an increased production of interleukin-1 (IL-1). We investigated the role of another cytokine which shares a number of biological properties with IL-1, tumour necrosis factor-alpha (TNF-alpha). In long-term haemodialysed patients, we found at the beginning of the dialysis increased plasma TNF-alpha levels and enhanced monocyte capacity to produce TNF-alpha spontaneously ex vivo. Non-haemodialysed uraemic patients also presented increased plasma TNF-alpha levels. During dialysis with cellulose acetate (CA) or polysulphone (PS) membranes, plasma TNF-alpha levels and the spontaneous and lipopolysaccharide-induced production of TNF-alpha by monocytes remained at predialysis levels. In contrast, when cuprophane membranes were used, there was a significant increase in plasma TNF-alpha levels and in both spontaneous (10-fold) and lipopolysaccharide-induced (seven-fold) ex vivo TNF-alpha production by monocytes. These results suggest that monocytes are stimulated during haemodialysis with the poorly biocompatible cuprophane membrane.     

The 80-kD fibronectin fragment increases the production of fibronectin and tumour necrosis factor-alpha (TNF-α) in cultured mesangial cells

The presence of fibronectin (FN) fragments has been demonstrated in several inflammatory processes, and they have been implicated in the recruitment of mononuclear cells. However, the interaction of these FN fragments with resident cells has hardly been studied. We have hypothesized that the 80-kD FN fragment, which includes the RGD cell binding domain of FN, could contribute to the pathogenesis of glomerular damage through the interaction with mesangial cells (MC) via α₅β₁ integrin. Since an increase in the glomerular deposit of matrix components, particularly FN, is frequently observed in progressive glomerulonephritis, we studied whether its synthesis is modulated by the 80-kD FN fragment and the native FN molecule. While the 80-kD FN fragment stimulated FN in a dose-dependent manner, both at the mRNA and protein level, the whole FN molecule exerted a dual effect. High doses produced FN inhibition, while low doses elicited a certain increase. This stimulation was abrogated by the presence of Sam-1, a MoAb against the α-subunit of the α₅β₁ integrin. Since cytokines play a fundamental role in glomerular injury, we studied the production of TNF-α, one of the most powerful mediators of inflammation. TNF-α synthesis was induced by the 80-kD FN fragment, in a dose-dependent manner, but not by native FN. When MC were incubated with the 29- and 31-kD FN fragments, which lack the RGD cell binding domain, TNF-α secretion was not detected. These results strongly suggest that in cultured MC, the 80-kD FN fragment induces the synthesis of matrix proteins such as FN, and cytokines such as TNF-α, via α₅β₁ integrin. This mechanism could contribute to the perpetuation of renal injury.     

Human polymorphonuclear leucocytes stimulated by tumour necrosis factor-alpha show increased adherence to extracellular matrix proteins which is mediated via the CD11b/18 complex.

The present study demonstrates that tumour necrosis factor (TNF) and FMLP, but not IL-1 or IL-8, enhanced the adherence of polymorphonuclear neutrophil (PMN) to fibronectin, an extracellular matrix protein. The adherence induced by FMLP was very rapid, within 5 min while the induction of adherence by TNF was much slower, reaching maximum at 60 min. TNF also enhanced an adhesion of PMN to other extracellular matrix proteins, such as laminin, collagen IV and gelatin II, but not to human serum albumin. Anti-CD18 MoAb completely inhibited the binding of TNF-stimulated PMN to fibronectin and partially inhibited the binding to laminin. Further investigation showed that adhesion of TNF-stimulated PMN to fibronectin and laminin was inhibited by anti-CD11b MoAb and to a lesser extent by CD11a MoAb. In contrast to TNF-stimulated PMN the binding of unstimulated PMN to fibronectin and laminin was only inhibited by anti-CD11a MoAb. Anti-CD11c had no effect on PMN adherence. These results suggest that unstimulated PMN adhere to extracellular proteins through the CD11a/18, while TNF-stimulated PMN adhere through the CD11b/18. These results suggest that TNF secreted at the site of inflammation may enhance the interaction of PMN with the extravascular environment through the CD11b/18 complex.     

Induction of tumour necrosis factor-alpha (TNF-alpha) mRNA in bladders and spleens of mice after intravesical administration of bacillus Calmette-Guérin.

Intravesical bacillus Calmette-Guérin (BCG) therapy is highly effective in the therapy of carcinoma in situ of the bladder, but the mechanism of BCG immunotherapy is not clearly understood. We studied the production of TNF-alpha in spleens and bladders of mice after intravesical BCG or BCG/interferon-gamma (IFN-gamma) instillation. Significant change of TNF-alpha mRNA expression of spleens and bladders of C3H/He mice was observed after intravesical BCG instillation, although intravesical IFN-gamma therapy 3 days after BCG instillation to maintain the activated state of monocyte/macrophage lineage cells did not show a significant change of TNF-alpha mRNA, compared with that of BCG therapy alone. Maximal production of TNF-alpha mRNA in spleens of mice was seen after the first or second intravesical BCG instillation, and production of TNF-alpha mRNA in bladders was also increased after intravesical BCG instillation. The increment of TNF-alpha production by BCG stimulation in HL-60, a promyelocytic leukaemic cell line, and peripheral blood mononuclear cells in vitro may support the in vivo effect of BCG therapy on the bladder. These data show that local production of TNF-alpha as well as systemic production by intravesical BCG treatment may correlate with one of the mechanisms of BCG immunotherapy of superficial bladder cancer.     

Tetrandrine, a plant alkaloid, inhibits the production of tumour necrosis factor-alpha (cachectin) hy human monocytes.

Human mononuclear leucocytes (MNL) or the adherent fraction (monocytes) produced tumour necrosis factor-alpha (TNF-alpha) (by ELISA) in culture when stimulated with killed Staphylococcus aureus. The bisbenzylisoquinoline alkaloid, tetrandrine inhibited the capacity of MNL and monocytes to produce TNF-alpha at a concentration range of 0.1 to 5 micrograms/ml. Tetrandrine may be potentially useful in the treatment of inflammatory diseases in which TNF-alpha plays a major role.     

Characterization of naturally occurring autoantibodies against tumour necrosis factor-alpha (TNF-alpha): in vitro function and precise epitope mapping by phage epitope library.

Naturally occurring autoantibodies against cytokines exist in the sera of patients with autoimmune diseases as well as in the sera of normal individuals. We report here that affinity-purified autoantibodies against human TNF-alpha from one rheumatoid arthritis (RA) patient inhibited the cytotoxic effect of TNF-alpha on the mouse fibrosarcoma cell line WEHI 164, by 50%. In an attempt to predict the autoantibodies' recognition site on TNF-alpha protein we screened a random nanopeptide phage library with the affinity-purified TNF-alpha autoantibodies. Among 63 random selected clones, 46 clones carried the sequence ASSLLASSP, NSSPYLNTK or PQSPGSSFP. Frequency analysis of the relative occurrence of the 20 amino acids in the nanopeptides displayed by 50 random bacteriophages picked before selection and 63 after selection to bind to TNF-alpha autoantibodies indicated that proline (P < 0.0003) and serine (P < 0.04) are involved in the binding of the autoantibodies to the phages. Furthermore, we demonstrated that three synthetic peptides (ASSLLASSP, NSSPYLNTK and PPLKPVIDE) displayed by the selected phages reduced the binding of the autoantibodies to TNF-alpha protein by 50%. Interestingly, the sera of mice (BALB/c) immunized with phages displaying ASSLLASSP and NSSPYLNTK peptide showed an anti-TNF-alpha response as detected by ELISA. This response was not found in mice immunized with the wild type phage. Thus, the recombinant phages selected from the phage libraries could be used as carrier for immunization, and therefore as a tool for vaccine development. This work sets the stage for experiments designed to isolate ligands for protective antibodies.     

Effects of interferon-gamma and tumour necrosis factor-alpha on the development of cytotoxic T lymphocytes in autologous mixed lymphocyte tumour cultures with human melanoma.

We have studied the influence of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on the development of cytotoxic T lymphocytes (CTL) against melanoma in mixed lymphocyte tumour cultures (MLTC). In these MLTC, TNF-alpha at 10(4) U/ml increased the expansion of the CTL up to 10(4)-fold over recombinant IL-2 (rIL-2) alone. IFN-gamma at 10(4) U/ml and combinations of TNF-alpha plus IFN-gamma at 10(2)-10(3) U/ml promoted the proliferation more variably. MLTC generated with rIL-2 showed a predominance of CD8+ cells, while 2 weeks of culture in the presence of IFN-gamma at 10(4) U/ml, or with IFN-gamma and TNF alpha at 1 x 10(2)-10(3) U/ml, favoured the emergence of CD4+ cell populations. The cytotoxic activity of the lymphocytes generated in these MLTC showed a consistent decline of K562 cytotoxic activity following exposure to the combination of IFN-gamma and TNF-alpha. Despite the altered T cell subset distribution with different combinations of cytokines, no consistent alteration in the specific anti-tumour cytotoxicity against melanoma was detected. These results suggest that TNF-alpha and IFN-gamma influence the activation, phenotypic, and functional outcome of MLTC-generated CTL, and may account for the phenotypic variations observed in T cell populations generated in vitro.     

Beneficial effects of tumour necrosis factor-alpha (TNF-alpha) blockade in rheumatoid arthritis (RA)

The biological properties of TNF-alpha make it a candidate therapeutic target in RA. Our studies have demonstrated that TNF-alpha and its receptors are up-regulated and co-expressed in the synovium and cartilage-pannus junction of RA joints. Neutralizing TNF-alpha antibodies reduce the production of the many pro-inflammatory cytokines, including IL-1 and granulocyte-macrophage colony-stimulating factor (GM-CSF), produced by mononuclear cells from RA in culture. When injected into DBA/1 mice with collagen-induced arthritis and TNF-alpha transgenic mice with arthritis, anti-TNF MoAbs decrease inflammatory damage of joints. Clinical trials employing cA2, a chimaeric anti-TNF-alpha MoAb, in open-label and randomized placebo-controlled studies have demonstrated a dose-dependent efficacy with impressive improvement in disease activity and acute-phase responses lasting several weeks. We conclude that TNF-alpha is a critical mediator of inflammation in RA, and is an important therapeutic target in this disease.     

Alterations in mononuclear cell tumour necrosis factor-alpha (TNF-alpha) response in patients on long term cuprophane haemodialysis.

We have investigated TNF-alpha secretory response of peripheral blood mononuclear cells (PBMC) from 13 uraemic patients undergoing regular haemodialysis with cuprophane membrane (CM). Sixteen healthy subjects and five uraemic patients under conservative therapy were also studied as controls. Cells of haemodialysis patients exhibited increased TNF-alpha release in vitro in the absence of activating stimuli other than culture conditions, as compared with normal and uraemic controls. In contrast to normal cells, this spontaneous secretion of TNF-alpha from dialysis PBMC could not be significantly reduced by addition of polymyxin B to culture medium, thus indicating its independence of trace amount of lipopolysaccharide (LPS) present in the medium as contaminant. Furthermore, predialysis PBMC were considerably more sensitive to stimulation with 10(7) pg/ml of LPS under in vitro culture conditions than normal and uraemic controls. To elucidate a role of direct contact with CM in stimulation of TNF-alpha release from monocytes, PBMC were cultured on CM in vitro. Contact with CM stimulated TNF-alpha secretion from PBMC above the level of cells cultured on tissue culture plastic. This response persisted with time in culture in contrast to a transient LPS-induced TNF-alpha release. Furthermore, PBMC stimulated by contact with CM for 2 days did not lose the capacity to secrete TNF-alpha in response to a subsequent LPS stimulation, while a 2-day treatment of cells with LPS was followed by LPS refractory state. Therefore, direct contact with CM induces in PBMC a long-lasting TNF-alpha response which is not down-regulated by the acquisition of refractoriness in a manner similar to that which occurs in the case of LPS stimulation. These in vitro findings provide a possible explanation of the observation that predialysis PBMC exhibit elevated TNF-alpha secretory capacity.     

The effects of live Neisseria meningitidis and tumour necrosis factor-alpha on neutrophil oxidative burst and beta2-integrin expression

The effects of human recombinant tumour necrosis factor-alpha (TNF-alpha) on neutrophil (PMNL) oxidative burst and on CD11b/CD18 and CD14 expression after stimulation with pathogenic or nonpathogenic Neisseria meningitidis were studied using chemiluminescence and flow cytometry. PMNL oxidative burst increased more when stimulated with the apathogenic 29E strain than with the pathogenic B strain both when studied by chemiluminescence and by flow cytometry. When TNF-alpha was added to whole blood or PMNL together with bacteria a significant increase in the oxidative burst was seen for the B strain only. When whole blood was preincubated for 30 min with TNF-alpha the increase in oxidative burst was significant for both meningococcal strains. TNF-alpha caused a significant increase in PMNL CD 11b/CD18 expression after 30 min of incubation at 37 degrees C. TNF-alpha added simultaneously with the bacteria induced a significant increase in PMNL CD11b/CD18 in both strains. Incubation with the B strain alone caused a low but significant increase in CD11b/CD18 expression, but the addition of TNF-alpha increased this expression to the same high level as incubation with TNF-alpha alone or the 29E strain alone. Only TNF-alpha and the 29E strain caused significant increases in CD14 expression. In conclusion, human PMNLs react differentially when stimulated with pathogenic and nonpathogenic N. meningitidis and the activating effect of TNF-alpha is variable depending on the bacteria involved.     

Activation of bovine neutrophil functions by interferon-gamma, tumour necrosis factor-alpha, and interleukin-1 alpha

Neutrophils are the first line of defence against microbial infections. They are important in protecting the bovine mammary gland against environmental and pathogenic bacteria such as Escherichia coli and Staphylococcus aureus. Neutrophils in the peripheral blood of healthy cows are functionally a heterogeneous population of cells with markedly varied phagocytic and oxidative activities. Several cytokines have been found to augment leucocyte functions in humans and animal studies. Therefore, the main objective of the present study was to determine if recombinant human interleukin-1 alpha (rhIL-1 alpha), tumour necrosis factor-alpha (rhTNF-alpha), and interferon-gamma (rhIFN-gamma) would potentiate the functional activity of bovine blood neutrophils, thereby providing a possible means to manipulate the resistance of cows to microbial infections.Neutrophils were isolated from the peripheral blood of nine Holstein-Friesian heifers, with a purity of 89%–96% and viability greater than 98%. Aliquots of neutrophils were incubated with five concentrations of rhIL-1 alpha (0.001–10 ng/ml), rhTNF-alpha (0.5–1000 ng/ml), and rhIFN-gamma (0.01-100 ng/ml) at 37°C for 1 h. Then, fluorescein-labelled opsonised zymosan particles were added and the neutrophil suspensions were incubated further for 30 min to evaluate phagocytic activity by fluorescence microscopy. Similarly, unlabelled opsonised zymosan particles and nitroblue tetrazolium (NBT) were added and incubation was carried out for 15 min to evaluate oxidative activity by a spectrophotometric method. Chemotactic activity of cytokine-treated neutrophils for E. coli lipopolysaccharide-activated fetal calf serum was evaluated using a modified Boyden chamber method. The results showed that pretreatment of neutrophils with various concentrations of each cytokine enhanced their phagocytic and NBT reductive activities but reduced chemotactic activity. The phagocytic and NBT reductive activities increased significantly (p0.05) with an increase in the concentration of the cytokines.     

Alveolar macrophages in AIDS patients: increased spontaneous tumour necrosis factor-alpha production in Pneumocystis carinii pneumonia.

In order to assess the role of alveolar macrophages and their products in the control of Pneumocystis carinii pneumonia (PCP) and other infections in AIDS, bronchoalveolar lavage cells and peripheral blood mononuclear cells from HIV-positive AIDS/ARC patients (with and without PCP) and HIV-negative patients were counted and cultured in vitro; spontaneous and LPS-induced tumour necrosis factor-alpha (TNF-alpha) production was measured. Markedly increased spontaneous TNF-alpha production by alveolar macrophages and, to a lesser extent, peripheral blood monocytes was found in HIV-positive patients with active PCP but not in patients without the infection. Higher TNF production was associated with lower counts of Pneumocystis in the bronchoalveolar lavage fluid. These results suggest that TNF-alpha production by macrophages may play an important role in the control of Pn. carinii infection in AIDS.     

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells.

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.     

Up-regulation of tumour necrosis factor-alpha receptors on monocytes by desferrioxamine.

The effect of endogenously generated reactive oxygen metabolites on the interaction of human blood monocytes with tumour necrosis factor-alpha (TNF-alpha) was investigated. Pre-exposure of unactivated human blood monocytes to dimethylthiourea, a scavenger of hydroxyl radical (OH.), or to desferrioxamine (DFX), an iron chelator preventing the synthesis of OH., enhanced the specific binding of 125I-TNF-alpha to its receptors. Scavengers of superoxide anion or hydrogen peroxide were without effect. DFX-induced up-regulation of 125I-TNF-alpha binding depended on the concentration of the drug (1-5 mM) and on the duration of the treatment (1-18 h). It was not due to a reduction of receptor occupancy by endogenously generated TNF-alpha. Scatchard analysis of binding data revealed that DFX caused an approximately two-fold increase in the number of type II TNF-alpha receptors, with no change in their affinity. This up-regulation, that did not require synthesis of new proteins, was associated with a decrease in the internalization rate of TNF-alpha receptors, the half-life of which was doubled. Conversely, these findings suggest that OH. generation by monocytes may have a physiological role in reducing the activity of membrane-associated TNF-alpha receptors.     

Defective tumour necrosis factor-alpha production in mother''s milk is related to cow''s milk allergy in suckling infants

BACKGROUND: The precise role of leucocytes in human milk is still unresolved. OBJECTIVE: To assist in clarifying the immune mechanisms involved in the development of CMA in suckling infants, we studied the role of immunoregulatory leucocytes and their mediators in human breast milk. METHODS: The study population consisted of 43 lactating mothers and their infants, aged 0.25-8.0 months, followed-up prospectively from birth. Of these mothers, 27 had an infant with challenge-proven cow's milk allergy manifested with either skin (n = 23), gastrointestinal (n = 2) or skin and gastrointestinal symptoms (n = 3). Sixteen mothers with a healthy infant served as controls. We evaluated the spontaneous and mitogen-induced tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) production of human milk leucocytes and isolated peripheral blood lymphocytes in vitro with a commercial ELISA kit. RESULTS: TNFalpha production of breast milk leucocytes was significantly lower in the mothers with a cow's milk-allergic infant, whereas IFNgamma production of these cells was comparable in the two groups. CONCLUSION: Our results suggest that in the breast milk of mothers having an infant with cow's milk allergy, the number and function of TNFalpha-producing cells is defective. This might lead to a disturbance in the development of oral tolerance and thereby to the development of CMA in suckling infants. These novel results may help in clarifying the etiopathogenesis of CMA.     

A tumour necrosis factor-alpha (TNF-α) promoter polymorphism is associated with chronic hepatitis B infection

Cytokines such as TNF-α and interferon gamma (IFN-γ) are important for the elimination of infected hepatocytes during acute hepatitis B virus (HBV) infection. Two G versus A transitions in the TNF-α promoter region at positions ?308 and ?238 possibly influence TNF-α expression. We investigated these TNF-α polymorphisms in 71 patients with chronic HBV infection, in 32 subjects that had spontaneously recovered from acute HBV infection, and in 99 healthy controls. The ?238 A promoter variant was present in 18 (25%) of 71 patients with chronic HBV infection compared with two (6%) of 32 subjects with acute infection (P < 0.04), and seven (7%) of 99 controls (P < 0.003). By contrast, the prevalence of the variant at position ?308 was similar in all investigated groups. The observed differences could not be explained by linkage disequilibrium to HLA-B or -DRB1* alleles. These findings suggest an association between the TNF-α promoter polymorphism at position ?238 and the development of chronic HBV infection. This promoter variant appears to be linked to defective viral clearance.     

Triggering of respiratory burst by tumor necrosis factor in neutrophils adherent to fibronectin. Evidence for a crucial role of CD18 glycoproteins

Human neutrophils, plated on fibronectin (FN)-coated wells, were found to release large quantities of superoxide anion (O ₂ ^– ) in response to tumor necrosis factor alpha (TNF-). The O ₂ ^– release was completely inhibited by two monoclonal antibodies (MoAbs, MHM23 and TS1/18) against CD18 glycoproteins. An independently derived anti-CD18 MoAb (60.3) was ineffective. These MoAbs failed to inhibit neutrophil adhesion to FN-coated surfaces. Moreover, neutrophils incubated for 30 min on FN and then washed to remove non-adherent cells, were responsive to TNF- in the presence of anti-CD18 MoAbs MHM23 and TS1/18. Consequently, the CD18-dependent capacitation of the respiratory burst can occur before TNF- triggering. Finally, neutrophils plated on FN in the presence of anti-CD18 MoAbs and then washed, i.e. adherent cells blocked in their surface CD18 molecules, released O ₂ ^– after adding TNF- but only in the absence of additional anti-CD18 MoAbs. This is consistent with a TNF- ability to induce rapid expression and activation of new oxidative burst-capacitating CD18 molecules. The results suggest that the anchorage of neutrophils to FN surfaces depends on adherence molecules apparently unrelated to CD18, probably the so-called fibronectin receptors (FNRs), whereas the capacitation of the respiratory burst in response to TNF- requires the intervention of CD18 glycoproteins, available on the membrane of resting neutrophils or mobilized to the cell surface by TNF-.