Platelet-activating factor: an inflammatory mediator in the acute phase of allergic conjunctivitis in a guinea-pig model

The role of platelet-activating factor (PAF) as a mediator of increased conjunctival vascular permeability was investigated in a guinea-pig model of immediate hypersensitivity. Vascular permeability of the conjunctiva was determined by measuring the albumin content in lavage fluid (LF) after topical challenge with either PAF or ovalbumin. PAF produced a dose-dependent increase of the vascular permeability within minutes. Topical pretreatment with levocabastine, a potent histamine H1-antagonist demonstrated no effect towards the vascular permeability in response to PAF provocation. Pretreatment with eyedrops containing the specific PAF antagonist BN 52021 (1%) showed a significant inhibition of the vascular permeability (60.2%) and the clinical score (27.5%) after PAF challenge. In sensitized guinea-pigs, levocabastine showed a marked inhibition of both the vascular permeability (80.5%) and the clinical score (70%) after topical challenge with ovalbumin. BN 2021, although to a lesser extent, showed a similar effect towards the vascular permeability (26.8%) and the clinical score (28%) after antigen provocation. When BN 52021 and levocabastine were administered in combination, the vascular permeability was significantly decreased after antigen challenge in comparison with eyes pretreated with levocabastine alone. These results indicate that PAF plays a role in the acute phase of allergic conjunctivitis in the guinea-pig.

Trm role of platelet-activating factor (PAF) as a mediator of increased conjunctival vascular permeability was investigated in a guinea-pig model of immediate hypersensitivity. Vascular permeability of the conjunctiva was determined by measuring the albumin content in lavage fluid (IF) after topical challenge with either PAF or ovalbumin. PAF produced a dose-dependent increase of the vascular permeability within minutes. Topical pretreatment with levocabastine, a potent histamine HI-antagonist demonstrated no effect towards the vascular permeability in response to PAF provocation. Pretreatment with eyedrops containing the specific PAF antagonist BN 52021 (1%) showed a significant inhibition of the vascular permeability (60.2%) and the clinical score (27.5%) after PAF challenge. In sensitized guinea-pigs, levocabastine showed a marked inhibition of both the vascular permeability (80.5%) and the clinical score (70%) after topical challenge with ovalbumin. BN 2021, although to a lesser extent, showed a similar effect towards the vascular permeability (26.8%) and the clinical score (28%) after antigen provocation. When BN 52021 and levocabastine were administered in combination, the vascular permeability was significantly decreased after antigen challenge in comparison with eyes pretreated with levocabastine alone. These results indicate that PAF plays a role in the acute phase of allergic conjunctivitis Introduction Allergic conjunctivitis is characterized by bilateral hyperaemia, itching, tearing and oedema. Increased vascular permeability has also been described as an important feature of allergic conjunctivitis. It is an immediate hypersensitivity reaction (type I); after interaction of the antigen-IgE antibody complex with the mast cell, the clinical signs and symptoms appear within minutes. Histamine, released from the granules of the mast cell, seems to play a key role but other mediators including leukotrienes (LT), prostaglandins (PG) and platelet-activating factor (PAF) seem to take part in the inflammatory process. 2 PAF has been shown to induce a wide variety of biological actions such as chemotaxis and activation of neutrophils and eosinophils; 4 moreover PAF induces increased vascular permeability. 5 On a molar basis, PAF appeared to be 1 000 to 10000 times more potent in inducing vascular permeability changes in skin compared with histamine. 6 Because of these effects, PAF has been implicated in the pathogenesis of aller-(C) 1995 Rapid Communications of Oxford Ltd gic diseases including allergic conjunctivitis.
In a number of studies PAF receptor antagonists have been shown to be very effective in inhibiting various effects after PAF challenge in different models. In the eye, PAF antagonists have been shown to be capable of inhibiting corneal oedema formation and pupillar constriction after intracameral injection of PAF' and they partially reduced the endotoxin-induced breakdown of the blood-aqueous barrier. 8 Furthermore, the specific PAF antagonist BN 52021 showed a significant inhibition of oedema, leucocyte infiltration and vascularization of the cornea in a rabbit model of immunogenic keratitis 9 after challenge with albumin. In view of these findings, we studied the inhibitory effect of BN 52021 towards microvascular permeability and clinical signs after PAF and antigen provocation in a guinea-pig model of immediate hypersensitivity.

Materials and Methods
Female Hartley strain guinea-pigs (weight range, 350-450 g) were sensitized to ovalbumin F. Meijer, J. L. van Delft and N. J. van  by i.p. injection of a mixture containing 10 l-tg Results ovalbumin and 0.1 g aluminium hydroxide suspended in 0.5 ml saline. Two to 3 weeks later, Effects of PAE.. Topical application of PAF (10 l.tg) ovalbumin (0.5%) or PAF (10 l.tg) were applied resulted, within 5 min, in an inflammatory reactopically in 25 1 eye drops. Ovalbumin eye tion consisting of hyperaemia, conjunctival drops were prepared as a 0.5% solution in phos-oedema and periorbital swelling which disphate buffered saline (PBS). Preliminary experi-appeared within 4h. The maximum CS was ments (not reported here) had shown that this observed after 30 min. dose of ovalbumin produced a useful response PAF provocation produced a dose related in terms of clinical signs and microvascular perincrease of the albumin concentration in LF ( 1). The minimal dose to provoke a leakage of PAF (C18) was dissolved in water and stored at albumin from the conjunctiva vessels was 3 I.tg/ 4C according to the manufacturer's instructions, eye. Maximum levels of albumin in LF were Just prior to the performance of the experiment, found after 30 min, showing a decline to zero 4 h the stock solution was further diluted in PBS, after challenge (Fig. 2). The increase of the vasusing polypropylene tubes and pipette tips. cular permeability with time showed a straight BN 52021 (1%) or levocabastine (0.05%) were correlation with the development of clinical signs dissolved in 0.3% hypromellose and applied at and symptoms. random to one eye l h before challenge with Pretreatment with BN 52021 showed a sig-PAF or antigen. The contralateral eye served as a nificant effect on both the CS (Fig. 3) and the control and received the solvent, albumin concentration in LF (Fig. 4). The mean Clinical score (CS) was determined and albumin content in LF after BN 52021 treatment lavage fluid (LF) was collected at 0.5, 1, 2 and was inhibited (by 60.2%) in comparison with 4h. LF was sampled by washing the eye with control eyes. PBS (25 btl) using a polypropylene micropipette, Levocabastine showed no effect on the CS and avoiding touching the eye. After three forced failed to reduce the conjunctival microvascular blinks the LF was collected and stored at-20C permeability after PAF administration. until used.
Ovalbumin was purchased from Sigma (St Effects of ovalbumin: Ovalbumin administration Louis, MO), PAF from Cayman (Ann Arbor, MI), produced a dose-dependent increase in vascular guinea-pig albumin anti-serum and guinea-pig permeability in sensitized animals (data not albumin from Nordic (Tilburg, the Netherlands). shown). As a useful concentration for our eyes of the challenged guinea-pigs were free from signs of inflammation. Levocabastine as a pretreatment significantly reduced the mean of the clinical score (Fig. 5) and showed a significant inhibition (80.5%) of the vascular permeability (Fig. 6). BN

Discussion
This study shows that PAF plays a role in the acute phase of allergic conjunctivitis. Topical application of PAF to the eyes of guinea-pigs resulted in an inflammatory response including hyperaemia, oedema and swelling of the con- partly mediated by PAF. Other reports also antagonists on the vascular permeability in gested that PAF employs its activity via the sec-*Significant diffrn from es reivin only th solvent {control) # < 0.01. ondary synthesis of lipoxygenase products. 9 It is "Significant differen from m/s receiving levoeabastine (l_vo) likely that many of the effects attributed to PAF < 0.O1. are dependent on the secondary generation of LT and PG. PAF has been shown to release LT, 2 junctiva. Moreover, PAF provocation showed a and in a rabbit conjunctival provocation model dose dependent increase in conjunctival vascular the inflammatory response after PAF challenge permeability. Our results coincide with the could be inhibited using a lipoxygenase inhibobservations of others who applied PAF to the itor. 2 Also, topical administration of LT results in 10 11 12 eyes of rats and guinea-pigs. The dose of a similar inflammatory response when compared PAF needed to produce an inflammatory with PAF provocation. 22 However, LT antagonists response of the conjunctiva seems very high appeared to be less effective in suppressing the when compared with the dose of PAF, less than conjunctival vascular permeability after antigen 0.1 l-tg, used in other models. In a rabbit model administration in comparison with exogenous of ocular hypertension, a dose of 250 tg per eye LT challenge in guinea-pigs. Because of the in the presence of 0.25% HSA was topically, significant effect of BN 52021 after ovalbumin administered without any systemic side effects. challenge, it seems that PAF plays a more promi-This may be explained by the unique characternent role compared to LT in the acute phase of istics of the eye towards topical administration of allergic conjunctivitis in guinea-pigs. PAF. (Possibly PAF is rapidly converted to lyso-Because levocabastine pretreatment showed PAF by enzymatic hydrolysis or PAF does not no effect on CS and vascular permeability after easily penetrate the outer surface of the eye). PAF administration, it seems that PAF does not Because albumin levels in LF and the CS both trigger the mast cell to release histamine from its reached a maximum after 30 min, we selected granules. This is confirmed by the observation this as a time point to test the effectiveness of that during in vitro experiments PAF was not the specific PAF antagonist BN 52021 and the H1-able to release histamine from human dispersed antagonist levocabastine after provocation with cutaneous mast cells. 23 antigen or PAF.
Because albumin is not produced by the lacri-Pretreatment with levocabastine reduced the mal gland of the guinea-pig, 24 the concentration inflammatory response after topical administra-of albumin on the ocular surface of the eye is a tion to antigen to sensitized guinea-pigs for the result of the flow of tissue fluids and other major part (Figs. 5 and 6). Woodward et al. 15 serum proteins across the epithelium to the observed a similar effect using a combination of ocular surface under the influence of several H1 and H2-antagonists in guinea-pigs, and in a mediators. The LF collected from the surface of human study levocabastine appeared to be very the eye at this stage is a mixture of 'leaking' effective in relieving the clinical signs and sympserum and reflex tearing from the lacrimal gland, toms when used by patients with allergic conresulting in some variation between animals but junctivitis. 16 These observations indicate the not between the eyes of one animal. Stock et dominant role of histamine in the acute phase of al. observed that in the conjunctival system the clinical evaluation was as good as an experimental estimate of the mediator effect measured by extravasation of Evans blue. Because, in our model, clinical score and albumin leakage showed a clear correlation, the observed differences in albumin leakage are due to pharmacological effects and not to the collection method of the LF.
Although the major response in the immediate hypersensitivity reaction seems to be histamine mediated, the effectiveness of BN 52021 in inhibiting the inflammatory response after ovalbumin challenge demonstrates the important role of PAF in the acute phase of allergic conjunctivitis.