Stephen J. Foster

Leukotriene B4 and inflammatory disease

Polymorphonuclear leukocytes (PMNL) are prominent at sites of acute inflammation. Their infiltration is stimulated under pathological conditions by a variety of agents which include bacteria, immune complexes and complement derived chemotactic peptides. Recently attention was focussed on the 5-lipoxygenase product leukotriene B4 (LTB4) which has been demonstrated to induce the key features associated with an acute inflammatory reaction. However, evidence supporting a pro-inflammatory role for LTB4, and therefore the anti-inflammatory efficacy of 5-lipoxygenase inhibitors, is largely circumstantial. Moreover, there are concerns that other chemotactic factors, notably C5a, may compensate for the absence of LTB4. Here we challenge this view and, on the basis of recent experimental and clinical data suggest that LTB4 does not simply duplicate the activity of C5a. Instead we propose that their predominant site(s) of action differ in such a way that they may synergise in mediating PMNL recruitment.

Leukotriene B4 and prostaglandin E2 mediate the inflammatory response of rabbit skin to intradermal arachidonic acid.

1 Acute inflammation was induced in rabbit skin by intradermal injection of arachidonic acid. 2 Inflammation was assessed by the local accumulation of intravenously‐injected 125I‐serum albumin (plasma extravasation) and histologically (polymorphonuclear leucocyte, PMNL infiltration). 3 Leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) were extracted from skin and fractionated using C18 mini‐columns. They were quantitated by specific radioimmunoassays and authenticated by reversed‐phase high performance liquid chromatography analysis. 4 Maximally elevated levels of LTB4 and PGE2 were detected in skin 5 min after arachidonic acid injection. At subsequent times the eicosanoid content of the skin decreased. 5 The decrease in the eicosanoid content of the skin was due to rapid clearance (t1/2 approximately 5 min) via the microvasculature and not a consequence of metabolism. 6 The concentration of LTB4 and PGE2 measured in inflamed skin was sufficient to account for the plasma extravasation and PMNL infiltration induced by arachidonic acid. The model therefore is useful for evaluating the anti‐inflammatory efficacy of inhibitors of arachidonic acid metabolism including 5‐lipoxygenase inhibitors. 7 The consequences of the rapid clearance of LTB4 from inflammatory sites are discussed with respect to its measurement in inflammatory disease and its role as an acute inflammatory mediator.

The inflammatory response of rabbit skin to topical arachidonic acid and its pharmacological modulation

1 The inflammatory reaction induced by the intradermal injection of arachidonic acid into the rabbit dermis has been investigated. Plasma extravasation was measured by the leakage of 125I‐albumin into the tissues and polymorphonuclear leukocyte (PMNL) accumulation was assessed histologically. 2 Arachidonic, 5,8,11,14,17‐eicosapentaenoic and 8,11,14‐eicosatrienoic acids, but not oleic, linoleic or linolenic acids, caused a concentration‐related plasma extravasation following their intra‐dermal injection. The plasma extravasation induced by arachidonic acid was dependent on PMNLs. 3 PMNL infiltration and plasma extravasation into arachidonic acid‐injected skin sites was inhibited by the mixed cyclo‐oxygenase‐lipoxygenase inhibitor, BW755C. 4 Arachidonic acid‐induced plasma extravasation was inhibited by cyclo‐oxygenase and 5‐lipoxygenase inhibitors but not by the Paf antagonist, kadsurenone. 5 The inflammation induced by arachidonic acid in the rabbit dermis may be a useful model for evaluating 5‐lipoxygenase inhibitors which could be potentially useful anti‐inflammatory agents for the treatment of psoriasis and other inflammatory diseases.

The contribution of cyclooxygenase and lipoxygenase products to acute inflammation in the rat

The efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is generally accepted to be due to the inhibition of cyclooxygenase (CO) and the subsequent reduction of blood flow and plasma exudation within the inflammatory lesion. It is speculated that drugs which prevent the accumulation of leukocytes into inflammatory lesions may also be clinically beneficial. One approach towards finding such a drug is based on the observation that leukotriene B4 (LTB4) is a potent leukotactic agent in several species including man. Therefore, reducing the levels of LTB4 through inhibition of 5-1ipoxygenase (LPO) may provide a means of inhibiting leukocyte migration. We have investigated the relationship between the effects of drugs on arachidonate metabolism and the inflammatory parameters of leukocyte migration and plasma exudation in the carrageenin-soaked sponge implant model in rats [1]. Our objectives were to investigate the role of LTB4 in the recruitment of leukocytes into the sponges and to assess the suitability of the model for evaluating the anti-inflammatory efficacy of LPO inhibitors. Agents were dosed p.o. in 1% carboxymethylcellulose one hour prior to the subcutaneous implant of sponges soaked in 0.5% 2 carrageenin and the various parameters were measured 4 hours post implant. E x vivo LPO and CO activity were determined by stimulation of the inflammatory exudate with the calcium ionophore A23187, as described by SALMON et al. [1]. Prostaglandin E 2 and LTB4 generated by ionophore were measured by radioimmunoassay [2]. To authenticate the nature of the immunoreactive LTB4, ionophore-stimulated exudate was extracted with methylformate [3] and the extracted lipids subjected to RPHPLC using a 5/~Spherisorb ODS2 column and linear gradient from 60-95% methanol in 0.1% acetic acid and water, pH 5.6. A single peak of immunoreactive material was identified which co-eluted with synthetic LTB4 standard. No other immunoreactive materials were evident. The NSAIDs flurbiprofen and indomethacin (table) inhibited leukocyte migration, exudate volume and CO activity but failed to inhibit LPO activity. In fact these agents actually elevated immunoreactive LTB4 levels, an observation noted elsewhere [1]. Similar anti-leukotactic profiles were observed for other NSAIDs. Nafazatrom (Bayer g6575), quercetin and NDGA, which inhibit LPO activity in RBL and human PMNL 105,000 x g cytosol [4, 5] had no effect on leukocyte migration, exudate volume, LPO or CO activity when dosed at 100 nag kg -1 p.o. These agents were also inactive when administered twice at 16 and 1 hour prior to sponge implant. The mixed cyclooxygenaselipoxygenase inhibitors BW755C and phenidone were equipotent inhibitors of LPO but BW755C was 10 times more potent than phenidone against CO (table). BW755C was also > 10 more potent than phenidone at inhibiting

Cyclic nucleotides, possible intracellular mediators of macrophage activation and secretory processes

Macrophages from various sources can be stimulated by a variety of substances to secrete a range of inflammatory mediators and degradative enzymes. The mechanisms involved in the activation and secretory processes are unknown. However, recent evidence suggests that cyclic AMP may play a role in the regulation of neutral protease secretion. Thus, it has been shown that agents known to increase intracellular cyclic AMP levels (cyclic AMP analogues, phosphodiesterase inhibitors, prostaglandin E1 and E2, catecholamines, cholera toxin and, indirectly, glucocorticosteroids) inhibit the secretion of the neutral protease plasminogen activator.It is speculated that macrophage activation may also be initiated by changes in the steady-state levels of cyclic nucleotides. A decrease in intracellular cyclic AMP and/or an increase in cyclic GMP levels would favour secretion. It is possible that these changes could be brought about by the action of various stimuli to modify the capacity of the macrophage to synthetize or degrade cyclic nucleotides.

Polymorphonuclear leucocyte accumulation in inflammatory dermal sites measured by a novel neutrophil specific marker protein

Acute inflammation is characterized by hyperaemia, plasma exudation and polymorphonuclear leucocyte (PMNL) accumulation. Blood flow and plasma exudation in skin can be accurately quantified using radiolabelling techniques [1] but methods available for assessing PMNL accumulation are sparse. Histology is time-consuming and inappropriate for estimating total cell numbers. Infusion of ex-vivo radiolabelled PMNL can be used qualitatively but the procedure is unpredictable and may modify cell behaviour [2]. Assay of the activity of myeloperoxidase, a naturally-occurring constituent of neutrophils, has also been used to assess PMNL content of skin [3]. Recently DARCY and DEAN [4] discovered that the cytoplasm of rat neutrophils contains a highly antigenic protein, specific for this cell type, which they called neutrophil-derived protein (NDP). We have found a similar highly antigenic cell specific