Dwight R. Robinson

Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function.

Abstract The effects of dietary fish-oil fatty acids on the function of the 5-lipoxygenase pathway of peripheral-blood polymorphonuclear leukocytes and monocytes were determined in seven normal subjects who supplemented their usual diet for six weeks with daily doses of triglycerides containing 3.2 g of eicosapentaenoic acid and 2.2 g of docosahexaenoic acid. The diet increased the eicosapentaenoic acid content in neutrophils and monocytes more than sevenfold, without changing the quantities of arachidonic acid and docosahexaenoic acid. When the neutrophils were activated, the release of [3H]arachidonic acid and its labeled metabolites was reduced by a mean of 37 per cent, and the maximum generation of three products of the 5-lipoxygenase pathway was reduced by more than 48 per cent. The ionophore-induced release of [3H]arachidonic acid and its labeled metabolites from monocytes in monolayers was reduced by a mean of 39 per cent, and the generation of leukotriene B4 by 58 per cent. The adherence of neutro...

Prostaglandin-stimulated bone resorption by rheumatoid synovia. A possible mechanism for bone destruction in rheumatoid arthritis.

Synovial tissue from patients with rheumatoid arthritis was maintained in organ culture for 3-14 days. Conditioned media from these synovial cultures contained bone resorption-stimulating activity, measured in vitro by using calcium release from mouse calvaria as the assay system. The synovial cultures also produce prostaglandin E2 (PGE2) as measured by serologic methods. The production of both the bone resorption-stimulating activity and PGE2 was inhibited by more than 90% by treatment of the synovial cultures with indomethacin (5 mug/ml). In contrast, treatment of the synovial cultures with colchicine (0.1 mug/ml) caused a marked and parallel increase in the concentration of both bone resorption-stimulating activity and PGE2 in the conditioned media. The bone resorption-stimulating activity was quantitatively extracted into diethyl ether. Within the limits of experimental error, all of the bone resorption-stimulating activity in medium was accounted for by its content of PGE2, itself a potent osteolytic factor. We conclude that the bone resorption-stimulating activity produced by rheumatoid synovia in culture is PGE2.

Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

Dietary enrichment with the polyunsaturated fatty acid eicosapentaenoic acid prevents proteinuria and prolongs survival in NZB x NZW F1 mice.

Prostaglandins and related compounds are active mediators of inflammation, but data concerning their role in the pathogenesis of the glomerulonephritis of New Zealand Black x New Zealand White (NZB x NZW) F1 mice are conflicting. Dietary eicosapentaenoic acid (EPA, C20:5), a fatty acid analogue of arachidonic acid (C20:4), has been shown to impair platelet aggregation in humans, apparently through inhibition of the synthesis of prostaglandins and thromboxanes from arachidonic acid. We report here the effects of a diet high in EPA on the development of renal disease and survival in female NZB x NZW F1 mice. Animals from 4--5 wk of age were fed diets containing 25% lipid, supplied either as beef tallow or menhaden oil, with fatty acid analysis of less than 0.05 and 14.4% EPA, respectively. In the first experiment, by 13.5 mo of age, mice on the beef tallow diet had all (9/9) developed proteinuria and the majority (6/9) had died, with renal histologic examination revealing severe glomerulonephritis. In contrast, none of 10 menhaden oil-fed animals had developed proteinuria, and all were alive at this time (P less than 0.005 for both proteinuria and survival). In a second experiment using 50 mice in each dietary group, 56% of the beef tallow group vs. none of the menhaden oil group had developed proteinuria at 9 mo of age (P less than 0.005). Native DNA binding at 6 mo of age was 23.9 +/- 14.7 vs. 10.1 +/- 9.7% in the beef and menhaden oil groups, respectively (P less than 0.01). Weights were similar in all groups, and there was no evidence of essential fatty acid deficiency in any group. These results demonstrate that a diet high in EPA protects NZB x NZW F1 mice from the development of glomerulonephritis.

Inhaled Nitric Oxide A Selective Pulmonary Vasodilator of Heparin-Protamine Vasoconstriction in Sheep

Nitric oxide (NO) has recently been discovered to be an important endothelium-derived relaxing factor and produces profound relaxation of vascular smooth muscle. To learn if NO could be a potent and selective pulmonary vasodilator, NO was inhaled by 16 awake lambs in an attempt to reduce the increase in pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR) induced by either the infusion of an exogenous pulmonary vasoconstrictor (the thromboxane analog U46619) or the endogenous release of thromboxane that occurs during the neutralization of heparin anticoagulation by protamine sulfate. Inhaling greater than or equal to 40 ppm of NO during a continuous U46619 infusion returned the PAP to a normal value, without affecting systemic blood pressure or vascular resistance. Pretreatment with the cyclooxygenase inhibitor indomethacin before infusing U46619 did not reduce the pulmonary vasodilatory effect of inhaled NO, and we conclude that the dilatory effect of NO on the lungs circulation is independent of cyclooxygenase products such as prostacyclin. Continuously inhaling NO at 180 ppm did not significantly reduce the mean peak thromboxane B2 concentration at 1 min after protamine injection; however, the mean values of pulmonary hypertension and vasoconstriction at 1 min were markedly reduced below the levels in untreated heparin-protamine reactions. Breathing NO at lower concentrations (40-80 ppm) did not decrease the mean peak PAP and PVR at 1 min after protamine but decreased the PAP and PVR values at 2, 3, and 5 min below those of control heparin-protamine reactions. Intravenous infusion of nitroprusside completely prevented the transient increase of PAP and PVR during the heparin-protamine reaction; however, marked concomitant systemic vasodilation occurred. Inhaled NO is a selective pulmonary vasodilator that can prevent thromboxane-induced pulmonary hypertension during the heparin-protamine reaction in lambs and can do so without causing systemic vasodilation.

PROSTAGLANDINS IN THE RHEUMATIC DISEASES

The prostaglandins may participate in the pathogenesis of the inflammatory rheumatic diseases by acting as mediators of inflammation and in promoting bone resorption. Levels of PGB (presumed to arise from PGE) in synovial fluids are elevated in the majority of a group of patients with inflammatory rheumatic diseases, as compared to similar patients treated with aspirin and indomethacin and patients with osteoarthritis. Rheumatoid synovium produces large amounts of PGE2 in organ culture. In addition, fibroblast cell lines derived from rheumatoid synovia synthesize more PGE and more cAMP than do cells from normal synovia or skin. The media from rheumatoid synovial organ cultures contain large quantities of bone-resorbing activity toward mouse calvaria in vitro. The bone resorption can be accounted for by PGE2 produced by the synovia, because the activity and PG synthesis are inhibited by more than 90% by incubation of the tissue with indomethacin, because it is quantitatively extractable into ether, and because it bears a relationship to the concentrations of PGE2 present, as measured by radioimmunoassay.

The protective effect of dietary fish oil on murine lupus

Dietary marine lipids markedly reduce the severity of glomerulonephritis and its associated mortality in inbred strains of mice developing autoimmune disease, a model for human systemic lupus erythematosus. We report here the influence of varying the dose of menhaden oil and the timing of its administration on the mortality of female (NZB x NZW) F1 mice. After ingesting 25 wt% menhaden oil (MO) for periods of 1.5 weeks to 12 months, there was a stable content of tissue n-3 fatty acids, with total n-3 fatty acids of 28% and 35% in spleen and liver, respectively. The extent of protection from mortality was dependent on the dose of MO with marked protection at doses of 11 to 25%, marginal protection at 5.5% and no protection at 2.5% MO. Delay in the institution of MO until ages 5 or 7 months still resulted in large reductions of mortality. Conversely, institution of a MO diet from 6 weeks until ages 5 to 7 months followed by a change to beef tallow resulted in little protection. Serum levels of 4 cyclooxygenase products were reduced ranging from 26 to 76% in mice fed MO diets, compared to mice fed beef tallow, based on radioimmunoassay. The degree of reduction of mortality on different doses of MO was correlated best with tissue levels of C22:5, and levels of C20:5 and C22:6 were similar at high and low doses of MO, suggesting that levels of 22:5 may be related to the protective effects of marine lipids on autoimmune disease.

Dietary marine lipids suppress continuous expression of interleukin-1β gene transcription1

Abstractn-3 Polyunsaturated fatty acids abundant in marine lipids suppress certain inflammatory and immune reactions, and dietary marine lipid supplements have antiinflammatory effects in experimental and human autoimmune disease. Previous work by other investigators demonstrated that dietary marine lipid supplements suppressed production of cytokines from stimulated human peripheral blood mononuclear cellsex vivo. The present study further documents the ability of n-3 fatty acids to inhibit cytokine formation, and in part defines the mechanism of the inhibition of production of interleukin-1β (IL-1β) by dietary n-3 fatty acid. Female BALB/c mice were each fed a fatfree balanced diet to which was added either a refined fish oil (FO) preparation as a source of n-3 fatty acid, or beef tallow (BT), which consisted primarily of saturated and monoenoic fatty acids. After ingesting the experimental diets for periods ranging from 3 to 12 wk, spleen cell preparations were stimulatedex vivo with either lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and prolL-1β mRNA (Il-1β mRNA) was measured by northern analysis. Levels of IL-1β mRNA in both LPS-and PMA-stimulated cells from BT-fed mice were elevated to a greater extent than in cells from FO-fed mice, at most concentrations of LPS and PMA. Stability of LPS-stimulated mRNA levels after actinomycin D was similar for BT and FO groups, indicating that lower levels of IL-1 mRNA with FO groups was related to suppressed IL-1 gene transcription and not due to accelerated transcript degradation. Nuclear run-on transcription assays revealed a more transient expression of the IL-1β gene in LPS-stimulated spleen cells from FO-fed mice compared to cells from BT-fed mice. We conclude that dietary marine lipids reduce transient expression of the IL-1β gene in stimulated splenic monocytic cells. Preliminary results from nuclear run-on transcription assays indicate that n-3 fatty acids may not change the initial rate of gene transcription but may promote more rapid shutting down of transcription of this gene after induction than do alternative lipids.

Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

ABSTRACT Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the α and βγ subunits of complex G proteins.

Acute polyarticular gout

Abstract The clinical features of gout, manifest as an acute polyarthritis, are described in 102 patients. In 34 of these the diagnosis was based on the finding of monosodium urate crystals in synovial fluid. The mean age of the patients was 60 years, and 90 per cent were male. Acute polyarthritis was the first manifestation of gout in 39 per cent of all patients. Serum uric acid concentrations were less than 7.0 mg/dl in 13 of 34 patients with documented crystal-induced synovitis and in 15 of 68 of the remainder at the time of the acute attack. Eighteen of the 20 normouricemic patients reexamined were hyperuricemic before or after the polyarticular attack. The distribution of joints involved was usually asymmetric. Of all joints involved, 83 per cent were in the lower extremity, but in one third of the patients the foot was spared. Fever, leukocytosis and dramatic response to therapy with colchicine or anti-inflammatory drugs were the rule. Gout should be considered in the differential diagnosis of acute polyarthritis regardless of the absence of hyperuricemia or foot involvement.