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We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the β-position, β-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the β-oxa-PUFA substantially depresses the production of leukotriene B4 (LTB4) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, β-oxa 21:3 n-6, is also a strong inhibitor of LTB4 production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB4. The parent β-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB4 formation. β-Oxa-21:3n-3 inhibits the conversion of [3H]20:4n-6 to [3H]5-hydroxyeicosatetraenoic acid and [3H]LTB4 by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. β-Oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of β-oxa-21:3n-3 to a 16-monohydroxy-β-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, β-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, β-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.

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-γ) 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-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of 3 H-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-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ 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.

The procoagulant response of endothelium to pathophysiological agents such as tumour necrosis factor α (TNFα) 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 TNFα (100 U) or PMA (10 −7 M) caused a significant inhibition of PCA. This inhibition was seen at 2–5 μM fatty acids. Dose response curves with TNFα indicated that the inhibition was greatest at higher concentrations of TNFα (≥250U TNFα/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 I 125 -labelled TNFα 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. There was no alteration in the cell surface expression of tissue factor by these fatty acids, suggesting that the inhibition of PCA seen with these fatty acids does not result from a decrease in the synthesis of tissue factor. These findings have important clinical consequences in cardiovascular disease and atherosclerosis.

TNF alpha is a cytokine which causes cytolysis of tumour cell lines in vitro as well as haemorrhagic necrosis of many transplanted tumours in vivo. In association with these activities, the cytokine manifests a high degree of toxicity in vivo. The in vitro and in vivo effects of a panel of 13 monoclonal antibodies against human TNF alpha have been investigated. Of these MAbs, eight neutralized TNF alpha activity in the WEHI-164 cytotoxicity assay as well as in the binding of TNF alpha to receptors on these cells. The effects of this group of antibodies on TNF alpha-induced regression of WEHI tumours in vivo correlated with their in vitro neutralizing activities. One MAb which inhibited cytotoxicity, receptor interaction and tumour regression in the WEHI model (MAb 37) failed to inhibit TNF alpha-receptor binding and tumour regression in Meth A models. This observation indicates that different classes of receptor specificity may exist on different tumour cells. Together the antibodies define six non-overlapping epitopic domains on TNF alpha and within these regions there are at least nine overlapping epitopes. Inhibitory MAbs, when co-injected into tumour-bearing mice with radiolabelled TNF alpha, resulted in the diversion of TNF alpha away from both tumour and lung, which correspond to the sites of highest TNF alpha uptake in control MAb-TNF alpha treated mice. In contrast, uptake of TNF alpha by the liver was increased and overall, biodistribution studies showed that very little TNF alpha reached the target tumour but was rapidly and widely dispersed throughout the body. Preliminary studies with these MAbs show that segregation of TNF alpha activities and receptor binding may be possible.

Several novel polyunsaturated fatty acids (PUFAs) that contain either an oxygen or sulfur atom in the beta-position were found to exhibit more selective antiinflammatory properties than their natural PUFA counterparts. One of these, beta-oxa-23:4n-6, unlike natural PUFAs, lacked ability to stimulate oxygen radical production in neutrophils but caused marked inhibition of agonist-induced upregulation of leukocyte adhesion to cultured human umbilical vein endothelial cells (HUVEC) and E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression. In addition, beta-oxa-23:4n-6 inhibited acute and chronic inflammatory responses in mice as well as the upregulation of adhesion molecule expression in arterial endothelium. This action of beta-oxa-23:4n-6 required a functional 12- but not 5-lipoxygenase or cyclooxygenases, consistent with its metabolism via the 12-lipoxygenase pathway. Whereas beta-oxa-23:4n-6 did not affect the activation of mitogen-activated protein kinases by tumor necrosis factor, activation of the IkappaB kinase/nuclear factor kappaB pathway was selectively inhibited. These novel PUFAs could form the basis for a potential new class of pharmaceuticals for treating inflammatory diseases, including atherosclerosis.

A novel polyunsaturated fatty acid (PUFA), β-oxa 21:3n-3, containing an oxygen atom in the β 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 β-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 (IC50 of 1.9 vs 5.2 μM, respectively). β-Oxa 21:3n-3 also inhibited the production of TNF-β, IFN-γ, 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 β-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, β-oxa 21:3n-3 significantly inhibited the delayed-type hypersensitivity response and carrageenan-induced paw edema in mice. In T lymphocytes, β-oxa 21:3n-3 inhibited the agonist-stimulated translocation of protein kinase C-βI and -ε, but not -α, -βII, or -θ to a particulate fraction, and also inhibited the activation of the extracellular signal-regulated protein kinase, but not c-Jun NH2-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.

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 0127: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.

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.

Interleukin 1 (IL-1), IL-6, and tumour necrosis factor (TNF) alpha are pleiotropic cytokines produced predominantly by macrophages which have been implicated in the pathogenesis of rheumatoid arthritis (RA). Sulphasalazine has been shown to have disease modifying properties and to inhibit the production of cytokines in vitro. To evaluate the effect of sulphasalazine on cytokine production in vivo, serum cytokine levels were measured in a group of patients with RA entered into a randomised controlled trial. Serum levels of IL-1 alpha, IL-1 beta, IL-6, and TNF alpha were measured at baseline and at two monthly intervals for six months in 17 patients receiving sulphasalazine and in 22 patients treated with placebo. The two groups of patients had a similar age and sex distribution, had had RA for less than a year, had no joint erosions, and had not been treated previously with any other disease modifying drugs. In the 39 patients studied IL-1 alpha was detected (> 0.1 ng/ml) at baseline in 14 patients (median 0.24 ng/ml), IL-1 beta in 25 patients (median 1.0 ng/ml), TNF alpha in 27 patients (median 1.2 ng/ml), and IL-6 in 33 patients (median 0.44 ng/ml). In the group treated with sulphasalazine there was a progressive and significant decline in serum IL-1 alpha, IL-1 beta, and TNF alpha levels over the six month period (median levels at six months were < 0.1, 0.12, and 0.44 ng/ml respectively). Interleukin 6 levels were significantly reduced only at the four month time point (median level of 0.23 ng/ml). These reductions were associated with improvements in clinical and laboratory measures of disease activity. In contrast patients receiving the placebo showed no changes in serum cytokine levels and no improvement in clinical and laboratory indices of disease activity. These results suggest that sulphasalazine may exert its disease modifying effect partly by suppressing cytokine production in vivo.

The primary aim of this work was to produce specific monoclonal antibodies to human growth hormone (hGH) for use in a diagnostic RIA of hGH levels in serum. Three different schedules were used for immunization of BALB/c mice and the splenocytes from each mouse were fused with myeloma cells Sp 2/0 Ag 14. Each fusion resulted in the production of hundreds of hybridomas secreting hGH-directed antibodies. Six antibodies have been fully characterized and have been grouped into pairs which recognize 3 different epitopes on the hGH molecule. One pair exhibits no cross reaction with the structurally related placental hormone, human placental lactogen (hPL), a second pair has low cross reaction with hPL (1.6–3%) and a third pair reacts equally well with hGH and hPL indicating binding to a common epitope in the 2 molecules. The highest affinity antibody, 746, which has an affinity constant of 4.4 × 1010 1/mol and 3% cross-reactivity with hPL, has been used to establish a RIA for serum hGH measurements. Evidence is provided that hGH levels measured in this assay correlate well with those obtained in a conventional rabbit antiserum assay.