A novel long chain polyunsaturated fatty acid, beta-Oxa 21:3n-3, inhibits T lymphocyte proliferation, cytokine production, delayed-type hypersensitivity, and carrageenan-induced paw reaction and selectively targets intracellular signals.

A novel polyunsaturated fatty acid (PUFA), beta-oxa 21:3n-3, containing an oxygen atom in the beta position, was chemically synthesized, and found to have more selective biological activity than the n-3 PUFA, docosahexaenoic acid (22:6n-3) on cells of the immune system. Although beta-oxa 21:3n-3 was very poor compared with 22:6n-3 at stimulating oxygen radical production in neutrophils, it was more effective at inhibiting human T lymphocyte proliferation (IC(50) of 1.9 vs 5.2 microM, respectively). beta-Oxa 21:3n-3 also inhibited the production of TNF-beta, IFN-gamma, and IL-2 by purified human T lymphocytes stimulated with PHA plus PMA, anti-CD3 plus anti-CD28 mAbs, or PMA plus A23187. Metabolism of beta-oxa 21:3n-3 via the cyclooxygenase and lipoxygenase pathways was not required for its inhibitory effects. Consistent with its ability to suppress T lymphocyte function, beta-oxa 21:3n-3 significantly inhibited the delayed-type hypersensitivity response and carrageenan-induced paw edema in mice. In T lymphocytes, beta-oxa 21:3n-3 inhibited the agonist-stimulated translocation of protein kinase C-betaI and -epsilon, but not -alpha, -betaII, or -theta to a particulate fraction, and also inhibited the activation of the extracellular signal-regulated protein kinase, but not c-Jun NH(2)-terminal kinase and p38. In contrast, 22:6n-3 had no effects on these protein kinase C isozymes. The increase in antiinflammatory activity and loss of unwanted bioaction through the generation of a novel synthetic 22:6n-3 analogue provides evidence for a novel strategy in the development of anti-inflammatory agents by chemically engineering PUFA.

Oxidation of oxa and thia fatty acids and related compounds catalysed by 5- and 15-lipoxygenase.

The modified fatty acids, (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid, 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid, N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine and N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid, all react with soybean 15-lipoxygenase. The products were treated with triphenylphosphine to give alcohols, which were isolated using HPLC. Analysis of the alcohols using negative ion tandem electrospray mass spectrometry, and by comparison with compounds obtained by autoxidation of arachidonic acid, shows that each enzyme-catalysed oxidation occurs at the omega-6 position of the substrate. In a similar fashion, it has been found that (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid, (Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid, (all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid and 3-[(all-Z)-(eicosa-5,8,11,14-tetraenylthio)]propionic acid each undergoes regioselective oxidation at the carboxyl end of the polyene moiety on treatment with potato 5-lipoxygenase. Neither (all-Z)-(eicosa-5,8,11,14-tetraenylthio)succinic acid nor N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]aspartic acid reacts in the presence of this enzyme, while N-[(all-Z)-(eicosa-5,8,11,14-tetraenoyl)]glycine affords the C11' oxidation product. The alcohol derived from (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid using the 15-lipoxygenase reacts at the C6' position with the 5-lipoxygenase.

Rapid degradation of articular cartilage proteoglycan by neutrophils: comparison with macrophages and synovial fibroblasts.

OBJECTIVE AND DESIGN: To determine and compare the proteoglycan degradative properties of neutrophils, macrophages and synoviocytes in cultures of articular cartilage. MATERIAL OF SUBJECTS: Bovine articular cartilage was aseptically isolated from metacarpopharyngeal joints. Neutrophils and macrophages were isolated from normal human blood and bovine synovial fibroblasts were isolated from explant cultures before being incubated with the cartilage. TREATMENT: Neutrophils, macrophages or synovial fibroblasts (1 x 10(6)-8 x 10(6)) were incubated with 35SO4 labelled cartilage for 2.5-72 h. METHODS: Cartilage degradation was measured as a loss of 35SO4 into the cartilage medium as a percentage of the total labelled proteoglycan in the cartilage slice. Statistical significances were determined using a 2-tailed unpaired Student's t-test. RESULTS: Neutrophils rapidly degraded articular cartilage. After 2.5 hours of culture, neutrophils degraded cartilage proteoglycan up to 28 times more than either macrophages or synovial fibroblasts. CONCLUSIONS: Neutrophils induce rapid damage to articular cartilage proteoglycan, whereas in comparison, macrophages and synovial fibroblasts degrade articular cartilage proteoglycans poorly. These findings indicate that at least under conditions where the influence of cellular-cellular interactions and soluble mediator action are excluded, adhesion of neutrophils to articular cartilage is sufficient to stimulate rapid and marked cartilage degradation compared to the other two cell types.

Tumor necrosis factor (TNF) and a TNF-mimetic peptide modulate the granulomatous response to Mycobacterium bovis BCG infection in vivo.

Tumor necrosis factor (TNF) is a critical mediator in the immune response to mycobacteria, particularly in the formation and maintenance of granulomas. Treatment of Mycobacterium bovis BCG-infected mice with TNF and a TNF-mimetic peptide (TNF(70-80)) altered the number and cellular composition of granulomas. This change was associated with a moderate decrease in the bacterial burden.

Stimulation of p38 phosphorylation and activity by arachidonic acid in HeLa cells, HL60 promyelocytic leukemic cells, and human neutrophils. Evidence for cell type-specific activation of mitogen-activated protein kinases.

Although it is well appreciated that arachidonic acid, a second messenger molecule that is released by ligand-stimulated phospholipase A2, stimulates a wide range of cell types, the mechanisms that mediate the actions of arachidonic acid are still poorly understood. We now report that arachidonic acid stimulated the appearance of dual-phosphorylated (active) p38 mitogen-activated protein kinase as detected by Western blotting in HeLa cells, HL60 cells, human neutrophils, and human umbilical vein endothelial cells but not Jurkat cells. An increase in p38 kinase activity caused by arachidonic acid was also observed. Further studies with neutrophils show that the stimulation of p38 dual phosphorylation by arachidonic acid was transient, peaking at 5 min, and was concentration-dependent. The effect of arachidonic acid was not affected by either nordihydroguaiaretic acid, an inhibitor of the 5-, 12-, and 15-lipoxygenases or by indomethacin, an inhibitor of cyclooxygenase. Arachidonic acid also stimulated the phosphorylation and/or activity of the extracellular signal-regulated protein kinase and of c-jun N-terminal kinase in a cell-type-specific manner. An examination of the mechanisms through which arachidonic acid stimulated the phosphorylation/activity of p38 and extracellular signal-regulated protein kinase in neutrophils revealed an involvement of protein kinase C. Thus, arachidonic acid stimulated the translocation of protein kinase C alpha, betaI, and betaII to a particulate fraction, and the effects of arachidonic acid on mitogen-activated protein kinase phosphorylation/activity were partially inhibited by GF109203X, an inhibitor of protein kinase C. This study is the first to demonstrate that a polyunsaturated fatty acid causes the dual phosphorylation and activation of p38.

A tumor necrosis factor mimetic peptide activates a murine macrophage cell line to inhibit mycobacterial growth in a nitric oxide-dependent fashion.

The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-gamma) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp. Therefore, we investigated the capacity of TNF(70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF(70-80) in the presence of IFN-gamma, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of 3H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF(70-80)-induced macrophage activation was dependent on IFN-gamma and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-gamma antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of L-arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF(70-80), demonstrating that the effect of TNF(70-80) is dependent on signaling through TNF receptor I. The mimetic effects of TNF(70-80) on macrophage activation in vitro suggest that treatment with TNF(70-80) may modulate mycobacterial infections in vivo.

Enhancement of lipopolysaccharide-induced neutrophil oxygen radical production by tumor necrosis factor alpha.

Although tissues become exposed to both exogenous and endogenous cell-activating mediators during infection, there is little appreciation of the effects of subjecting cells to multiple mediators. We examined the hypothesis that the response of neutrophils to bacterial lipopolysaccharide (LPS) is significantly altered in the presence of the endogenous mediator tumor necrosis factor alpha (TNF). The data showed that human neutrophils pretreated with TNF for 10 to 30 min, displayed significantly enhanced superoxide production in response to LPS (from either Escherichia coli K-235 or E. coli O127:B8), measured as lucigenin-dependent chemiluminescence (CL), seen as an increase in the initial peak rate as well as the total CL accumulated over the incubation period. TNF amplified the response to LPS at 1 to 100 U of TNF/10(6) neutrophils and was able to enhance the response to a wide range of concentrations of LPS (0.01 to 1,000 ng/ml). The TNF-induced increase in the LPS response was paralleled by an increase in LPS binding to the neutrophils, which could be abrogated by an anti-CD14 monoclonal antibody. The results demonstrate that TNF significantly increases the LPS-induced release of oxygen radicals in neutrophils through the upregulation of cell surface CD14.

N-6 and n-3 polyunsaturated fatty acids stimulate translocation of protein kinase Calpha, -betaI, -betaII and -epsilon and enhance agonist-induced NADPH oxidase in macrophages.

The polyunsaturated fatty acids (PUFA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were poor inducers of oxygen-dependent respiratory activity (chemiluminescence) in human monocytes and macrophages, but markedly enhanced the response to the tripeptide, N-formylmethionyl-leucyl-phenylalanine. The effects of these fatty acids were seen at concentrations of 1 microg/ml. A similar enhancement was seen with PMA, a stimulus that acts on protein kinase C (PKC), or calcium ionophore (A23187), which increases intracellular calcium, suggesting that the effect of the fatty acids was post-surface receptor binding. HL-60 cells, differentiated to macrophage-like cells by culture in the presence of vitamin D3, were similarly affected by the fatty acids. In experiments in which the time of pre-exposure of the monocytes to PUFA was varied, it was found that the priming effect induced by AA, EPA and DHA was maximal at 5 min. The ability of these fatty acids to synergize with other agonists was completely lost if the fatty acids were either methylated or oxidized to the hydro and hydroperoxy derivatives. Saturated fatty acids were inactive. Western blot analysis demonstrated that the PUFA induced the translocation of PKCalpha, -betaI, -betaII and -epsilon isoenzymes to a particulate fraction. The synergistic response between fatty acids and A23187 was completely inhibited by pretreating the cells with a PKC inhibitor, GF-109203X, or by pretreatment of monocytes with PMA for 18 h, to deplete PKC levels. From these investigations it is evident that PUFA prime macrophages, causing increased/synergistic oxidative respiratory burst activity to other stimuli and that this priming is dependent on PKC translocation and activation.

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

Inhibition of gap junctional communication by polyunsaturated fatty acids in WB cells: evidence that connexin 43 is not hyperphosphorylated.

Polyunsaturated fatty acids have attracted much interest due to their wide spectrum of biological activities which include the modulation of gap junctional communication (GJC). Since gap junctions play critical roles in maintaining the functional integrity of organs and tissues, and loss of intercellular communication is associated with a number of pathological conditions, we investigated the effects of the n-6 and n-3 series of polyunsaturated fatty acids and their derivatives on GJC in WB cells as determined by the ability of Lucifer Yellow-loaded cells to transfer the dye to neighbouring recipient cells. Studies were also conducted to investigate the possible mechanisms of action of the fatty acids. Treatment of cells with 10 microM arachidonic acid (20:4 n-6) resulted in a rapid and transient loss of communication competence. The response to 20 microM 20:4 (n-6) was prolonged (> 210 min) but was readily reversible by washing the cells with fatty acid-free bovine serum albumin. Cells which had regained their communication competence responded to further additions of 20:4 (n-6). The fatty acids, 18:3 (n-6), 20:5 (n-3), 22:6 (n-3) and the 15-hydroxy- and the 15-hydroperoxy-derivatives of 20:4 (n-6) were also powerful inhibitors of GJC, while 23:4 (n-6) was a relatively weak inhibitor. The saturated 20 carbon fatty acid, 20:0, and the methyl ester of 20:4 (n-6) were without effect. This illustrates the importance of unsaturation and the carboxyl group as structural requirements for activity. 20:4 (n-6)-induced inhibition of dye transfer was not attenuated by pretreating the cells with either phorbol-12-myristate-13-acetate (PMA) or indomethacin, suggesting that regulation of gap junctional permeability by 20:4 (n-6) in WB cells was neither dependent on PMA-responsive isozymes of protein kinase C nor required the metabolism of the fatty acids by cyclo-oxygenase. However, the effect of 20:4 (n-6) was antagonized by preincubating WB cells with either nordihydroguaiaretic acid or (+/-)-isoproterenol and isobutylmethyl-xanthine. Western blot analysis of connexin 43 (Cx43), the major gap junctional protein expressed in these cells, revealed no detectable changes to the electrophoretic mobility of Cx43 even after 60 min of incubation in the presence of 20:4 (n-6). As expected, other inhibitors of gap junctional permeability including epidermal growth factor, phorbol ester or lysophosphatidic acid induced a retardation in the mobility of Cx43, indicating an enhancement in the phosphorylation of Cx43 protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitory effects of arachidonic acid (20:4,n-6) and its monohydroperoxy- and hydroxy-metabolites on procoagulant activity in endothelial cells.

The procoagulant response of endothelium to pathophysiological agents such as tumour necrosis factor alpha (TNF alpha) and phorbol myristate acetate (PMA) alters the expression of proteins such as tissue factor. The modulation of such procoagulant activity (PCA) by the polyunsaturated fatty acid arachidonic acid (20:4,n-6) and its 15-hydroperoxy (15-HPETE) and 15-hydroxy (15-HETE) metabolites was examined since this may have important implications in cardiovascular disease and atherosclerosis. Treatment of human umbilical vein endothelial cells (HUVEC) for 30 min with 20:4, 15-HPETE or 15-HETE before induction of PCA with TNF alpha (100 U) or PMA (10(-7) M) caused a significant inhibition of PCA. This inhibition was seen at 2-5 microM fatty acids. Dose response curves with TNF alpha indicated that the inhibition was greatest at higher concentrations of TNF alpha (> or = 250U TNF alpha/ml). The mode of administration of the fatty acid was not critical as fatty acids presented as DPC-fatty acid micelles or solubilised in ethanol gave similar inhibitions of PCA. 20:4, 15-HPETE or 15-HETE did not alter the binding of I125-labelled TNF alpha to its surface receptors on HUVEC, suggesting that the effect of these fatty acids was not mediated by events at the cell surface receptor level. In support of this, these fatty acids were found to inhibit PCA induced by PMA which bypasses cell surface receptors to activate protein kinase C directly.(ABSTRACT TRUNCATED AT 250 WORDS)

A synthetic tumor necrosis factor-alpha agonist peptide enhances human polymorphonuclear leukocyte-mediated killing of Plasmodium falciparum in vitro and suppresses Plasmodium chabaudi infection in mice.

A peptide corresponding to residues 70-80 of the TNF-alpha polypeptide was synthesized and shown to enhance human PMN-mediated killing of Plasmodium falciparum in vitro and reduced the Plasmodium chabaudi parasitemia in mice. Studies of the mechanism of action showed that the peptide, TNF(70-80), stimulated and primed PMN for an increased respiratory burst and release of granule constituents in response to a second agonist. The PMN-stimulatory activity of the peptide was inhibited by mAbs against the p55 and p75 TNF receptors and a TNF-neutralizing mAb. Analysis of PMN receptor expression showed that CR3 (CD18/CD11b) and Fc gamma RIII were upregulated by TNF(70-80), which was consistent with the peptide's ability to enhance parasite killing by PMN. The peptide, unlike TNF, did not increase the expression of adhesion molecules on endothelial cells and failed to promote binding of P. falciparum-infected erythrocytes to endothelial cells. TNF(70-80) also inhibited the TNF-induced increase in adhesion of P. falciparum-infected erythrocytes to endothelial cells. The results demonstrate that the host-protective effects of TNF can be retained while toxic effects are eliminated using a selected, characterized subunit of the cytokine.

Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism.

Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 microM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, delta, and epsilon to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, delta and epsilon. In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.

Interaction of Staphylococcus aureus with human neutrophils and the down-regulation of TNF receptors.

We have shown previously that pre-exposure of neutrophils to TNF significantly enhanced their killing of opsonized Staphylococcus aureus. We now demonstrate that the ability of TNF to enhance the bactericidal activity is dependent on preincubation time; enhancement was still evident when TNF and bacteria were added simultaneously to neutrophils but if TNF addition was delayed by 5 min, no enhancement was seen. Evidence is presented that suggests that this could be related to a down-regulation of TNF receptors by the bacteria, but in addition, the release of TNF receptor fragments may contribute to the inhibition observed. Scatchard analyses demonstrated a decrease from approximately 3000 TNF receptor (receptor binding) sites per cell to 450 following treatment with S. aureus, but essentially no change in receptor affinity. Using mAb directed against the type A (75 kDa) receptor (utr-1) and the type B (55 kDa) receptor (htr-9), it was found that the expression of both receptors was decreased following treatment with the bacteria. The time course of loss of these receptors showed that the surface expression of both molecules was markedly decreased by 5 min which correlated with the loss in ability of TNF to enhance the bactericidal activity. In contrast to changes seen in the binding of TNF, similarly treated neutrophils showed essentially no change in the binding of radiolabeled tripeptide FMLP and, if anything, an increase in the expression of the CD11b Ag (CR3 receptor). When another phagocytic stimulus was used, opsonized fungi (Torulopsis glabrata), a similar depression of TNF binding was also found, but opsonized sheep erythrocytes had no effect on the TNF binding, suggesting that the effects on the TNF receptor cannot be explained simply on the basis of particle phagocytosis.

The effect of enhancing antibodies on TNF interactions with its specific receptor: consequences for in vitro TNF antiviral activity.

This study examines the interaction of TNF with its receptor(s) in the presence of antibodies which have been previously found to enhance the in vivo antiviral and antitumor activities of this cytokine. The presence of Mab 32 has been previously shown to enhance the binding of 125I-TNF to the surface L929 cells (13), and this property was also found in the present study for HeLa cells. In addition to this, Mab 32 was found to enhance the internalization of the TNF ligand into both L929 and HeLa cells compared to control treated cultures. The consequences of such enhanced TNF-binding on TNF antiviral activity were examined in both L929 cells and HeLa cells. It was discovered that the similarities in antibody-enhanced TNF binding and internalization contrasted dramatically with the sensitivities of these two cell lines to the antiviral actions of TNF, both with and without Mab 32 (viz., L929 cells were sensitive; HeLa cells were resistant). It has been proposed that the modulation of TNF-R expression, particularly by IFNs, is an important factor in TNF's biological effects. It has been shown that the presence of IFN-gamma, with TNF plus specific enhancing antibodies, further augmented antiviral activity in vivo (13). This finding stimulated interest in examining IFN-gamma modulation of TNF-R as a factor in the antiviral activity of TNF. The expression of TNF receptor(s) in TNF- and/or IFN-gamma-exposed cells, both with and without HSV-1 infection, was therefore examined. TNF alone could induce a dose-dependent increase in receptor expression which was not significantly increased by Mab 32. Exposure of L929 cells to IFN-gamma alone also induced TNF receptor expression in mock-infected cells. HSV-1 infection of L929 cells resulted in a significant upregulation of TNF-R expression which was reversed if the cells had been preexposed to IFN-gamma. The inclusion of TNF with IFN-gamma before infection restored TNF-R expression but did not show any further synergistic or additive effects on TNF-R expression. Some minor increases in TNF-R expression were seen if Mab 32 was included with these two cytokines.

Killing of Staphylococcus aureus by tumor necrosis factor-alpha-activated neutrophils. The role of serum opsonins, integrin receptors, respiratory burst, and degranulation.

We have examined the effects of TNF priming on the killing of Staphylococcus aureus by human neutrophils. In the absence of serum opsonins, neutrophils failed to kill S. aureus, and TNF priming did not induce the cells to become bactericidal. Normal human serum, containing complement activity, promoted the killing of the bacteria by neutrophils. Pretreatment of neutrophils for 30 min with TNF significantly enhanced their bactericidal activity. The effects of TNF on neutrophil bactericidal activity was dependent on serum concentration and the degree of enhancement induced increased up to a concentration of 1%. The kinetics of bacterial killing showed that TNF-only enhanced the initial rate of killing, over the first 30 min. Little killing of bacteria occurred in the presence of complement-inactivated serum, and TNF did not stimulate this killing. These results suggest that TNF enhances the neutrophil complement-dependent killing of S. aureus. TNF increased the expression of CR3 (CD11b/CD18) and CR4 (P150, 95; CD11c/CD18) adhesion receptors but not LFA-1 (CD11a/CD18); and mAb against the alpha-chain of either CR3 or CR4 but not LFA-1 prevented the enhancing effects of TNF on the neutrophil bactericidal activity.

Differential effects of small tumour necrosis factor-alpha peptides on tumour cell cytotoxicity, neutrophil activation and endothelial cell procoagulant activity.

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.

The enhancement of human tumor necrosis factor-alpha antiviral activity in vivo by monoclonal and specific polyclonal antibodies.

This report describes the potentiation of the antiviral effects of human tumour necrosis factor-alpha (TNF-alpha) in vivo by specific antibodies. Complexes of TNF with either an anti-TNF monoclonal or polyclonal antibody at optimal doses induced a significantly greater antiviral (vaccinia virus) state in CBA/H mice than TNF alone. Furthermore, an antiviral synergy between murine gamma-interferon and TNF was found in vivo when the latter was in complex with enhancing antibody. Two other inbred mouse strains, C57/B6 and BALB/c, were also examined under these conditions. These antibodies greatly increase the binding of TNF to the surface of cells, which may account for the observed enhancement of TNF activity. Such antibodies may be of value clinically in viral diseases and cancer therapy where an increase in TNF activity, in the absence of side effects, would lead to more effective use of this cytokine in human therapy.