Tumour necrosis factor-alpha and interleukin-8 inhibit neutrophil migration in vitro and in vivo

Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-alpha) and/or interleukin-8 (rIL-8), but not with either transforming growth factor-beta, interleukin-6 or interferon-gamma, rendered these cells less responsive to FMLP, in microchemotaxis assays. This inhibitory effect was dose dependent and more powerful when neutrophils were pretreated with a mixture of both cytokines. Intravenous injection of human rIL-8 (hrIL-8) and/or murine rTNF-alpha (mrTNF-alpha) also significantly reduced in vivo neutrophil migration into peritoneal cavities of rats stimulated with carrageenan. These data suggest that the defect in neutrophil migration during septicaemia or endotoxaemia may be the result of the continuous release of IL-8 and TNF-alpha into the circulation. Thus, either the selective control or blockade of releasing of these cytokines as well as of its effects on neutrophils may be clinically useful in reestablishing the cell defence mechanisms.     

Inhibition of migration of tumor cells in vitro by lymphokine-containing supernatants.

Lymphokine-containing supernatants derived from seven different human lymphoid cell lines and lymphokine-containing supernatants from concanavalin A-stimulated murine lymphocytes were found to be capable of reversibly inhibiting the migration of tumor cells in vitro. The tumor cell lines used in these studies were the P815 mastocytoma, Ehrlich ascites, Walker carcinosarcoma, Hepatoma 129, and Sarcoma 37. Preliminary physiochemical evidence suggests that the mediator, here termed TMIF, is distinct from MIF. In any case, these results suggest the possibility that lymphokines other than lymphotoxin or macrophage-activating factors may play a role in tumor immunity.     

Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor

U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.     

Lipoxygenase inhibitors suppress formation of tumor necrosis factor in vitro and in vivo

Injection of endotoxin in D-galactosamine sensitized mice resulted in increased serum levels of tumor necrosis factor as determined in a fibroblast cytolysis assay. Concomitant injection of different lipoxygenase inhibitors decreased the titer of TNF. When the lipoxygenase inhibitors were tested in macrophage cultures stimulated with LPS, they prevented the production of TNF. Indomethacin, a blocker of cyclooxygenase was neither in vivo nor in vitro effective in the prevention of the endotoxin-induced synthesis of TNF. The involvement of superoxide anion in this effect was excluded by use of superoxide dismutase which did not influence the formation of TNF in macrophages.     

Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor

Tumor necrosis factor (TNF) is a 17,000-Da protein which is produced by mononuclear cells upon exposure to endotoxin. Increases in adherence, phagocytosis, hydrogen peroxide release, and lysozyme secretion have been demonstrated after prolonged incubation of human neutrophils with TNF. In this study, the ability of highly purified recombinant human TNF to modulate neutrophil responses to soluble stimuli was evaluated. Tumor necrosis factor alone (0.1 to 10,000 units/ml) failed to induce neutrophil superoxide anion (O2-) production, granule release, or aggregation when incubated for up to 25 min at 37 degrees C. TNF did, however, stimulate a significant time-, dose-, and temperature-dependent increase in neutrophil F-actin content. Although exposure of neutrophils to TNF alone caused no superoxide anion production, it enhanced the O2- production in response to the chemotactic peptide, f-methionyl-leucyl-phenylalanine (FMLP) or the tumor promotor, phorbol myristate acetate, by as much as 278%. The enhancement was time-, dose-, and temperature-dependent and was due to a more rapid initial rate of O2- production. The TNF enhancement of FMLP-induced O2- production was blocked when an anti-TNF monoclonal antibody 241-1H11, is present during the preincubation period. TNF preincubation also enhanced FMLP-induced lysozyme release, but had no effect on aggregation and actin polymerization by FMLP. The kinetics of NADPH oxidase activation by arachidonic acid was unaltered by TNF. These results indicate that brief exposures to recombinant human TNF are able to enhance or prime the neutrophil oxidative burst in response to a second stimulus.     

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-α synthesis has been studied in vitro and in vivo. The synthesis of TNF-α in J774 macrophages stimulated with LPS (0.l μg/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 μM) and reduced by either L-arginine (3-30-300 μM) or the NO donor SIN-1 (1-10-1OOμM). The level of TNF-α in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L- arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-α synthesis suggesting an important regulatory link between NO and TNF-α in pathological processes.     

In vivo and in vitro induction of natural killer cells by cloned human tumor necrosis factor

Summary The natural killer (NK) cell activity of mice in the peritoneal cavity is very low or undetectable and testing peritoneal NK cells is a useful model for studying the influence of activating substances upon local injection. Injection of tumor necrosis factor (TNF) at doses of 10–200 ng caused a marked activation of NK cell activity which was maximal after 24 h and declined rapidly on day 2. A similar effect was observed when interferons alpha and beta were injected, and there were additive results when interferon was injected together with TNF. The NK cell nature of the effector cells activated by TNF was substantiated by the finding that previous injection with anti-asialo GM 1 antibody prevented activation. Interferon could not be detected in the peritoneal wash fluid after injection of TNF suggesting interferon-independent activation. In further experiments after i.p. injection of TNF peritoneal exudate cells (PECs) only killed YAC-1 targets in a 4-h assay. There was no additional killing in an 18-h assay towards neither YAC-1 cells or P815 cells, suggesting that macrophages were not involved. Furthermore TNF was also active in vitro by activating NK cells in isolated human peripheral blood cells. However in the PECs stimulated in vitro no significant induction of cytotoxic capacities by TNF was measured. Our data suggest that the action of TNF is not restricted to the lysis of tumor cells but can also induce immunological properties in the host defense against virus infections and neoplasms.     

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.     

Modulation of IL-1, tumor necrosis factor, and C5a-mediated murine neutrophil migration by alpha-melanocyte-stimulating hormone

This study was designed to examine the effects of i.p.-injected alpha-melanocyte stimulating hormone (MSH) on murine neutrophil migration into subcutaneously implanted sponges in response to IL-1-alpha, TNF-alpha, and C5a. The results show that as little as 0.1 ml of 5 x 10(-7) M MSH injected i.p. significantly blocked the accumulation of neutrophils in sponges in response to IL-1. This action of MSH was dose dependent, reversible, and was maximally effective if MSH was given at the same time as the injection of IL-1. This effect of MSH was not restricted to IL-1-induced neutrophil emigration, because MSH also antagonized the accumulation of neutrophils in response to both TNF and C5a. The proopiomelanocortin-derived peptide ACTH which contains the MSH sequence also significantly reduced neutrophil accumulation in response to IL-1, although less effectively than MSH. Similar studies with beta-endorphin showed that it had no effect on neutrophil accumulation in this system. The direct injection of MSH, beta-endorphin and ACTH into sponges or i.p. did not stimulate a neutrophil emigration and eliminated the possibility that MSH or ACTH suppressed the neutrophil influx in response to IL-1, TNF, or C5a by competing for circulating neutrophils. The action of MSH on IL-1, TNF, and C5a-induced neutrophil emigration suggests that this peptide may be an important regulator of the inflammatory response.     

Enhancement of in vivo immune response by tumor necrosis factor

Interleukin 1 (IL-1) has been shown to regulate several immunologic functions. Since tumor necrosis factor (TNF) shares many biologic properties with IL-1, we have investigated here the role of TNF in the modulation of the immune response. We have thus tested low doses of human recombinant TNF-alpha (hu rTNF-alpha) for its capacity to enhance the in vivo antibody responses evaluated at the cellular level in the hemolytic plaque assay. It was found that hu rTNF-alpha, like human IL-1 beta, is able to enhance the immune response to a T cell-dependent antigen (sheep red blood cells). Interestingly, at variance with human recombinant IL-1 beta, hu rTNF-alpha was not able to enhance the in vivo antibody response to a T cell-independent antigen (type III pneumococcal polysaccharide). These results suggest that low levels of TNF may have a role in the modulation of the immune response in vivo and shed new light on the biologic significance of this mediator.     

Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.     

The neutrophil migration induced by tumour necrosis factor alpha in mice is unaffected by glucocorticoids

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites.     

Anti-tumor activity of recombinant tumor necrosis factor on mouse fibrosarcoma in vivo and in vitro

The experiments presented were designed first to determine the effects of rTNF on the methylcholanthrene-induced fibrosarcoma (FSA-1) in C3H/JSed mice and second to determine whether the observed effects are the result of direct action by rTNF on the tumor or whether rTNF acts as a mediator of other effector mechanisms. Mice received syngeneic FSA-1 fibrosarcoma cells either s.c. or i.v. in order to evaluate growth of transplantable solid tumor or lung metastases, respectively. The range of dosages, from 10(2) to 2 x 10(5) U of rTNF, was administered i.v. at different intervals after the tumor cell injection. Early injection of 10(3) to 10(4) U of rTNF reduced the growth of s.c. injected tumor and the number of lung metastases in i.v. injected mice. In both cases, survival of mice was also prolonged. However, in vitro treatment of FSA-1 tumor cells with rTNF did not result in the reduction of their proliferating activity after injection into mice, although direct cytostatic and moderate cytotoxic activity of rTNF in vitro was demonstrated. To identify whether other cellular mechanisms are involved in the effects observed in vivo, the anti-tumor activity of rTNF-treated spleen cells was evaluated in vitro using a 75Se release assay. Whereas nontreated spleen cells demonstrated very low cytotoxic activity in this system, the cells from rTNF-treated mice showed marked increase in the cytotoxicity against syngeneic tumor cells. These results suggest that the anti-tumor activity of rTNF represents a combination of its direct effect on tumor cells and indirect effects involving host immune mechanisms.     

Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor.

The ability of human tumor necrosis factor-alpha (TNF-alpha) and human granulocyte colony stimulating factor (G-CSF) to induce phosphorylation of protein tyrosyl residues in human peripheral neutrophils (PMN) was investigated by Western blot analysis with antiphosphotyrosine antibody. Both TNF-alpha and G-CSF increased the tyrosyl phosphorylation of various proteins, such as species of 54-, 63-, 72-, 83-, 98-, 108-, and 115-kDa proteins. The ligand-stimulated tyrosyl phosphorylation of the 115-kDa protein was time- and concentration-dependent. When the 115-kDa protein was phosphorylated, it was recovered from membrane fractions. The phosphorylation of the 115-kDa protein was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamide (ST 638), inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl) methyl-piperazine dihydrochloride (H-7) and staurosporine, inhibitors of Ca(2+)- and phospholipid-dependent protein kinase (PKC). Similar inhibition by the TK inhibitors and stimulation by the PKC inhibitors were also observed with formylmethionyl-leucyl-phenylalanine (FMLP)-induced superoxide (O2.-) generation by TNF-alpha- or G-CSF-primed PMN. Phosphorylation of the 115-kDa protein occurred in parallel with the ligand-dependent generation of O2.-. These and other observations suggested that substrate proteins for tyrosine kinase, such as the 115-kDa protein, might play critical roles in the mechanism for priming of neutrophils. This is the first report describing that tyrosyl phosphorylation is involved in the priming of neutrophils by G-CSF and TNF-alpha.     

Cyclosporin a modulation of tumor necrosis factor gene expression and effects in vitro and in vivo

We investigated, in vitro and in vivo, the cyclosporin A (CsA) regulation of LPS-induced TNF gene expression and subsequent pathophysiologic changes. In vitro dose-response kinetics data showed that CsA inhibited TNF bioactivity in the supernatant without delaying its production, whereas Northern blot and in situ hybridization analysis demonstrated that CsA did not inhibit TNF mRNA expression. We then sought to examine the in vivo effects of CsA (75 mg/kg) in CBA/J mice that were primed with CFA, and injected 2 wk later with LPS. CsA demonstrated suppression of local levels (ascites) of TNF as measured by either bioactivity or an anti-murine TNF ELISA. However, CsA did not decrease mRNA for TNF, or cell-associated TNF. In vivo kinetics studies were performed to show that CsA blocked both local (ascites) and systemic (plasma) LPS-induced TNF production without delaying these effects. CsA inhibited the neutrophilia and lymphopenia that developed after the LPS challenge, but did not block the lung neutrophilic infiltrate. These observations are helpful in understanding the role of the macrophage in CsA immunosuppression, particularly with regard to the ability of CsA to block LPS-induced TNF secretion.     

Inhibition of tumor necrosis factor alpha secretion in rat Kupffer cells by siRNA: in vivo efficacy of siRNA-liposomes

Tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease. In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-alpha by anti-TNF-alpha antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-alpha. The efficacies of various siRNA duplexes were tested against TNF-alpha mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-alpha by 55% (p<0.005) with a proportionate reduction in TNF-alpha mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 microg/kg body weight/day for 2 days) of liposome-encapsulated SSL3. The LPS-induced TNF-alpha secretion was inhibited by >60% (p<0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-alpha, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-alpha.     

Inhibition of target cell mitochondrial electron transfer by tumor necrosis factor

Using digitonin permeabilization to assay mitochondrial electron transfer, we have found that respiratory activity (succinoxidase and cytochrome oxidase) in three mouse fibroblast lines is completely eliminated by incubation with human recombinant tumor necrosis factor-alpha (hrTNF). As with cytotoxicity, hrTNF-induced mitochondrial dysfunction occurs in resistant cells upon inhibition of protein synthesis, whereas sensitive cells exhibit spontaneous respiratory inhibition. In C3HA cells, inhibition is detectable 1.5-2 h after hrTNF addition, preceding cell lysis by at least 5 h (as measured by dye exclusion), and is approximately coincidental with morphological changes we have previously reported for this cell line. LM cells also exhibit inhibition of electron transfer, coincidental with morphological changes. These results suggest that bioenergetic dysfunction may be involved in the cytotoxic mechanism of TNF.     

Reactive oxygen species facilitate the in vitro and in vivo lipopolysaccharide-induced release of tumor necrosis factor

Secretion of TNF from mouse peritoneal macrophages exposed to LPS in vitro was enhanced in the presence of H2O2 or sodium periodate. Neither of these agents induced release of TNF in the absence of LPS. Both iron chelators and free radical scavengers inhibited this enhanced secretion of TNF, implying the involvement of free radicals via a Fenton-type reaction. Oxidant stress, in the form of alloxan or divicine, also enhanced serum levels of TNF in mice made sensitive to LPS by low-level infection with malaria, and then given i.v. LPS. Pretreatment with the iron chelator, desferal, or the free radical scavenger, BHA, inhibited TNF release in these animals. Less TNF was also detected in mice given desferal before LPS in the absence of exogenous radical generator. These results could have implications for understanding the details of the MLR, the adherence of neutrophils to the walls of pulmonary vessels in free radical-induced lung pathology, and the side effects of bleomycin.