Kenneth E. Eakins

Chemokinetic activity of arachidonic acid lipoxygenase products on leuocyctes of different species

A number of hydroperoxy (HPETE) and hydroxy (HETE) products of the lipoxygenase pathway of arachidonic acid metabolism are chemotactic and chemokinetic for human neutrophils. We have investigated the relative chemokinetic potency of some of these products on human, rat and rabbit neutrophils. The most potent lipoxygenase product studied was 5,12-dihydroxy-6,8,10-14-eicosatetraenoic acid (5,12-diHETE), which was maximally chemokinetic and chemotactic between 0.1 and 1.0ng/ml for the three species. The 5, 11 and 12-HPETEs and HETEs were chemokinetic, but less active by at least two orders of magnitude, for human and rabbit neutrophils at concentrations between 0.1 and 10micrograms/ml. 15-HPETE and 15-HETE were inactive on human leucoctes, and none of the monosubstituted products studied were chemokinetic for rat neutrophils. These results indicate that 5,12-diHETE may be an important mediator in the local accumulation of leucocytes in the inflammatory response.

The effects of non-steroid anti-inflammatory drugs on leukocyte migration in carrageenin-induced inflammation

Some non-steroid anti-inflammatory drugs which inhibit arachidonate cyclo-oxygenease have been examined for their effects on leukocyte migration, prostaglandin production and oedema formation in carrageenin-induced inflammation in the rat. At doses which inhibited oedema, all the drugs tested caused a dose-dependent reduction in numbers of leukocytes and prostaglandin concentrations in 24-h inflammatory exudates. At lower doses, indomethacin, aspirin, sodium salicylate, flurbiprofen and phenylbutazone significantly potentiated leukocyte migration by 20-70%. Ibuprofen, naproxen and BW755C reversed the indomethacin-induced increase in leukocyte accumulation. BW755C inhibits the generation of chemotactic lipoxygenase products and it is possible that the effects of all these drugs on leukocyte migration are mediated through the lipoxygenase pathway of arachidonic acid metabolism.

Chemotactic response to some arachidonic acid lipoxygenase products in the rabbit eye.

The effects of arachidonic acid, its cyclo-oxygenase and lipoxygenase products and the synthetic chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP) on leukocyte accumulation in the aqueous humour and intraocular pressure in the rabbit were studied in vivo. Substances were injected into the anterior chamber of the eyes of anaesthetised rabbits using a closed circuit perfusion system. Injection of arachidonic acid, prostaglandins E1 and E2, and the monohydroperoxy and hydroxy acids of the lipoxygenase pathway did not result in any significant accumulation of leukocytes in the anterior chamber. In contrast, FMLP and 5,12-diHETE (Leukotrine B4) resulted in significant dose dependent accumulation of leukocytes into the aqueous humour. Leukocytes appeared in the aqueous humour between 2 and 3 h after the injection of either FMLP or LTB4 and the response was maximal at 4 h. None of the lipoxygenase products tested had any effect on intraocular pressure in contrast to the profound effects observed with arachidonic acid and the E type prostaglandins. FMLP had a small but significant effect on intraocular pressure at the highest dose tested for leukocyte accumulation. These results indicate that the effects of the cyclo-oxygenase products of arachidonate metabolism are mainly vascular in the rabbit eye in contrast to the predominantly cellular effects of lipoxygenase products. Thus in the eye, the interaction of cyclo-oxygenase and lipoxygenase products of arachidonate metabolism may be important in the development of both acute and chronic ocular inflammation.

Increased intraocular pressure produced by prostaglandins E1 and E2 in the cat eye

The effects of intracameral injections of prostaglandins E 1 and E 2 on intraocular pressure were studied in cats under pentobarhital anesthesia. Doses of 1 μg or more of either of these prostaglandins were found to produce a variable increase in intraocular pressure accompanied by vasodilation and miosis. Smaller doses were found to produce miosis without any change in intraocular pressure. 1 μg of either prostaglandin produeed an increase in the protein content of aqucous humor which was related to the magnitude of the sustained increase in intraocular pressure. The intraocular pressure response showed marked tachyphylaxis to both prostaglandins. Much larger doses of prostaglandins were required to elevate intraocular pressure in the cat than in the rabbit. Animal to animal variations in cats were much greater than those found previously in rabbits. It is concluded that the production of local ocular vasodilation and increased permeability of the blood-aqueous barrier play an important part in the effect of prostaglandins on cat intraocular pressure. The similarities between the responses of the cat eye to prostaglandins and irritation are discussed.

Biosynthesis of lipoxygenase products by ocular tissues

The metabolism of arachidonic acid via the lipoxygenase pathway has been investigated in conjunctival and iris tissue taken from eyes of various species. In addition we have also studied two inhibitors of arachidonate metabolism, BW 755 and indomethacin, on albino rabbit ocular tissues. The ocular tissues of most species (monkey, dog, cat, rabbit, guinea-pig and rat) formed lipoxygenase products from exogenous arachidonic acid. The exception was the albino rabbit iris, where no lipoxygenase product was detected. The major lipoxygenase product found was 12-HETE, although 5-HETE and 5,12-diHETE were formed to a lesser extent by the conjunctiva and iris of the Dutch rabbit. The rat ocular tissues and guinea pig conjunctiva also formed 5-HETE. In the conjunctiva of the albino rabbit, indomethacin was a relatively specific inhibitor of the cyclo-oxygenase pathway whereas BW 755 inhibited both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In view of the possible roles of lipoxygenase products in inflammatory reactions and the ability of ocular tissues to synthesize these products, dual inhibitors of cyclo-oxygenase and lipoxygenase pathways may be useful agents to control ocular inflammatory responses.

The reepithelialization of rabbit cornea following partial and complete epithelial denudation.

Abstract The rate of reepithelialization of rabbit corneas following partial and complete epithelial denudation was studied by fluorescein staining, photography, and electron microscopy. Partially denuded corneas healed smoothly but some completely denuded corneas exhibited delayed or regression of healing which was independent of the methods used to denude corneas. Electron microscopy of reepithelializing corneas suggested that polymorphonuclear leucocytes which are present in denuded corneas may influence the course of reepithelialization.

Ascorbic acid inhibits the activity of polymorphonuclear leukocytes in inflamed ocular tissues

Myeloperoxidase (MPO) is present at high levels in polymorphonuclear leukocytes (PMNs) and has been used as a marker to quantify the accumulation of PMNs in inflamed tissues. MPO activity in inflamed ocular tissues was inhibited by aspirates of aqueous humor. This inhibition could be duplicated by the addition of ascorbic acid at concentrations equivalent to those present in the aliquots of aqueous humor. Similarly, aqueous humor and ascorbic acid inhibited MPO from isolated rabbit leukocytes. Therefore, ascorbic acid appears to inhibit the functional activity of the peroxidase in PMNs, thus preventing potential tissue damage by this enzyme when released during leukocyte degranulation in inflammation. Ascorbic acid might fulfill a role as an endogenous anti-inflammatory agent in the eye.

Polyphloretin phosphate — A selective antagonist for prostaglandins F1a and F2a

Abstract The effect of polyphloretin phosphate (PPP) on the smooth muscle stimulating action of prostaglandins on the jird colon has been studied. PPP in concentrations of 5–10 μm/ml was found to selectively antagonise the actions of F prostaglandins on the jird colon. The effects of E prostaglandins and of acetylcholine and 5-hydroxytryptamine were unaltered.

The mechanism of the antagonism of experimentally induced ocular hypertension by polyphloretin phosphate

Abstract The ability of polyphloretin phosphate to antagonize the rise in intraocular pressure produced by prostaglandins E2 (PGE2), F2α (PGF2α) and formaldehyde has been studied in the conscious rabbit. Topical instillation of polyphloretin phosphate antagonized only the response to PGF2α. In contrast, subconjunctival injections of polyphloretin phosphate antagonized the response of the intraocular pressure to all 3 irritants. Polyphloretin phosphate did not prevent the conjunctival vasodilation produced by all 3 irritants. Polyphloretin phosphate was separated into high and low molecular weight fractions by fractionation on Sephadex G-25 columns. Fraction I (the high molecular weight fraction) had almost no prostaglandin-blocking activity, but was a potent antagonist of hyaluronidase. Conversely Fraction III (the low molecular weight fraction) had very little anti-hyaluronidase activity, but was 2 to 5 times more potent than polyphloretin phosphate as a prostaglandin antagonist. Subconjunctival injections of Fraction I did not antagonize the response of the rabbit intraocular pressure to PGE2 whereas Fraction III produced a significant inhibition of this response. Both Fractions I and II were found to antagonize the rise in intraocular pressure produced by formaldehyde. The present results suggest that the antiinflammatory actions of polyphloretin phosphate in the rabbit eye are due to both the prostaglandin-blocking and anti-hyaluronidase actions. It is concluded that both prostaglandin-antagonists and anti-hyaluronidase agents should be evaluated more thoroughly for use as ocular anti-inflammatory agents.

The reepithelialization of rabbit cornea following single and multiple denudation.

Abstract The reepithelialization of rabbit corneas completely denuded once by abrasion with a dental stone was compared with the rate of reepithelialization of the contralateral eye following repeated but gentle repeeling of the epithelium at 2-day intervals for 6 days after an initial complete denudation by abrasion. Two or three times repeeled corneas showed a more rapid initial reepithelialization than observed after the first denudation. Polymorphonuclear leukocytes (PMNs) were found in the corneal stroma and tear fluid following both single and repeated denudation, but only up to 24 hours after the first, more traumatic, denudation were large numbers of PMNs found adherent to the exposed basement membrane of the corneal epithelium. It was hypothesized that PMNs on the corneal surface may interfere with reepithelialization.