Identification of an antagonist that selectively blocks the activity of prostamides (prostaglandin-ethanolamides) in the feline iris.

BACKGROUND AND PURPOSE: The prostamides (prostaglandin-ethanolamides) and prostaglandin (PG) glyceryl esters are biosynthesized by COX-2 from the respective endocannabinoids anandamide and 2-arachidonyl glycerol. Agonist studies suggest that their pharmacologies are unique and unrelated to prostanoid receptors. This concept was further investigated using antagonists. EXPERIMENTAL APPROACH: The isolated feline iris was used as a key preparation, where prostanoid FP receptors and prostamide activity co-exist. Activity at human recombinant FP and other prostanoid receptors was determined using stable transfectants. KEY RESULTS: In the feline iris, AGN 204396 produced a rightward shift of the dose-response curves for prostamide F2alpha and the prostamide F2alpha analog bimatoprost but did not block the effects of PGF2alpha and synthetic FP receptor agonists. Studies on human recombinant prostanoid receptors confirmed that AGN 204396 did not behave as a prostanoid FP receptor antagonist. AGN 204396 exhibited no antagonism at DP and EP1-4, but was a highly effective TP receptor antagonist. Contrary to expectation, the FP receptor antagonist AL-8810 efficaciously contracted the cat iris. AGN 204396 did not affect AL-8810 induced contractions, demonstrating that AL-8810 and AGN 204396 are pharmacologically distinct. Unlike AL-8810, the ethylamide derivate of AL-8810 was not an agonist. Al-8810 did not block prostamide F2alpha activity. Finally, AGN 204396 did not block PGE2-glyceryl ester activity. CONCLUSIONS AND IMPLICATIONS: The ability of AGN 204396 to selectively block prostamide responses suggests the existence of prostamide sensitive receptors as entities distinct from receptors recognizing PGF2alpha and PGE2-glyceryl ester.

Comparison of leukotriene B4 and D4 effects on human eosinophil and neutrophil motility in vitro.

The motility of isolated normal human peripheral blood eosinophils and neutrophils in response to exogenous leukotrienes B4 and D4 was examined by means of a modified under-agarose technique and a novel quantitative sampling strategy. Leukotriene D4 was a potent chemoattractant for eosinophils, with a significant threshold chemotactic effect evident at 10(-10) M. The abolition of eosinophil chemotaxis by the potent and selective peptide-leukotriene-antagonist SK&F 104353 indicated the pharmacological specificity of the leukotriene D4-induced response. The chemokinetic response of eosinophils to leukotriene D4 generally did not differ significantly from spontaneous migratory activity of unstimulated cells. Leukotriene D4 did not, however, alter directed neutrophil motility until a very high concentration (10(-5) M) was achieved, although significant neutrophil chemokinesis relative to unstimulated movement was observed over the tested concentration range. Directional emigration of both eosinophils and neutrophils was induced by leukotriene B4 at concentrations as low as 10(-8) M. Analysis of leukocyte orientations provided evidence that chemokinetic responses were not being interpreted as indications of chemotactic behavior. These studies suggest that leukotriene D4 may behave as a potent and selective chemoattractant for human eosinophils at physiologically relevant concentrations.

Determination of leukotriene effects on human neutrophil chemotaxis in vitro by differential assessment of cell motility and polarity.

The effects of leukotriene (LT) B4 and D4 on the motility of human peripheral blood neutrophils were investigated employing a novel analytical method. Using the under-agarose technique, migration distance and vectorial orientation of neutrophils in response to selected LT concentrations were determined with the aid of digital image processing. Neutrophil polarization induced by a chemotactic gradient was very apparent even at fields taken adjacent to the cell seeding well where little directional cell motility had occurred. Thus, cell polarization appeared to be the earliest response to chemoattractive LTs. Cell motility occurred in a dose-dependent manner to LTB4 according to determination of the leading edge. LTD4 produced similar effects on neutrophil polarization and motility, but these occurred only at very high concentrations. These data support the view that vectorial orientation is a prerequisite for directional migration of cells and it is also feasible that these are separately regulated events. Furthermore, our studies confirm that LTB4 and, to a much lesser extent, LTD4 are chemotactic for human peripheral blood neutrophils.

Conjunctival immediate hypersensitivity: re-evaluation of histamine involvement in the vasopermeability response.

The recent development of a technique for quantitative measurement of conjunctival microvascular permeability has permitted detailed pharmacological evaluation of H1- and H2-receptor involvement in histamine-induced increases in conjunctival microvascular permeability and the role of histamine in microvascular permeability changes associated with immediate hypersensitivity responses in the conjunctiva. The conjunctival microvascular permeability response to histamine appears to be entirely mediated by H1-receptors. Pyrilamine (H1-receptor antagonist) virtually abolished the increase in conjunctival extravascular albumin content produced by graded doses of histamine, whereas cimetidine (H2-receptor antagonist) was ineffective. Moreover, selective histamine H2-receptor agonists did not elicit a dose-dependent vasopermeability response in the conjunctiva. Although H1-receptor blockade essentially abolished the microvascular permeability response to histamine, it only partially attenuated the conjunctival microvascular permeability response associated with immediate hypersensitivity and compound 48-80. It appears that conjunctival inflammation caused by mast cell degranulation comprises both a histaminergic and a nonhistaminergic component.

Conjunctival eosinophil infiltration evoked by histamine and immediate hypersensitivity. Modification by H1- and H2-receptor blockade.

The present histological studies have demonstrated that histamine causes dose-dependent eosinophil infiltration into the conjunctiva. A highly directional movement toward the conjunctival epithelium was observed, and the presence of large numbers of degranulating eosinophils appeared to result in epithelial cell damage and goblet cell discharge. Blockade of H2-receptors by systemic cimetidine pretreatment significantly inhibited the eosinophil infiltration elicited by an intermediate histamine dose, whereas the H1-receptor blockade produced by systemic pyrilamine pretreatment markedly reduced the response to all histamine doses. The pyrilamine-insensitive residual eosinophil infiltrate was not affected by administering a cimetidine-pyrilamine combination. In animals presensitized to ovalbumin, antigen challenge evoked extensive and directional emigration of eosinophils toward the conjunctival epithelium with resultant exfoliation and depletion of goblet cell populations. In conjunctival immediate hypersensitivity, neither cimetidine nor pyrilamine alone produced an inhibitory effect, but a cimetidine-pyrilamine combination caused a significant reduction in the number of infiltrating eosinophils and prevented epithelial damage and goblet cell depletion. These results suggest that histamine may participate in the recruitment of eosinophils during immediate hypersensitivity reactions. The differential effect of pyrilamine on the eosinophil infiltration evoked by histamine or immediate hypersensitivity may, perhaps, reflect the importance of increased microvascular permeability in facilitating eosinophil emigration.

Characterization of the conjunctival vasopermeability response to leukotrienes and their involvement in immediate hypersensitivity.

The microvascular permeability response of the guinea pig conjunctiva to sulfidopeptide leukotrienes (LTs) was quantified as extravasation of radiolabeled bovine serum albumin. The LTs were potent inducers of increased microvascular permeability, with relative potencies LTE4 greater than or equal to LTD4 greater than LTC4. The response to LTs was unaffected by indomethacin or a pyrilamine/cimetidine combination, but the LT antagonists FPL 55712 and SKF 102922 significantly inhibited the response to LTC4, LTD4 and LTE4. In guinea pigs actively sensitized to ovalbumin, topical ocular administration of ovalbumin markedly increased conjunctival microvascular permeability; this response was reduced by approximately 50% following histaminergic blockade by pyrilamine/cimetidine. FPL 55712 and SKF 102922 and the 5-lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) had no effect on the response to antigen when used alone. However, each agent significantly reduced the non-histaminergic component of the response when given in conjunction with pyrilamine/cimetidine. Thus, it appears that the immediate hypersensitivity response in guinea pig conjunctiva has a possible non-histaminergic component which is at least partly mediated by LTs.

Studies on the ocular pharmacology of prostaglandin D2.

Prostaglandin D2 (PGD2) exerts a variety of biologic actions in the eye; these include ocular hypotension and inflammatory effects on the conjunctiva. The profile of activity of PGD2 in ocular tissues was compared to that of BW 245C, a selective agonist for the PGD2-sensitive (DP) receptor, and to that of the biologically active metabolites of PGD2, 9 alpha,11 beta-prostaglandin F2 (9 alpha,11 beta-PGF2) and prostaglandin J2 (PGJ2). PGD2 produced a dose-dependent decrease in intraocular pressure and in the conjunctiva it caused increased conjunctival microvascular permeability, eosinophil infiltration and goblet cell depletion. Although BW 245C was equipotent to PGD2 as an ocular hypotensive agent, it did not cause pathological effects in the conjunctiva. Thus, the ocular hypotensive effect of PGD2 may be separated from inflammatory effects on the conjunctiva by employing a selective DP-receptor agonist such as BW 245C. 9 alpha,11 beta-PGF2 was a weak ocular hypotensive and did not cause conjunctival inflammation. PGJ2 produced no significant effect on intraocular pressure. PGJ2 did not elicit a microvascular permeability response in the conjunctiva, but was inflammatory in other respects and caused eosinophil infiltration and goblet cell depletion similar to PGD2. Thus, both the ocular hypotensive actions and the conjunctival pathology of PGD2 may be replicated individually by employing PGD2 analogues and metabolites.

Platelet-activating factor causes goblet cell depletion in the conjunctiva.

Platelet-activating factor (PAF) (1-O-hexadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) produced dose-dependent depletion of the goblet cell population associated with the conjunctival epithelium. Reductions in goblet cell numbers did not correspond to leukocyte infiltration and were consistent with a direct effect of PAF. In contrast, LTD4 and LTE4 did not affect the goblet cell population although they caused massive eosinophil infiltration into the conjunctival epithelium. Histamine also produced conjunctival goblet cell depletion, but this appeared secondary to eosinophil degranulation and resultant epithelial desquamation. In addition to goblet cell expulsion, PAF produced an increase in conjunctival microvascular permeability over an identical dose-range. PAF-induced leukocyte emigration was small or absent and comprised a neutrophil infiltrate which exhibited no clear dose-dependent relationship. Lyso-PAF produced effects only at the highest dose employed where pathological changes and a distinct increase in conjunctival microvascular permeability were evident. Lyso-PAF- and PAF-induced increases in conjunctival microvascular permeability were virtually abolished by the PAF antagonist CV-6209. The pronounced inhibitory activity of CV-6209 suggests that high doses of lyso-PAF may either weakly stimulate conjunctival PAF receptors or that there may be sufficient conversion of lyso-PAF to biologically active levels of PAF.

Interactive effects of peptidoleukotrienes and histamine on microvascular permeability and their involvement in experimental cutaneous and conjunctival immediate hypersensitivity.

The involvement of peptidoleukotrienes (LTs) in mediating the increase in microvascular permeability associated with experimental cutaneous immediate hypersensitivity was studied by examining the effect of SK&F 104353, a potent and selective LT-antagonist, on the response evoked by graded, intradermal injections of antigen. SK&F 104353, employed at doses that profoundly blocked LTC4, LTD4 and LTE4 responses, significantly reduced the response produced by experimental cutaneous immediate hypersensitivity. The response to the lowest antigen dose (0.1 microgram) was, however, entirely insusceptible to SK&F 104353. The effect of SK&F 104353 was also examined in combination with a pyrilamine-cimetidine dosing regimen sufficient to remove the histaminergic component of cutaneous immediate hypersensitivity. The non-histaminergic component associated with higher antigen doses (10 and 100 micrograms) was significantly reduced but not abolished by SK&F 104353; the non-histaminergic component associated with low antigen doses (0.1 and 1 microgram) was not susceptible to SK&F 104353. Thus, the increase in cutaneous microvascular permeability evoked by immediate hypersensitivity appears to comprise three components: (1) A histaminergic response apparent for all antigen doses; (2) a LT-mediated component which is manifest in response to high antigen doses; (3) a third, unidentified component that is present for the entire antigen dose-range but contributes less to the overall response when high antigen doses are used. A distinct non-histaminergic, non-leukotriene mediated component was not a feature of conjunctival immediate hypersensitivity. SK&F 104353 administered in combinatio with pyrilamine-cimetidine virtually abolished the response with a small residual remaining only for the highest antigen dose. In further contrast to cutaneous immediate hypersensitivity, SK&F 104353 alone was comparatively ineffective in type 1 allergic conjunctivitis. This difference in susceptibility to SK&F 104353 appears to reflect the type of histamine-LTD4 interactive effect on microvascular permeability. Histamine and LTD4 were additive in terms of cutaneous microvascular permeability. In the conjunctiva, histamine and LTD4 appeared mutually exclusive in that the level of response produced by the combination tended not to exceed that of the single component which caused the greater effect.

Further studies on ocular responses to DP receptor stimulation.

Prostaglandin D2 (PGD2) and the selective DP receptor agonist BW 245C have been previously shown to lower intraocular pressure in rabbits, while PGD2, but not BW 245C, caused plasma extravasation, eosinophil infiltration, and goblet cell depletion. In these present studies definition of the ocular pharmacology of prostaglandin D2 (PGD2) has been extended by using a further selective DP receptor agonist SQ 27986 and a potent and selective DP receptor antagonist BW A868C. In cats and rabbits SQ 27986 caused ocular hypotension. The ocular hypotensive effect of PGD2 in rabbits was blocked by pretreatment with the DP receptor antagonist BW A868C, whereas the activities of PGE2 and PGF2 alpha remained unaltered. The singular involvement of the DP receptor in changes in rabbit intraocular pressure evoked by PGD2 was thereby verified by using the antagonist BW A868C. In terms of effects on the ocular surface, SQ 27986 caused no increase in conjunctival microvascular permeability, no eosinophil infiltration, and no depletion of the goblet cell population. These findings reinforce the concept that selective DP receptor agonists may be useful for lowering intraocular pressure without causing ocular surface pathology. PGD2 induced increases in conjunctival microvascular permeability were inhibited by BW A868C, despite the fact that DP receptor agonists failed to evoke a plasma exudation response. This finding was unexpected and suggests a possible subdivision of the DP receptor designation.

Effects of leukotrienes B4 (LTB4) and D4 (LTD4) on motility of isolated normodense human eosinophils and neutrophils.

The effects of exogenous leukotrienes B4 and E4 (LTB4, LTD4) on the under-agarose motility of isolated normodense human eosinophils and neutrophils were examined using a novel sampling strategy for quantitation of leukocyte migration distance and vectorial orientation. Eosinophil chemotaxis to LTD4 was evident at a 10(-10)M threshold. The selective peptide-LT antagonist, SK&F 104353, abolished LTD4-induced eosinophil migration, indicating pharmacological specificity of the response. Neutrophil chemotaxis was apparent only with a very high (10(-5)M LTD4 concentration. LTB4 was a potent eosinophil and neutrophil chemoattractant over a 10(-9)M to 10(-4)M dose range. Analysis of leukocyte orientations provided evidence that chemokinetic responses were not being interpreted as indications of chemotactic behavior. LTB4 and LTD4 significantly altered neutrophil vectorial orientation. Comparison of migration distance and orientation at the leading edge and at the periphery of the well seeded with cells suggested that cell polarization appeared to be the earliest response to chemoattractive LTs. These results indicate that chemoattractant responses to LTs may be identified by utilizing the under-agarose technique and computer assisted analysis of cell orientation.

Studies on leukotriene D4 as an eosinophil chemoattractant.

The effects of LTD4 on eosinophil motility were studied in order to indicate its potential role as an eosinophil chemoattractant. The guinea pig was selected as a suitably sensitive species for in vivo studies. Eosinophil infiltration was quantified by digital image analysis of 6 microns paraffin ocular and cutaneous tissue sections stained by Luna's method. LTD4, applied topically to the ocular surface, caused pronounced eosinophil infiltration into the conjunctival epithelium and was more potent and efficacious than a variety of other putative mediators of allergy. Pretreatment with the LT-antagonist SK&F 104353 (i.v. 1 mg/kg) abolished LTD4-induced eosinophil infiltration into the conjunctiva. Eosinophil infiltration did not occur in other ocular anterior segments structures such as the cornea, iris and ciliary body after either topical application or intracameral injection. LTD4 did not cause eosinophil emigration into skin following intradermal injection, despite causing an increase in cutaneous microvascular permeability at identical doses. These studies indicate that LTD4 may cause eosinophil emigration in vivo according to tissue-dependent regulation.

Differential effects of alpha-adrenoceptor agonists on human retinal microvessel diameter.

The effects of locally administered brimonidine, clonidine, and p-aminoclonidine on microvessel caliber were compared in human retinal tissues grafted into the hamster cheek pouch. Clonidine and p-aminoclonidine, but not brimonidine, potently constricted human retinal microvessels over a broad concentration range. All three agonists elicited significant vasoconstriction in naive hamster cheek pouch microvasculature. The alpha2-adrenoceptor antagonist, rauwolscine, inhibited p-aminoclonidine-induced constriction in naive hamster cheek pouch microvessels, but not p-aminoclonidine-induced effects in retinal grafts. Selective alpha1-adrenoceptor agonists evoked vasoconstriction in retinal grafts only at relatively high concentrations. These differential effects on the retinal microvasculature could not be readily explained solely on the basis of alpha1- or alpha2-adrenoceptor involvement. Clonidine, p-aminoclonidine and brimonidine are also imidazoline derivatives that interact with putative non-adrenergic imidazoline-sensitive binding sites, the so-called I1-imidazoline binding site subtype implicated by some investigators in mediation of peripheral vasoconstriction. As with p-aminoclonidine, the potent vasoconstriction in human retinal microvasculature elicited by moxonidine, an alpha-adrenergic agonist that has also been reported to exhibit selectivity for putative I1-imidazoline binding sites, was not inhibited by the selective alpha-adrenoceptor antagonist, rauwolscine, nor by idazoxan, an antagonist characterized as having substantial activity at putative I2-imidazoline binding sites. These data suggest the possible involvement of an unconventional non-adrenergic imidazoline-sensitive pathway in regulation of microvascular responses in the inner retina, and that drug activity mediated via such an imidazoline-sensitive component could potentially evoke deleterious effects in the retinal microvasculature.

The pruritogenic and inflammatory effects of prostanoids in the conjunctiva.

The therapeutic utility of cyclooxygenase (CO) inhibitors, such as ketorolac, in reducing the inflammatory events associated with allergic conjunctivitis is not unexpected since prostanoids (PG) elicit conjunctival redness (PGD2, PGE2, PGF2 alpha), edema (PGD2, TxA2), eosinophil infiltration (PGD2, PGJ2) and mucous cell discharge (PGD2, PGJ2, TxA2). Recently, topically administered ketorolac has also been reported to alleviate the itching associated with allergic conjunctivitis. This was viewed as intriguing since CO inhibitors are not regarded as useful for treating itching dermatoses and PGs do not elicit itching when applied to the skin. In order to investigate the antipruritic activity of ketorolac, we developed a model for reproducibly measuring ocular surface itch responses. The model involves itch-scratch responses to pruritogens applied locally to the ocular surface. Painful and foreign body stimuli do not produce an itch-scratch response. Unlike reported skin studies, PGE2 was a potent itch-producing substances in the conjunctiva. PGD2 was weakly pruritogenic but PGF2 alpha and the TxA2-mimetic U-46619 were inactive. The PG precursor arachidonic acid was also a potent pruritogen and its effects were inhibited by ketorolac pretreatment. Ketorolac also dose-dependently inhibited the itching associated with experimental allergic conjunctivitis. It appears that PGs are potent itch-producing substances in the conjunctiva and the anti-itch efficacy of ketorolac in allergic conjunctivitis appears to involve inhibition of conjunctival PG biosynthesis from arachidonic acid.

Topical anti-inflammatory activity of beta 2-adrenoceptor agonists in the conjunctiva.

Increased microvascular permeability is believed to involve the contraction of venular endothelial cells, which results in the formation of leaking gaps in the postcapillary segment of the microvasculature. Several reports have suggested that interendothelial cell gap formation caused by permeability factors may be functionally antagonized by locally administered beta 2-adrenoceptor agonists. This represents a potentially novel approach for reducing inflammatory episodes in a variety of externally accessible tissues. The studies described herein extend these findings to the conjunctiva, where the relatively selective beta 2-adrenoceptor agonists salbutamol and terbutaline reduced the microvascular permeability response to histamine. Similarly, pretreatment with salbutamol or terbutaline inhibited the increase in conjunctival microvascular permeability associated with experimental immediate hypersensitivity, which indicates potential utility for allergic conjunctivitis.

Thromboxane A2-mimetics are potent microvascular permeability factors in the conjunctiva.

These studies demonstrate that the thromboxane (Tx) A2 mimetics U-46619, U-44069 and carbocyclic-TxA2 elicit a microvascular permeability response in the conjunctiva. U-46619 and U-44069 are among the most potent microvascular permeability factors described to date for the conjunctiva; their potency is exceeded only by that reported for leukotrienes D4 and E4. The conjunctival microvascular permeability response to U-46619 was inhibited by the TxA2-antagonists daltroban (BM 13505) and SQ 29548. Prostaglandin (PG) D2 also increased conjunctival microvascular permeability, but was less potent than U-46619 and far less susceptible to pretreatment with daltroban or SQ 29548. PGE2, PGF2 alpha and the prostacyclin analog carbocyclin did not increase conjunctival microvascular permeability. It appears that the conjunctiva exhibits a unique microvascular permeability response to TxA2-mimetics: this view is experimentally supported by the absence of a cutaneous microvascular permeability response to U-46619, U-44069 and carbocyclic-TxA2.

Characterization of receptor subtypes involved in prostanoid-induced conjunctival pruritus and their role in mediating allergic conjunctival itching.

Topically administered ketorolac (Acular), a cyclooxygenase inhibitor, has recently been reported as clinically beneficial for treating allergic conjunctivitis. The ability of ketorolac to relieve the itching associated with allergic conjunctivitis is intriguing because cyclooxygenase inhibitors are not regarded as useful in treating allergic dermatoses and prostaglandins (PG) do not elicit an itch response in human skin. To gain further insight into the mechanisms involved in the antipruritic activity of ketorolac, we used a method of reproducibly assessing ocular surface itch responses in the guinea pig. The measurement of conjunctival pruritus involved a recently developed behavioral model whereby hind limb scratching episodes directed toward the afflicted area were quantified. Itch-scratch episodes have previously been delineated from foreign body and pain sensations, which do not evoke such a behavioral response. Ketorolac significantly inhibited the itching associated with experimental allergic conjunctivitis. The basis of this antipruritic activity may be ascribed to preventing the biosynthesis of itch-producing PGs because ketorolac inhibited arachidonic acid-induced pruritus. In contrast to skin studies, PGE2 and PGI2 were found to be potent pruritogens at the guinea pig ocular surface. PGD2 was a weak pruritogen, and PGF2 alpha and the thromboxane-mimetic U-46619 produced no meaningful response. Further studies involving selective agonists and antagonists suggested that EP1 receptors, IP receptors and PGD2-sensitive receptors may mediate prostanoid-induced conjunctival itching. No evidence for the involvement of other prostanoid receptor subtypes was obtained. Although the EP1 receptor antagonist AH 6809 and the DP receptor antagonist BW A868C inhibited PGE2- and PGD2-induced itching, respectively, neither antagonist alone significantly affected the itching associated with experimental allergic conjunctivitis. A combination of AH 6809 and BW A868C, however, did exhibit antipruritic activity. It appears that for effective relief of itching in allergic conjunctivitis, it is not sufficient to block the effects of a single pruritogenic PG. It is preferable to reduce the participation of all pruritogenic PGs by either using combined receptor antagonists or by using a cyclooxygenase inhibitor such as ketorolac to block their biosynthesis.

Histamine H1- and H2-receptor involvement in eosinophil infiltration and the microvascular changes associated with cutaneous anaphylaxis.

Several substances alter eosinophil motility, but the relative importance of these putative mediators in immediate hypersensitivity remains unclear. The present study has re-investigated the role of histamine in type I allergic eosinophil infiltration, and the temporally associated microvascular events, by examining the effect of H1- and H2-receptor antagonist pretreatment. A combination of cimetidine and pyrilamine significantly reduced eosinophil accumulation, whereas neither antagonist alone was effective. Similarly, cutaneous hyperemia, measured indirectly as ear surface temperature, was reduced only by the cimetidine-pyrilamine combination. Pyrilamine partially attenuated the increase in microvascular permeability, but the addition of cimetidine provided no further reduction. It appears that histamine participates significantly in mediating both the microvascular changes and the eosinophil infiltration evoked by cutaneous anaphylaxis. The histaminergic component of increased microvascular permeability appears to be an H1-receptor mediated phenomenon. However, blockade of both H1- and H2-receptor subtypes is required to inhibit the hyperemia and eosinophil infiltration responses.

Leukotrienes cause eosinophil emigration into conjunctival tissue.

The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.

Synergistic effects of LTB4 and LTD4 on leukocyte emigration into the guinea pig conjunctiva.

Leukotrienes (LT) B4 and D4, alone and in combination, were topically applied to the eyes of guinea pigs, and their effects on conjunctival leukocyte infiltration studied. LTD4 potentiated the neutrophil response to LTB4, even though no neutrophil emigration was evoked by LTD4 itself over a dose range of 10-1000 ng. LTB4 alone at the 1-ng and 10-ng doses failed to evoke any leukocyte emigration, but significant numbers of neutrophils were observed at these concentrations when LTD4 (1-1000 ng) was present. Although a dose-dependent increase in neutrophil infiltration was observed for the 100-ng and 1000-ng doses of LTB4, cell counts were substantially higher with these doses in the presence of LTD4. Eosinophil numbers increased in a dose-related manner in response to LTB4 and LTD4 alone, with a greater response to LTD4. The addition of either 10 ng or 100 ng of LTB4 to graded doses of LTD4 (10-1000 ng) caused increased eosinophil numbers, the lower dose of LTB4 potentiating the response to LTD4 and the higher LTB4 dose showing no significant effect. The effects on leukocyte infiltration that were evoked by the LT combinations could not be explained simply on the basis of an increase in vascular permeability. Bradykinin (BK), a potent conjunctival microvascular permeability factor that does not elicit any leukocyte infiltration, did not significantly potentiate LTB4-induced eosinophil or neutrophil emigration. The synergistic effects of LTs on leukocyte emigration are also difficult to ascribe to hyperemia (ie, increased blood volume in the conjunctiva), because both LTB4 and LTD4 caused only very modest increases in conjunctival blood content, and BK, which did not potentiate the leukocytic responses to LTB4, caused marked increases in tissue blood content. High-dose LT combinations caused eosinophils, but not neutrophils, to migrate into the conjunctival epithelium and fragment, resulting in overt tissue damage. These results further suggest a synergistic interaction between LTB4 and LTD4 that directly alters leukocyte function. The relevance of these observations to a number of disease and trauma states is discussed.