Intravitreally injected platelet activating factor induces retinitis in experimental animals.

PURPOSE: Platelet activating factor is a lipid which has been strongly implicated in anterior uveitis. In order to investigate further the role of platelet activating factor in intraocular inflammation, we have characterized the histological changes associated with the intravitreal injection of platelet activating factor, PAF analogs, or lyso-PAF in laboratory rabbits and rats. METHODS: Initial studies utilized a PAF analog (rac 1-0-octadecyl 2-0-ethyl glycero phosphoryl choline or ethoxy PAF), because this compound is relatively resistant to degradation by hydrolase, the major degradative enzyme for PAF. Doses ranging from 1 ug to 5 mg and time points from 6 hours to 7 days after injection were studied. RESULTS: In either rats or rabbits, 100 ug of ethoxy PAF consistently induced a marked uveitis with the predominance of inflammation focused in the retina and choroid. Retinitis was also induced in rabbits by either 1 mg PAF injected intravitreally or a similar dose of the PAF precursor/metabolite, lyso PAF. Retinal inflammation was not induced by an inactive lipid, 1,1-0,0-dihexadecyl-rac-glycero-3-phosphocholine, although this compound resulted in mild vitreous inflammation. The histological changes induced by PAF could be readily distinguished from the predominantly anterior inflammation induced by intravitreal injections of substances such an interleukin-1 or endotoxin. CONCLUSIONS: Recent studies indicating that PAF antagonists inhibit a variety of retinal toxicities and our own observations suggest that PAF could be a major mediator of retinal inflammation.

Failure to inhibit endotoxin-induced uveitis with antibodies that neutralize tumor necrosis factor.

Many of the effects of bacterial endotoxin are attributed to the cytokines that it induces. Tumor necrosis factor alpha (TNF alpha) may be responsible for many of the pathophysiologic effects of endotoxin because antibodies that neutralize TNF alpha reduce endotoxin-induced mortality in experimental animals and many of the activities of endotoxin can be mimicked by the infusion of TNF alpha. Accordingly, we tested the ability of an antibody that neutralizes TNF alpha in a well characterized rat model, endotoxin-induced uveitis. Rats receiving subcutaneous endotoxin had a prompt rise in serum TNF alpha that was eliminated by the monoclonal antibody. Small quantities of TNF alpha were detectable in aqueous humor after either subcutaneous or intravitreal injection of endotoxin. Endotoxin-induced uveitis as quantitated by cells and protein in aqueous humor was, however, not affected by the antibody. Similarly, intraocular inflammation was not reduced by the intravitreal injection of the neutralizing antibody. Although TNF alpha may mediate many of the systemic effects of endotoxin, its role in the intraocular inflammatory effects of endotoxin should be questioned.

Efficacy of antibodies to adhesion molecules, CD11a or CD18, in rabbit models of uveitis.

Adhesion molecules play a critical role in leukocyte emigration to a site of inflammation. In order to assess the potential therapeutic benefit of blocking adhesion molecule function in anterior uveitis, the efficacy of antibodies to specific adhesion molecules was tested in 3 separate rabbit models of anterior uveitis. Antibodies to two different leukocyte molecules, CD11a and CD18, and antibodies to the endothelial ligand for CD11a/CD18, ICAM-1 (intercellular adhesion molecule-1, CD54), were studied in inflammation after intravitreally injected interleukin-1, intravitreally injected endotoxin, or an ocular reversed passive Arthus reaction. The CD18 antibody (2 mg/kg intravenously) reduced the cellular infiltrate with each of these 3 models. The antibody to CD11a was equally effective but was tested only in the IL-1-induced model. The antibody to ICAM-1 reduced the cellular infiltrate associated with this model, but the results did not reach statistical significance. None of the antibodies was able to reduce the associated increase in vascular permeability as measured by protein in the aqueous humor. The antibody to CD18 failed to reduce the inflammation if it was administered 24 hours after the intravitreally injected endotoxin. These observations demonstrate that leukocyte migration into the anterior segment of the eye is dependent on the CD11a/CD18 complex.

Ocular inflammatory effects of intravitreally injected interleukin-2.

The primary, known physiologic effect of interleukin-2 (IL-2) is to act as a T lymphocyte growth factor. We investigated the potential contribution of IL-2 to intraocular inflammation by studying the inflammation resulting from the intravitreal injection of recombinant, human IL-2 in New Zealand white rabbits. Serial slit lamp observations indicated that 40 microgram of intravitreally injected IL-2 induced an anterior uveitis which was maximal 5 days after the injection. Inflammation was less marked but still significant with amounts of IL-2 as low as 400 ng. Direct examination of aqueous humor confirmed elevations of protein, prostaglandin E2, and mononuclear cells which correlated with the clinical observations. The kinetics of the response to intravitreal IL-2 distinguished it from the responses to other intravitreally injected cytokines such as interleukins 1, 6, or 8 as well as tumor necrosis factor. Intramuscular injection of cyclosporine A significantly reduced the protein extravasation associated with IL-2 injection, but cyclosporine had no effect on inflammation secondary to an intravitreal injection of interleukin-1. These observations implicate IL-2 as a potential contributor to uveitis. In addition, the studies with cyclosporine indicate the heterogeneity of inflammation such that pharmacologic agents which affect one cause of uveitis are not necessarily efficacious in another model.

Activity of an interleukin 1 receptor antagonist in rabbit models of uveitis.

Interleukin 1 has been implicated in intraocular inflammation. The availability of a cloned, recombinant interleukin 1 receptor antagonist has enabled us to test the role of interleukin 1 in specific models of uveitis in New Zealand white rabbits. Seventy-five micrograms of interleukin 1 receptor antagonist injected intravitreally resulted in a 97% reduction in aqueous humor cells present 6 hours after intravitreal injection of 10 ng of human interleukin 1 alpha. Disruption of the blood aqueous barrier was prevented by the receptor antagonist (mean +/- SD aqueous humor protein of 0.6 +/- 0.1 g/L in rabbits treated with interleukin 1 receptor antagonist vs 32.2 +/- 9.9 g/L in controls). Lower doses of interleukin 1 produced more modest but significant inhibition. Despite the activity of interleukin 1 receptor antagonist in inhibiting interleukin 1-induced inflammation, interleukin 1 receptor antagonist did not produce significant reduction in inflammation subsequent to an active Arthus reaction or subsequent to the intravitreal injection of 125 ng of endotoxin. A potential explanation of these observations is that cytokines in addition to interleukin 1 may be present in sufficient quantities to produce intraocular inflammation or that the effects of interleukin 1 may be primarily intracellular (intracrine) and therefore resistant to the activity of exogenously administered receptor antagonist.

Use of a soluble interleukin-1 receptor to inhibit ocular inflammation.

Interleukin-1 (IL-1) has been strongly implicated as an inflammatory mediator in anterior uveitis. Recently, solubilized receptors have been utilized to block the binding of viruses to cell membranes or to inhibit cytokine activity. We have tested the activity of an intravitreally injected soluble, human interleukin-1 receptor in a rabbit model of IL-1-induced inflammation. 3 ug of the soluble receptor markedly inhibited both the cellular infiltration and the protein extravasation that followed 6 hours after an intravitreal injection of 10.5 ng of recombinant human interleukin-1 alpha. The efficacy of the soluble receptor was less marked 24 hours after the IL-1 injection. The cellular infiltrate was not reduced at all if the IL-1 receptor was injected 2 hours after the IL-1. The activity of the soluble receptor deserves further study as a therapeutic modality for inflammatory eye disease.

Synthesis of platelet activating factor by ocular tissue from inflamed eyes.

Platelet activating factors (PAFs) are a family of ether lipids with properties that suggest a major role in inflammation. We have previously implicated PAFs in ocular inflammation based on the inhibition of several rabbit models of iritis with a specific PAF receptor antagonist. We have tested ocular tissues for the ability to synthesize PAF. Iris, ciliary body, cornea, and/or retina were carefully dissected from New Zealand white rabbits, and tissue from four eyes was pooled. Tissues were stimulated with calcium ionophore (10 mumol/L), and supernatants were extracted with chloroform-methanol. Platelet-aggregating activity was found in the chloroform phase in 2 of 9, 1 of 8, 0 of 9, and 3 of 9 studies involving iris, retina, ciliary body, or cornea, respectively. Twenty-four hours after the intravitreal injection of 125 ng of endotoxin, aggregating activity was consistently detectable from supernatants of stimulated iris and ciliary body, occasionally present from stimulated retina but not detectable from cornea. The shape of the aggregation curve resembled that produced by 0.5 to 2.0 ng of authentic PAF. Moreover, the aggregation could be completely inhibited by a PAF receptor antagonist and the aggregating activity chromatographed identically on high-performance liquid chromatography to a PAF standard. These studies indicate that PAF-like activity could be detected from several ocular tissues subsequent to inflammation. Iris, ciliary body, retina, vascular endothelium, and/or leukocytes could each contribute to the presence of this inflammatory mediator.

The effects of topical S(+)-ibuprofen on interleukin-1 induced ocular inflammation in a rabbit model.

Topical non-steroidal anti-inflammatory drugs may serve as an alternative or adjunct to topical corticosteroid therapy for iritis. We have tested the efficacy of topically given S(+)-ibuprofen in a rabbit model of uveitis secondary to the intravitreal injection of human recombinant interleukin 1-alpha. Topically administered S(+)-ibuprofen was found to inhibit increased vascular permeability associated with this model. These results with topical S(+)-ibuprofen compare favorably to the results seen with topical prednisolone and are significantly superior to the results seen with topical flurbiprofen. Topical S(+)-ibuprofen did not significantly reduce the cellular infiltration associated with interleukin-1 induced inflammation. These findings suggest a potential role for topical S(+)-ibuprofen in the treatment of ocular inflammation and, in this animal model, it appears to be superior to an alternative non-steroidal compound, but further studies are indicated to assess its activity in alternate models of ocular inflammation.