M. J. H. Smith

LEUKOTRIENE B4: A MEDIATOR OF VASCULAR PERMEABILITY

1 Intradermal injection of leukotriene B4 (LTB4) or prostaglandin E2 (PGE2), 1 to 100 ng per skin site produced little or no change in plasma exudation in the rabbit, guinea‐pig and rat. 2 Intradermal injection of LTB4 together with PGE2 produced a significant increase in plasma exudation in the rabbit, guinea‐pig and rat. 3 Intradermal injection of LTB4 or PGE2 together with bradykinin (500 ng) resulted in a significant potentiation of the plasma exudation produced by bradykinin alone in the rabbit and guinea‐pig. 4 LTB4 (1 to 10 ng) had no effect on blood flow in the rabbit skin, in contrast to PGE2 which was a potent vasodilator in this species. 5 It is concluded that LTB4 is a mediator of vascular permeability and that this effect can only be observed in the presence of a vasodilator such as PGE2.

Leukotriene B4: an inflammatory mediator in vivo.

Leukotriene B4 (LTB4 isomer III), which promotes the movement and aggregation of leucocytes in vitro also stimulates the chemo-attraction of leucocytes and their adherence to vascular endothelium in vivo. These effects were observed directly in the hamster cheek pouch preparation and on histological examination of sections from the rabbit mesentery. Intravenous injection of LTB4 (isomer III) into the rabbit resulted in a profound but transient neutropenia. Intradermal injection of LTB4 (isomer III) in the rabbit produced a rapid accumulation of neutrophils and this effect was also observed in skin chambers applied over abrasions on the rabbit back or the human forearm.

Leukotriene B4, an inflammatory mediator in gout.

Leukotriene B4 (LTB4), a metabolite of arachidonic acid and a potent cytotaxin, is generated by human peripheral polymorphonuclear leucocytes (PMNs) exposed to monosodium urate crystals (MSU). The leukotriene is present in gouty effusions in concentrations significantly greater than those found in synovial fluid from patients with active rheumatoid arthritis or osteoarthritis. Further metabolism and biological deactivation of LTB4 by PMNs are partly inhibited by MSU. It is concluded that LTB4 is an important chemical mediator in an acute gouty attack.

Anti-inflammatory drugs, prostaglandins and leucocyte migration

The action of some non-steroidal acidic anti-inflammatory drugs, aspirin, phenylbutazone and indomethacin, in reducing leucocyte migration into the exudates of inert porous sponges implanted subdermally in the rat has been shown to be distinct from their effect in reducing the content of prostaglandins in the exudates. It is concluded that a component of the anti-inflammatory and antirheumatic actions of the drugs is concerned with a mechanism other than inhibition of prostaglandin biosynthesis.

Salicylate and enzymes

Salicylate inhibits the activities of a number of cellular enzymes and in some instances the mechanisms of inhibition have been established (Smith, 1968a). Reported inhibitory actions of the salicylate ion on important enzyme systems in vitro are now reviewed and assessed in relation to the known clinical and toxic effects of the drug.

Isomers of leukotriene B4 possess different biological potencies

Rat elicited polymorphonuclear leucocytes (PMNs), when exposed to the ionophore A23187, release three isomers of leukotriene B4. The three isomers have been purified and tested for their ability to induce the chemokinesis of human PMNs in vitro, the aggregation of rat PMNs in vitro and changes in vascular permeability in rabbit skin in vivo in the presence of PGE2. The results demonstrate that all three isomers are biologically active and that the enzymatically produced isomer, in which the conjugated triene contains one cis and two trans double bonds, is more potent than the two diastereoisomers of LTB4 which contain all trans double bonds in the conjugated triene and which are produced by non-enzymatic hydrolysis.

The stimulation of lysosomal enzyme secretion from human polymorphonuclear leucocytes by leukotriene B4

chemical properties to cimetidine also failed to raise proCare , P F., Martin, L. E. (1979) J. Liq. Chromatogr. 2: lactin in healthy volunteers after intravenous injection. 12q1-1.303 They speculated that the specific effect of cimetidine on Carlson. H. H., Ip liti, A. F. (1977) J. Clin. Endocrinol. prolactin was due to a particular feature of the cimetidine Metab. 45: 367-gjb molecule itself. Domschke, S., Domschke. W. (1980) HepatoGastroenterol. 27: 163-168 We thank Research Ltd for and for Macaron, C., Kyncl, M., Singh, S. P, (1979) Clin, providing the trial material. This project was also funded Endocrinol, 11: 371-375 by the South African Medical Research Council and the Nelis, G, F., Van de Meene, J , G, C. (1980) Postgrad, University of Cape Town Staff Research Fund. We are Med. J. 56: 478-480 grateful to ~ i s t e r ~ ~ e n d ~ Lucke for her willing assistance Sharpe, P. C., Melvin, M. A., Mills, J. G., Burland, W. L., and to Mr R. Sayed for the statistical analysis. Groom, G. V. (1980) in: Torsoli, A,, Lucchelli, P. E., Brimblecombe. R. W. (eds) Further Experience with Hz-Receptor ~ntagonisis in Peptic Ulcer Disease, REFERENCES Excerpta Medica, Amsterdam, pp 366-370 Berstad, A., Kett, K., Aadland, E., Carlsen, K., Frislid, Van Thiel, D. H.. Gavaler, J. S., Smith, W. I., Paul, G. K., Saxhaug, K., Kruse-Jensen, A. (1980) Scand. J. (1979) New Eng. J. Med. 30: 1012-1015 Gastroenterol. 15: 637-639 Yeo, T., Delitala,,G., Besser, G. M., Edwards, C. R. W. Burland, W. L., Gleadle, R. I., Lee, R. M., Rowley-Jones, (1980) Br. J. Cl~n. Pharmacol. 10: 171-173 D., Groom, S. V. (1979) Br. J. Clin. Pharmacol. 7: 1e21

THE EFFECTS OF SOME ANTIRHEUMATIC DRUGS ON AN in vitro MODEL OF HUMAN POLYMORPHONUCLEAR LEUCOCYTE CHEMOKINESIS

1 A rapid, reproducible in vitro assay for studying the chemokinetic movement of human polymorphonuclear leucocytes (PMNs) is described. Two synthetic peptides, formyl methionyl‐leucylphenylalanine (FMLP) and formyl methionyl‐phenylalanine (FMP), were used as standard chemokinesins. 2 Maximal chemokinetic movement was observed with peptide concentrations of 2.5 nm (FMLP) and 100 μm (FMP). EC50 values of 650.0 ± 60.0 pm and 27.0 ± 3.5 μm respectively are similar to those reported for chemotactic activity of the peptides in micropore filter assays. 3 The PMN chemokinetic response to FMLP was enhanced by histamine (100 nm) and vitamin C (2.5 μm). 4 Human serum albumin was shown to induce chemokinesis but to antagonize the response to FMLP in a dose‐related fashion. Fibrinogen similarly antagonized the cell response to peptide. 5 Levamisole (250 nm to 2.5 μm) significantly potentiated the chemokinetic responses to FMLP and FMP in a dose‐related manner. The chemokinetic response to FMLP was unaffected by d‐penicillamine (250 μm to 10 mm) while alclofenac (500 μm to 1 mm), salicylic acid (250 μm to 10 mm) and indomethacin (100 μm to 1 mm) caused dose‐related inhibition.

The binding of paracetamol to plasma proteins of man and pig

The binding of N‐acetyl‐4‐aminophenol (paracetamol) to human and porcine plasma at both toxic and therapeutic concentrations was investigated by ultrafiltration and equilibrium dialysis over the range 50–300 μg ml−1. Plasma protein binding occurred at paracetamol concentrations greater than 60 μg ml−1. The extent of protein binding at a plasma concentration of 280 μg ml−1 of the drug is between 15 and 21% for both pig and man. There is no appreciable binding to erythrocytes in either species over the whole concentration range studied.

Leukotriene B4 in synovial fluid.

Leukotriene B, (LTB,) is generated enzymatically from arachidonic acid by human leucocytes in vitro (Smith 1981). Recent work suggests that LTB, is a putative mediator of inflammation in vivo since it both increases leucocyte infiltration and, in the presence of vasodilatory prostaglandins, causes increased vascular permeability (Smith 1982). A second criterion is whether it is present at sites of inflammatory reactions and here the only quantitative evidence is that of Klickstein et al (1980). The LTB4 was separated from 2 to 3 ml samples of synovial fluid by solvent extraction, column chromatography and reverse phase high performance liquid chromatography (h.p.1.c.); recognized by a comparison of retention times with standard material and measured by ultraviolet absorption techniques. In synovial fluid from eighteen patients with rheumatoid arthritis the reported values (mean f s.e.m.) were 141 k 34 ng ml-1 (Klickstein et a1 1980). There are objections to relying on physicochemical methods only for the measurement of LTB,. There are a number of isomers of the leukotriene which are not only difficult to separate completely from the authentic material but also are either much less active or devoid of the relevant biological activities. Also, more recent work (Hansson et al 1981) has shown that LTB, is metabolized by human leucocytes to more polar hydroxy and carboxy compounds which are far less potent as leucotactic agents. We have therefore repeated the work measuring the LTB, in the final h.p.1.c. fraction both by ultraviolet absorption and a sensitive and specific bioassay method (Cunningham et a1 1980). In synovial fluid specimens, ranging in volume from 10 to 24 ml, from 12 patients with rheumatoid arthritis we could find no LTB4 using the ultraviolet absorption method. In our hands the lower limit of detection with this technique is 50 ng per whole specimen i.e. approximately 2.5 ng ml-1. The more sensitive bioassay procedure yielded a mean value of 0.34 f 0.14 ng ml-1 of