Exp. Eye Res.(1990) 51, 93-96

93

Further Investigation into the Ocular Effects Leukotriene 6, and Form+Methionyl-Leucyl PARIMAL

BHATTACHERJEE

AND

of Prostaglandin Phenylalanine

CHRISTOPHER

E,,

A. PATERSON

KY Lions Eye Research Institute, University of Louisville, Louisville, KY 40292, U.S.A. (Received 21 September 1989 and accepted in revised form 17 November 1989) The effect of indomethacinand dexamethasoneon the inflammatory responseto intraocularly injected prostaglandinE, (PGE,),leukotrieneB, (LTB,), and formyl-methionyl-leucyl phenylalanine(PMLP)has been examined in rabbits. PGE,, in nanogram quantities, dose-dependentlyincreasedintraocular pressureand protein concentrationin the aqueoushumor. Theseresponses werenot affectedby an antiinflammatory doseof dexamethasone,but indomethacinhad a smallinsignificantinhibitory effect.LTB, and FMLP causedsubstantialleukocyte accumulationin the anterior chamberand did not disrupt the blood-aqueousbarrier. Neither indomethacin nor dexamethasonehad any effect on the leukocyte responseto LTB, However, dexamethasonesignificantly reducedthe FMLP-inducedleukocyte response. Theseresultssuggestthat PGE,and LTB, do not elicit a further releaseof AA metabolitesand that their actionsaredirect. However,the chemofacticresponse of polymorphonuclearleukocytesto FMLPappears to be mediatedby lipoxygenaseproducts. Keywords: prostaglandins ; leukotrieneB, ;FMLP;intraocular pressure: indomethacin; dexamethasone.

1. Introduction

2. Materials and Methods

Prostaglandins and leukotrienes

Intraocular Injections

elicit vascular and cellular inflammatory responses in the rabbit eye. The actions of PGs and peptido-leukotrienes are predominantly vascular. Leukotriene B,, on the other hand, causes infiltration of polymorphonuclear leukocytes (PMN) (Bhattacherjee et al., 1981). Intraocular or topical administration of PGE, disrupts the blood-aqueous barrier and elevates or reduces intraocular pressure in rabbits (Beitch and Eakins,

1969; Bhattacherjee, 1974; Bite, Nichols and Barody, 1982). These responsesare not only dose and type of PG dependent, but also species dependent (Bito et al., 1989). The ocular response to PGs is thought to be the result of their direct actions on target elements in the

ocular tissues. However, there is evidence that in some non-ocular tissues the response to exogenous PGs and peptido-leukotrienes involves a secondary release of

cyclooxygenase products. For instance, in perfused rat heart PGF,, release PGE, and prostacyclin (de Deckere and ten Hoor, 1980). In the guinea-pig lung, the constrictor effects of LTC, and LTD, are thought to be mediated by endogenously released thromboxane and PGs (Piper and Samohoun, 1981). Whether such a secondary release also occurs after ocular administration of PGs and LTs is unknown. In the present study, we have examined whether an intraocular injection of PGE,, LTB, and FMLP release arachidonic acid metabolites using pharmacological interventions with the cyclooxygenase inhibitor indomethacin and the anti-inflammatory steroid,

dexamethasone.

Male adult albino rabbits weighing l-S-2.5 kg were used in this study treated in accordance with the ARVO resolution on the care and use of animals in research. Animals with no ocular abnormalities were

anesthetized with an i.v. injection of 40 mg kg-’ sodium pentobarbitone. Prostaglandin E,, LTB, or FMLP dissolved in saline were injected in a 10-/A volume into the anterior chamber of one eye using a 30-g needle attached to a polyethylene tubing and a microsyringe. The needle introduced through the sclera, 2-3 mm posterior to the liibus, into the posterior chamber was guided under an operating microscope through the pupil into the anterior chamber. To determine that injections are made into the anterior chamber, solutions of injected substance contained a trace amount of sodium fluorescein. The contralateral eyes received an equal volume of saline.

Treatment Schedule

In separate groups of rabbits one eye was topically treated with either indomethacin or dexamethasone sodium phosphate. Indomethacin (100 yg, dissolved with equimolar concentration of Na,CO,) in 50 ,A saline was instilled onto the cornea at 60, 30 and

15 min before intraocular

at 24, 20, 4 and 2 hr before injection of either PGE,, LTB, or FMLP. The selection of these doses for the study was made on the basis of the previous reports (Srinivasan

Srinivasan, 00144835/90/070093+04

%03.00/0

injections. Dexamethasone

(50 pg) in 50 ,~l saline was given topically, beginning

and Kulkarni,

1980;

Kulkarni

and

1985) and our own experience. 0 1990 AcademicPressLimited

94

P. BHATTACHERJEE

AND

C.A.

PATERSON

lntruocular Pressure Intraocular pressure (IOP) was measured using a digital pneumatonometer (Digilab, Model 30D) at 30 and 0 min before and 15, 30 and 60 min after PGE,, LTB, or FMLP injections. We did not routinely measure IOP at 5 min, but in animals exhibiting meiosis immediately after the injection, IOP was measured at this interval. Animals showing meiosis and an abrupt increase in IOP by more than 10 mmHg within 5 min of intraocular injections were excluded from the experiments: such responses with a rapid onset are due to traumatization of the iris by the needle in the process of injections. The animals were killed with an overdose of sodium pentobarbitone 60 min after PGE, and 240 min after LTB, or FMLP. Aqueous humor was then aspirated, added to heparinized saline for leukocyte count in a hemocytometer and for estimation of protein concentration according to the method of Lowry et al. (1951).

Time (mln)

FIG. 1. Time courseand the magnitudeof the response of IOP to various dosesof PGE, injected into the anterior

chamber.The changein IOP is calculatedasthe difference between the readings immediately before (0 min) and various time periods after the injection. The values are mean+s.~.~. of four to six experiments.

O-O Soline AA Untreoted A-AINDO (100 O-0 DExA (50

Statistical Analysis The difference between mean responses of the control and the groups treated with anti-inflammatory agents was analyzed by unpaired Students t-test.

pg) pf$

3. Results

The response of the IOP and PGE, over a period O-60 min is shown in Fig. 1. The change in IOP was calculated as the difference between the saline and PGE, injected eyes (test-control). Within 15 min of injection, IOP increase reached the peak and remained at that level for another 15 min. The magnitude and the duration of IOP elevation were dose-dependent. The effects of indomethacin or dexamethasone on the IOP response to 250 ng of PGE, are shown in Fig. 2. Dexamethasone had no effect, while indomethacin reduced the IOP rise by 24% which was not significantly different from the untreated group. Prostaglandin E, dose-dependently caused influx of protein into the anterior chamber, due to the breakdown of the blood-aqueous barrier, as shown in Fig. 3. Indomethacin (100 pg) given topically at 60, 30 and 15 min before PGE, did not significantly inhibit the influx of protein into the anterior chamber. Similarly, 50 ,ug dexamethasone administered 24, 20, 4 and 2 hr before PGE, had no effect on the protein influx (Fig. 3). Leukotriene B,, at a dose of 200 ng per eye, caused substantial accumulation of PMN 4 hr after its injection into the anterior chamber. FMLP (250 ng) also caused PMN accumulation at a similar time interval. The doses of these two compounds used in the present study have previously been shown to cause maximal PMN infiltration of ocular fluids and tissues at 4 hr (Bhattacherjee et al., 1981). The

15

30

45 Tune

FIG.

60

i 75

(mm)

2. The effect of indomethacin or dexamethasone

pretreatment

on the responses

of IOP to 250 ng PGE,

injected into the anterior chamber of the rabbit eye. The changein IOPwas calculatedasthe differencein IOPof the separategroups of untreated and treated animals. The values are mean+s.E.M. of four to six experiments.

i

5

e a

FIG. 3. The increase in protein concentration in the aqueoushumor 60 min after various dosesof PGE,and the effect of indomethacinand dexamethasoneon the response to 250 ng PGE,.The columnsaremean~s.E.M. of four to six

experiments.

AA

METABOLITES

AND

OCULAR

mpa mpg lND0 DE**

EFFECTS

95

mJP4 mpg IN00 DEXA

FIG. 4. Effects of indomethacin and dexamethasone pretreatment on the leukocyte accumulation in response to

LTB, and FMLP injected into the anterior chamber. The columns are the mean+~.~.~. of four to six experiments

(*P > 005). increase in aqueous humor protein by leukotrient B, and FMLP was not significantly diierent from that after saline. Of these two agents, only FMLP increased IOP by less than 3 mmHg. Neither indomethacin nor dexamethasone had an inhibitory effect on leukocyte infiltration induced by LTB,. Dexamethasone, but not indomethacin, significantly inhibited the leukocyte response to FMLP. 4. Discussion

The results of this study confirm the previous observations that the early phase of PGE, effect on IOP is hypertensive (Camras, Bito and Eakins, 1977). Whether the ocular hypertension is followed by a hypotensive response was not examined as the experiments were terminated at 60 min after PGE,. The hypertensive effect appears to be the direct action presumably on PGE, receptors in the iris-ciliary body (Bhattacherjee, Csukas and Paterson, 1989) and does not involve a further release of cyclooxygenase products. The lack of inhibitory effect by indomethacin or dexamethasone supports this observation. However, the small inhibitory effect of indomethacin on the IOP response to PGE, may not be related to a further release of cyclooxygenase products. If this were so, dexamethasone, which inhibits the release of PG substrate arachidonic acid, did not attenuate this response. Probably, exogenous PGE, released a substance which is not a PG. but is sensitive to indomethacin by a mechanism unrelated to its cyclooxygenase inhibition. Leukocyte accumulation in the anterior chamber in response to LTB, was not affected by indomethacin or dexamethasone. Thus, it appears that the in vivo chemotactic effect of LTB, does not involve a secondary release AA metabolites. In contrast, chemotaxis of leukocytes by FMLP was significantly reduced by dexamethasone, but not by indomethacin. This obser-

vation suggests that FMLP effect is indirect, probably mediated by 5-lipoxygenase product(s). We have not investigated this speculation further using a selective 5-lipoxygenase inhibitor because all these inhibitors are unsuitable for topical administration or not available in sufficient amount for systemic use. There are studies, however, to indicate that leukocyte response to FMLP could involve other mechanisms. FMLP has been shown to bind to the surface receptors in PMN and this binding can be inhibited by indomethacin an 5, 8, 11, 14-eicosatetraenoic acid (Williams et al., 1977: Cost, Gespatch and Abita, 1981; Atkinson et al., 1982). Since in our study indomethacin is ineffective, interference with receptor binding can be ruled out. There is also evidence that FMLP stimulates AA and

LTB, release (Hirata et al., 1979; Palmer, 1983). Dexamethasone inhibits AA release by blocking phospholipase A, (Blackwell et al., 1982). Therefore, it seems the attenuation of PMN response to FMLP by dexamethasone could be the result of inhibition of the release of AA via phospholipase A,, which is metabolized by 5-lipoxygenase pathway to LTB, and other products. Acknowledgments This study was supported by USPHS research grant number EY-06918, the Kentucky Lions Eye Research Foundation, and an unrestricted grant from Researchto Prevent Blindness,Inc. We thank DeborahJeanDean and CeceliaWroblewskifor manuscriptpreparation.

References Atkinson, J. P., Simchowitz.L., Mehta, J. and Stenson,W. F. (1982). 5, 8. 11, 14Eicosatetraynoic acid (ETYA) inhibits binding of N-formyl-methionyl-leucyl-phenylalanine(FLMP)to its receptoron human granulocytes. lmmunopharmacologg

4, 1-9.

Beitch. B. R. and Eakins, K. E. (1969). The effect of prostaglandms on the intraocular pressureof the rabbit. Br. J. Pharm. 37. 158-67. Bhattacherjee,P. (1974). Autoradiographic localization of intravitreally injected[aH]prostaglandins. Exp. Eye Res. 18181-8. Bhattacherjee, P., Csukas. S. and Paterson, C. A. Prostaglandin E, binding sites in bovine iris-ciliary body. Invest. Ophthalmol. Iris. Sci. (in press). Bhattacherjee.P., Hammond,B. R., Salmon,J. A., Shepney. R. and Eakins, K. E. (1981). Chemotacticresponseto somearachidonic acid lipoxygenaseproducts in the rabbit eye. Eur. J. Phurmucol. 73, 21-8. Bito. L. Z., Camras.C. B., Gum, G. G. and Resul.B. (1989). The ocular hypotensive effects and side effects of prostaglandinson the eyesof experimentalanimals.In The Ocular Efects of Prostaglandins and OtherEicosanoids (EdsBito. L. 2. and Stjernshantz,J.). Pp. 349-68. Alan R. Liss:New York. Bito, L. Z., Nichols, R. R. and Barody, R. A. (1982). A comparisonof the miotic and inflammatory effectsof biologicallyactive polypeptidesand prostaglandinE, on the rabbit eye. Exp. Eye Res. 34, 325-37. Blackwell, G.J.. Carnuccio, R., di Rosa,M., Flower. R. J..

96

Langham, C. S. J., Parente, L.. Persico, P., KussellSmith, N. C. and Stone, D. (1982). Glucocorticoids induce the formation and release of anti-inflammatory and anti-phospholipase proteins into the peritoneal cavity of the rat. Br. 1. Pharmacol. 76, 185-94. Camras, C. B., Bito, L. Z. and Eakins. K. E. (1977). Reduction of intraocular pressure by prostaglandins applied topically to the eyes of conscious rabbits. Invest. Ophthahnol. Vis. Sci. 16, 112 5-34. Cost, H., Gespach. C. and Abita, J. (1981). Effect of indomethacin on the binding of the chemotactic peptide formyl-met-leu-phe on human polymorphonuclear leukocytes. FEBS Left. 132, 85-8. de Deckere, E. A. M. and ten Hoor. F. (1980). PGF,, stimulates release of PGE, and PGI, in the isolated perfused rat heart. Adv. Prostaglandin Thromboxane Res. 7, 655-8. Folco, G. E.. Ourini. E., Vigano, T., Iantorno, G. and Berti, F. (1980). Pharmacologic control of Thromboxane A, generation in lungs. Adv. Prostaglandin Thromboxane Res. 7. 917-25. Hirata. F., Corcoran, B. A., Venkatasubramanian, K., Schiffman. E. and Axelrod, J. (19 79). Chemoattractants stimulate degradation of methylated phospholipids and

P. BHATTACHERJEE

AND

C.A.

PATERSON

release of arachidonic acid in rabbit leukocytes. Pm. Nat!. Acad. Sri. U.S.A. 76. 2640-3. Kulkarni. P. and Srinivasan. B. D. ( 1985). Comparative in antiviva inhibitory effects of non-steroidal inflammatory agents on PG synthesis in rabbit ocular tissues. Arch. Ophthalmol. 103, 103-6. Lowry. 0. H.. Rosebrough, N. J., Farr. A. L. and Kandall, R. J. (1951). Protein measurement with the Folin phenol reagent. 1. Biol. Chem. 193, 265-75. Palmer, R. M. J. (1983). Some Actions of Non-steroidal AntiInflammatory Drugs on Human Neutrophils In Vitro. Ph.D. Thesis, University of London. Piper, P. J. and Samhoun. M. N. (1981). The mechanism of acting Leukotriene C, and D, in guinea pig isolated perfused long and parenchymal strips of guinea pig, rabbit and rat. Prostaglandins 21, 793-803. Srinivasan. B. D. and Kulkarni. P. (1980}. The role of arachidonic acid metabolites in the mediation of the polymorphonuclear leukocyte response following corneal injury. Invest. Ophthalmol. Vis. Sci. 19, 1087-93. Williams, L. T.. Snyderman, R., Pike, M. R. and Lefkowitz, R. T. (1977). Specific receptor sites for chemotactic peptides in human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. U.S.A. 74. 1204.

Further investigation into the ocular effects of prostaglandin E2, leukotriene B4 and formyl-methionyl-leucyl phenylalanine.

The effect of indomethacin and dexamethasone on the inflammatory response to intraocularly injected prostaglandin E2 (PGE2), leukotriene B4 (LTB4), an...
418KB Sizes 0 Downloads 0 Views