Retinoids are potent inhibitors of the generation of rat leukocyte leukotriene B4-like activity in vitro

European Journal of Pharmacology, 98 (1984) 61-67

61

Elsevier

R E T I N O I D S ARE P O T E N T I N H I B I T O R S OF T H E G E N E R A T I O N OF RAT L E U K O C Y T E L E U K O T R I E N E B4-LIKE ACTIVITY IN V I T R O MICHAEL A. BRAY

Department of Inflammation, Ciba- Geigy A G, R - 1056. P.27, 4002 Basel, Switzerland Received 29 August 1983, revised MS received 20 October 1983, accepted 8 November 1983

M.A. BRAY, Retinoids are potent inhibitors of the generation of rat leukocyte leukotriene B4-1ike activity in vitro, European J. Pharmacol. 98 (1984) 61-67. Several retinoids (tretinoin, retinol, retinal, retinyl acetate, etretinate and RO 10-1670) were tested in vitro for their ability to inhibit the generation of the leukocyte chemotactic factor leukotriene B4 (LTB4) from calcium ionophorestimulated rat PMN in vitro. LTB4 activity was measured in vitro via bioassay using rat PMN aggregation and chemokinesis. Tretinoin, retinol, RO 10-1670 and retinal were active inhibitors of LTB4 generation with IDs0s (~tM) of 0.55-1.5, 5.9-6.5, 4.8-5.9 and 19-28.4 respectively whilst retinyl acetate and etretinate were inactive. Inhibition of the generation of LTB4 by tretinoin was confirmed by reverse phase-high pressure liquid chromatography. These results are discussed in the context of retinoid treatment of skin diseases such as psoriasis where inhibition of neutrophil accumulation during retinoid treatment has been reported. Retinoids

Leukotriene B4

Neutrophil chemotaxis

1. Introduction Retinoids have been reported to be potent inhibitors of neutrophil activation both in vitro, for example, in terms of superoxide anion generation and lysosomal enzyme release (Camisa et al., 1982) and in vivo in terms of polymorphonuclear leukocyte (PMN) emigration into skin chambers (Dubertret et al., 1982). These actions of Vitamin A compounds have been proposed as part of the explanation for the efficacy of such preparations in some forms of inflammatory skin disease (for example inflammatory and pustular psoriasis) where neutrophil accumulation is an important clinical symptom (Cormane et al., 1976; Jablonska et al., 1979). Neutrophils are known to metabolise the C-20 fatty acid, arachidonic acid via both a cyclooxygenase enzyme pathway leading to the generation of prostaglandins and a lipoxygenase enzyme pathway leading to the formation of leukotrienes (Samuelsson, 1983). One of the most prominent P M N leukotriene products is leukotriene B4 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.

Psoriasis

( 5 (S), 12( R)dihydroxy- 6,8,10,14-(cis, tran s,trans,cis)-eicosatetraenoic acid) and this material has been shown to be an extremely potent stimulator of leukocyte migration both in vitro and in vivo (Smith, 1981; Bray, 1983). It therefore seemed appropriate to examine the ability of retinoids to modify P M N generation of LTB4. This paper demonstrates that all trans-retinoic acid (tretinoin) and several other retinoids are potent inhibitors of rat P M N leukotriene B4 generation in vitro as assessed by both bioassay (rat P M N aggregation and chemokinesis) and physicochemical criteria (reverse phase - high pressure liquid chromatography - RP-HPLC).

2. Materials and methods 2.1. Cells

Rat P M N leukocytes ( > 85% neutrophils) were obtained from the peritoneal cavity of male outbred Wistar rats (400-600 g) 24 h after in-

62 traperitoneal (i.p.) injection of 16 ml of a 12% ( w / v ) sodium caseinate solution. The cells were removed by peritoneal washout, washed in Eagles minimal essential medium (E-MEM) buffered to p H 7.4 with 30 m M N'-2-hydroxyethylpiperazineN'-2-ethane sulfonic acid (HEPES) and resuspended in buffered E-MEM to a concentration of ] 0 7 viable cells/ml for aggregation and - 2 × l0 s cells/ml for chemokinesis (viability greater than 95%, as assessed by Trypan blue dye exclusion). 2.2. P M N aggregation assay

PMN aggregation was assessed via nephelometry as previously described (Cunningham et al., 1980). Briefly, 0.5 ml of cell suspension was placed into 1.0 ml glass cuvettes in a two-well Born platelet aggregometer (Payton Associates Ltd, Canada). The cells were stirred at 800-900 rpm and warmed to 37°C for 2 min prior to the addition of drugs. Aggregation responses were monitored via a change in light transmittance measured in m m on a pen recorder (W + W, Switzerland). Generation of LTB4-1ike activity was stimulated by the addition of 2 /~1 of the calcium ionophore A23187 (CaI) dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 10 6 M for 4 min. Supernatants from the CaI-stimulated P M N cultures were tested as 5 /~1 aliquots (1 : 100 dilution) in a fresh cell culture. Drugs were added 5 min prior to the addition of ionophore. Remaining Cal had no intrinsic aggregatory actiyity at the dilution of supernatant used to test for LTB4-1ike activity.

results were expressed as area of cell emigration in m m 2. Supernatants from PMNs stimulated with CaI in the presence or absence of drugs were used to stimulate cell emigration. 2.4. R P - H P L C

Ten ml of rat PMNs (10 7 cells/ml) in E-MEM were prewarmed to 37°C prior to a 10 min incubation with tretinoin or vehicle. The cells were then stimulated for 5 min with 10 -6 M CaI and the reaction terminated by addition of 1.5 vol of methanol. Cell debris was removed by centrifugation and the supernatant pH adjusted to 3.0-3.5 with 1 N HCI. Supernatants were partially purified by passage over a C18 silicic acid column (Sep Pak; Waters Associates, U.S.A.) and the leukotriene-containing fraction eluted off with methyl formate (Powell, 1980). After drying in a rotary evaporator the samples were resuspended in 50/~1 of a 70 : 30 : 0.01 mixture of methanol : water : acetic acid. A 25 /~1 aliquot of each sample was chromatographed using a 250 x 7.6 mm Beckman Cl~ Ultrasphere column (Beckman Ass., U.S.A.) at a flow rate of 1 m l / m i n with 70 : 30 : 0.01, methanol : water : acetic acid as elution buffer. Using this system synthetic standard LTB 4 eluted after - 23 min. Samples were monitored at a wavelength of 270 nM and fractions eluting at 22-24 min were collected, U.V. spectra obtained and the samples dried, resuspended in E-MEM and tested for biological activity in the rat PMN aggregation and chemokinesis assays. 2.5. Drugs

2.3. P M N ehemokinesis assay

PMN chemokinesis was modified from the method of Smith and Walker (1980). Briefly rat peritoneal PMN ( - 2 × 10S/ml) were mixed with agarose (Indubiose A 37; Pharmindustrie, France) (final concentration of agarose was 0.8%) and 2/~1 droplets placed into the centre of wells in a 96 well sterile microtitre plate (Sterilin, UK). The droplets were overlaid with E-MEM and incubated at 37°C for 2-3 h. The image of the resulting cell emigration was projected and the leading edge of the cellular emigration outlined via planimetry. The

All trans-retinoic acid (3,7-dimethyl-9-(2,6,6-trimethyl-l-cyclohexen-l-yl)-2,4,6,8-nonatretraen- 1carbonic acid: tretinoin), the all trans alcohol (vitamin A: retinol), the all trans aldehyde (retinal) and the all trans acetate (retinyl acetate) were all obtained from Fluka A G (Switzerland). The trimethylmethoxy phenyl analogue of retinoic acid ethyl ester (etretinate, RO 10-9359) was obtained from Hoffmann La Roche A G (Switzerland) and the carboxylic acid metabolite of etretinate (RO 10-1670) was prepared from etretinate. The calcium ionophore A23187 was obtained from the

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Calbiochem-Behring Corp. (U.S.A.). All compounds were stored as directed and dissolved in DMSO immediately prior to use (the maximal concentration of DMSO present during the experiments was 0.8%)• Synthetic LTB4 was supplied by Professor E.J. Corey (Harvard University).

3. Results 3.1. Effects on in vitro rat P M N aggregation and chemokinesis

Retinoids dissolved in DMSO (or DMSO itself as control) were added in 2/~1 aliquots at various

TABLE 1 Effect of retinoids on the generation of LTB4-1ike activity. IDs0 data derived graphically from figs. 1 and 2. Compound

PMN aggregation n~

Tretinoin Retinol Retinal Retinyl acetate Etretinate RO 10-1670

10-14 4-5 4 4-6 4-6 4

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12-18 6-12 6-12

1.5 6.5 28.4

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Fig. 2. Effect o f several r e t i n o i d s o n the g e n e r a t i o n o f LTB4-1ike activity f r o m C a I - s t i m u l a t e d rat P M N in v i t r o m e a s u r e d b y r a t P M N c h e m o k i n e s i s . E a c h p o i n t r e p r e s e n t s the m e a n % c h a n g e (% S.E.M. < 1 0 % ) f r o m a r a n g e of o b s e r v a t i o n s (see t a b l e 1). - . . . . . Tretinoin, - ..... retinol, - retinal, - . . . . . retinyl acetate, • . . . . . e t r e t i n a t e , - . . . . . R O 10-1670.

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65 doses to 0.5 ml cultures of PMNs 5 min prior to stimulation with 10 -6 M Cal. Resulting supernatants were tested for LTB4-1ike activity in fresh cell suspensions to test for aggregation or added to the microtitre plates to measure chemokinetic activity. In the absence of drugs, CaI-stimulated cell supernatants caused a substantial increase in P M N aggregation (mean r e s p o n s e = 52.53 + 2.86 m m (standard error of the mean, S.E.M.) (n = 46)) and enhanced P M N chemokinesis (mean response ( + S.E.M.) = 4.83 + 0.096 m m 2 (n = 52)). Pretreatment with tretinoin caused a dose-related inhibition of the generation of LTB4-1ike activity (figs. 1 and 2). Treatment with the other retinoids caused a similar or lesser degree of inhibition of activity with a rank order of potency of tretinoin > RO 10-1670 > retinol > retinal >> retinyl acetate and etretinate. IDs0 values derived from the data in figs. 1 and 2 are shown in table 1. The results indicate that tretinoin, retinol, retinal and RO-101670 are potent inhibitors of the aggregatory and chemokinetic bioactivities thought to be primarily due to the generation of LTB4 (Ford-Hutchinson et al., 1979; Bray et al., 1980; Ford-Hutchinson et al., 1980). Tretinoin was - 4 times as active in these assays as nor-dihydroguaretic acid ( N D G A ) a lipoxygenase inhibitor with an IDs0 of - 2 × 10 6 M in our hands. At the doses tested none of the compounds either caused P M N aggregation directly, modified chemokinesis or caused cell death (as measured by Trypan blue dye exclusion). By varying the time of pre-incubation of PMNs with tretinoin it could be shown that this compound was able to inhibit CaI-generated bioactivity up to 1 h prior to stimulation. Fig. 3 shows the effects of a 5 min incubation of P M N with tretinoin (5 >( 10 -6 M) after which the cells were washed and subsequently stimulated with CaI at the times indicated.

3.2. Effect of tretinoin of the generation of LTB 4 In order to confirm that the effects of retinoids shown above by bioassay were due to inhibition of the formation of LTB4, P M N were preincubated with varying amounts of tretinoin, stimulated with CaI and the resulting supernatants analysed via RP-HPLC. Tretinoin (10-6-10 -5 M) caused a

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dose-related inhibition of the U.V. absorbtion peak corresponding to LTB 4 (fig. 4). The peak eluting at 22-24 min in the absence of tretinoin (fig. 4a) was shown to have the characteristic triple peak U.V. spectrum of LTB4 (fig. 4f) with U.V. maxima of 259, 270 and 281 nM which correspond to the U.V. maxima obtained with synthetic LTB4 (data not shown). This fraction was also able to stimulate P M N aggregation and chemokinesis (data not shown).

66

4. Discussion The data presented above indicate that several retinoids are potent inhibitors of CaI-induced PMN LTB 4 generation in vitro. It seems possible that these compounds are acting primarily via inhibition of the lipoxygenase enzyme system although this conclusion could not be confirmed by the results of the present study as only LTB4 generation was examined. Retinoids have been shown to be active free radical scavenging agents in vitro (Witz et al., 1980; Camisa et al., 1982) and thus may reduce the activity of the lipoxygenase enzyme by inhibiting the availability of oxygen. Interestingly Camisa and his colleagues noted that etretinate was not a good superoxide anion inhibitor. This observation would tend to support the above explanation, however they also reported that RO 10-1670 was a poor inhibitor, a result inconsistent with the data from this study. Another explanation for the ability of retinoids to inhibit LTB4 generation could be that these compounds reduce the availability of arachidonic acid substrate. This possibility is suggested as tigason (etretinate) treatment has been reported to e n h a n c e p h o s p h o l i p i d levels and m o d i f y arachidonic acid binding to phospholipids in human skin (Binazzi and Papini, 1981). The ability of tretinoin to inhibit the formation of LTB4-1ike activity up to 1 h prior to stimulation with CaI (fig. 3) implies that this compound may irreversibly inhibit the leukotriene-generating lipoxygenase activity or provide long-term inhibition of substrate availability as it seems unlikely that simple free radical scavenging activity could account for the effects observed. The results of the present study indicate that retinoids may exert some of their anti-psoriatic activity via inhibition of the formation of neutrophil chemotactic LTB4. Such a conclusion is consistent with the clinical data showing a major reduction in infiltrating PMNs following retinoid treatment in guinea-pigs and man (Tsambaos and Orfanos, 1981; Schultz-Ehrenburg and Orfanos, 1981). Interestingly, there is also a recent report that benoxaprofen (a relatively weak lipoxygenase inhibitor) caused some remission of the psoriatic lesions of patients with psoriatic arthritis (Allen

and Littlewood, 1982). Similarly the present results provide an explanation for the ability of local or oral treatment with etretinate and RO 10-1670 to inhibit the serum-stimulated infiltration of neutrophils into skin chambers in man (Dubertret et al., 1982). The authors showed that the metabolite RO 10-1670 was more active than the parent etretinate. This result is consistent with the data presented here, a fact which implies that the etretinate may need to be metabolised to the active carboxylic acid derivative before it becomes effective as an inhibitor of neutrophil accumulation. This process occurs rapidly in human plasma (Paravicini, 1981). There is also a tentative link between the effects of retinoids on epithelial cells from psoriatic lesions and inhibition of LTB4 formation as a recent report has indicated that bovine epithelial lens cells may have the capacity to generate LTB4 in vitro (measured via RP-HPLC) (Lonchampt et al., 1983), however whether LTB4 generation by such cells has a pathophysiologicai role is not clear. In conclusion, the ability of retinoids to inhibit the generation of chemotactic LTB4 should be considered as an explanation for the activity of such compounds as inhibitors of inflammatory cell accumulation in diseases such as psoriasis.

Acknowledgements The author gratefully acknowledgesthe technical assistance of Fr~iulein Cornelia Saladin and Fr~iulein Elvira Dittli. Thanks are also due to Dr. Andreas von Sprecher for the preparation of RO 10-1670.

References Allen, B.R. and S.M. Littlewood, 1982, Benoxaprofen: effect on cutaneous lesions in psoriasis, Br. Med. J. 285, 1241. Binazzi, M. and M. Papini, 1981, Effects of retinoid RO 10-9359. Alone or in combined therapy in several skin diseases - clinical observations and biochemical data, in: Retinoids: Advances in Basic Research and Therapy, eds. Orfanos, C.E. et al. (Springer-Verlag, Berlin) p. 317. Bray, M.A., 1983, The Pharmacologyand Pathophysiologyof Leukotriene B4, Br. Med. Bull. 39, 249. Bray, M.A., A.W. Ford-Hutchinson, M.E. Shipley and M.J.H. Smith, 1980, Calcium ionophore A23187 induces release of

67 chemotactic and aggregating factors from polymorphonuclear leucocytes, Br. J. Pharmacol. 71,507. Camisa, C,, B. Eisenstat, A. Ragaz and G. Weissman, 1982, The effects of retinoids on neutrophil functions in vitro, J. Am. Acad. Dermatol. 6, 620. Cormane, R.H., J. Hunyadi and F. Hamerlinck, 1976, The role of lymphoid cells and polymorphonuclear leukocytes in the pathogenesis of psoriasis, Proceedings of the Second International Symposium, eds Farber, E.M. and A.J. Cox (Yorke Medical Books, New York) p. 47. Cunningham, F.M., M.E. Shipley and M.J.H. Smith, 1980, Aggregation of rat polymorphonuclear leucocytes in vitro, J. Pharm. Pharmacol. 32, 377. Dubertret, L., C. Lebreton and R. Touraine, 1982, Inhibition of neutrophil migration by etretinate and its main metabolite, Br. J. Dermatol. 107, 681. Ford-Hutchinson, A.W., M.A. Bray and M.J.H. Smith, 1979, The aggregation of rat neutrophils by arachidonic acid: a possible bioassay for lipoxygenase activity, J. Pharm. Pharmacol. 31,868. Ford-Hutchinson, A.W., M.A. Bray, M.V. Doig, M.E. Shipley and M.J.H. Smith, 1980, Leukotriene B: a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes, Nature 286, 264. Jablonska, S., E.H. Beutner, W.L. Binder, M. Jarzabek-Chorzelska, G. Rzesa and O. Chowaniec, 1979, Immunopathology of psoriasis, Arch. Dermatol. Res. 264, 65. Lonchampt, M.-O., C. Bonne, F. Regnault, J.-P. Masse, C. Coquelet and D. Sincholle, 1983, Evidence of Leukotriene B4 biosynthesis in epithelial lens cells, Prostagl. Leukot. Med. 10, 381.

Paravicini, U., 1981, Pharmacokinetics and metabolism of oral aromatic retinoids, in: Retinoids: Advances in Basic Research and Therapy, eds. C.E. Orfanos et al. (SpringerVerlag, Berlin) p. 13. Powell, W.S., 1980, Rapid extraction of oxygenated metabolites of Arachidonic Acid from biological samples using octadecylsilyl Silica, Prostaglandins 20, 974. Samuelsson, B., 1983, Leukotrienes: Mediators of immediate hypersensitivity reactions and inflammation Science, 220, 568. Schultz-Ehrenburg, U. and C.E. Orfanos, 1981, Light and electron microscopic changes of human epidermis under oral retinoid treatment, in: Retinoids: Advances in Basic Research and Therapy, eds, C.E. Orfanos et al. (SpringerVerlag, Berlin) p. 85. Smith, M.J.H., 1981, Leukotriene B4, Gen. Pharmacol. 12, 211. Smith, M.J.H. and J.R. Walker, 1980, The effects of some antirheumatic drugs on an in vitro model of human polymorphonuclear leucocyte chemokinesis, Br. J. Pharmacol. 69, 473. Tsamboas, D. and C.E. Orfanos, 1981, Effects of oral retinoid on dermal components in human and animal skin, in: Retinoids: Advances in Basic Research and Therapy, eds. C.E. Orfanos et al. (Springer-Verlag, Berlin) p. 99. Witz, G., B.D. Goldstein, M. Amoruso, D.S. Stone and W. Troll, 1980, Retinoid inhibition of superoxide anion radical production by human polymorphonuclear leukocytes stimulated with tumor promoters, Biochem. Biophys. Res. Commun. 97, 883.