Creticos PS, Peters SP, Adkinson NF Jr, Naclerio RM,. Hayes EC, Norman PS, .... Gary RK, Woodward DF, Nieves AL, Williams LS,. Gleason JG, Wasserman ...
American Journal of Pathology, Vol. 130, No. 2, February 1988 Copyright X American Association of Pathologists
Synergistic Effects ofLTB4 and LTD4 on Leukocyte Emigration Into the Guinea Pig Conjunctiva
CLAYTON S. SPADA, DAVID F. WOODWARD, STEPHEN B. HAWLEY, AMELIA L. NIEVES, LINDA S. WILLIAMS,
From the Pharmacology Department, Discovery Research, Allergan Eye and Skin Care Group, Irvine, California
and BARBARA J. FELDMANN
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 dosedependent 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 LTB, 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. (AmJ Pathol 1988, 130:354368)
THE 5-LIPOXYGENATION of arachidonic acid leads to the formation of leukotrienes (LTs). These lipid compounds are able to reproduce biologic effects which are characteristic of immediate hypersensitivity reactions, including leukocyte chemotaxis,'i3 increased microvascular permeability,4'5 bronchoconstriction, 6-8 and altered mucociliary clearance.9-'0 Secretion of LTs is observed during bronchial asthma episodes' 1.12 and after antigen challenge in patients with allergic rhinitis,'3 and specific allergen provocation produces elevated peptidoleukotriene levels in the tear fluid of patients suffering from allergic conjunctivitis.'4"15 LTs have also been identified in atopic cutaneous inflammatory lesions.'5'17 These findings implicate LTs as important mediators of immediate
Many acute reactions in the bronchopulmonary system,18" 9 ocular tissue,20'21 and skin23'24 are characterized by significant numbers of leukocytes that have infiltrated into inflamed areas. LTB4 can cause reversible adhesion of leukocytes to the endothelium of the postcapillary venules25'26 and induces eosinophil and neutrophil chemotaxis in vitro and in vivo.27-" A recent study has demonstrated that LTD4 and LTE4 can also elicit eosinophil emigration into conjunctival tissues in vivo.3' Both tissue-resident and infiltrating
Accepted for publication September 25, 1987. Address reprint requests to Clayton S. Spada, Pharmacology Department, Discovery Research, Allergan Eye and Skin Care Group, 2525 Dupont Drive, Irvine, CA 92715.
hypersensitivity. 354
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LTB4/LTD4 SYNERGISM IN THE CONJUNCITIVA
leukocytes have the ability to respond to a range of stimuli with the generation and release of LTs into the extracellular milieu. Mast cells,32 macrophages,3334 neutrophils, and eosinophils35-37 have been shown to produce the slow-reacting substance of anaphylaxis (SRS-A), now known to be composed of the sulfidoLTs, LTC4, LTD4, and LTE4.38 These cells also produce LTB4.35,39-42 Thus, LTs appear to be candidates as primary mediators of the cellular phase of the inflammatory process. Given that LTs and various other mediators of inflammation are found in inflammatory exudates, there has been an increased awareness of the likelihood that these substances might also act to augment or offset the effects of one another. There are reports concerning the synergistic actions of LTs and the prostaglandins (PGs) in terms of increased leukocyte adhesion and movement.29,43A45 The common origin and extensive array of biologic activities ofthe LTs led us to investigate whether LTB4 acts in concert with the peptido-LTs in vivo. Conjunctival tissue was chosen for these studies to provide an established and well-characterized model of inflammation in which agents may be introduced topically. The effects of such challenges can be evaluated without interference from mechanical trauma, as may occur with injection.46 The conjunctiva is a readily accessible tissue that manifests inflammatory states that appear relevant to a range of clinically signficant disorders other than ocular diseases. We report here that the numbers of eosinophils and neutrophils that infiltrate into the guinea pig conjunctiva in response to topically administered combination treatments of LTB4 and LTD4 may be considerably greater than that achieved with either LT alone. Moreover, evidence is provided that shows that the synergistic interaction between LTB4 and LTD4 on leukocyte responses is apparently independent of effects on the microvasculature.
pared in distilled water. Bradykinin (BK, Sigma Chemical Company, St. Louis, Mo) was prepared in distilled water. LTB4/LTD4 and LTB4/BK combinations were prepared by mixing the appropriate solutions to result in the correct final working concentrations of each component. All solutions were prepared fresh on a daily basis.
Materials and Methods Animals and Materials All studies utilized conscious, female, Hartley strain, albino guinea pigs weighing 400-600 g. The 5S,l12R LTB4 stereoisomer and the 5S,6R stereoisomer of LTD4 were kindly supplied by J. G. Gleason (Smith Kline & French Medicinal Chemistry, Philadelphia, Pa) and were stored dry under vacuum in liquid nitrogen until use. LT solutions were analyzed by ultraviolet spectroscopy in aqueous solution before preparing serial dilutions in distilled water for topical application. The sodium salt of LTB4 was pre-
355
Quantitation of Leukocyte Infiltration All dose-response studies were performed by topically administering 20 ,ul ofthe test solution to the left eye and 20 ,ul of distilled water to the right eye as a contralateral control. Animals were sacrificed 6 hours after treatment time by intracardiac injection of 0.5 ml T-6 1 Euthanasia Solution (Hoechst, Sommerville, NJ). The histologic and counting techniques that were employed in this study have been previously described.47 Briefly, the eyes with attached eyelid rings were quickly removed and fixed in 10% neutralbuffered formalin for 24 hours at room temperature. Two nonserial 6-u paraffin sections were obtained from each specimen and stained by Luna's technique for eosinophil granules.48 Neutrophils are also easily distinguished with this staining method. Cell counts were taken in the epithelial and subepithelial regions of the conjunctiva under brightfield illumination at X 500 magnification with a Zeiss Photomicroscope III. Results are expressed as the contralateral difference (left eye minus right eye) in mean cell count per high-power field (HPF), ( ± standard error of the mean [SEM]). Although cell counts were obtained only in the conjunctiva, other tissue areas were qualitatively examined for the presence of leukocytes. Representative fields were recorded on Kodak Kodachrome 25 color slide film at an exposure index of 16.
Quantitation of Albumin Extravasation and Tissue Blood Content Twenty microliters of LT or BK solutions were topically applied to the left eye and 20 1 of distilled water were applied to the right eye as contralateral control. Animals were sacrificed by intracardiac injection of 0.5 ml T-6 1 Euthanasia Solution at 15 minutes posttreatment time. Quantitation of tissue blood content and extravascular albumin leakage was performed according to the methods of Woodward and Ledgard.49 Briefly, this method employed 1251-labeled bovine serum albumin and 5'Cr-labeled erythrocytes to enable quantitation of both extravascular albumin content and tissue blood content. Evans blue dye was also used to visualize plasma exudation. The radiola-
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SPADA ET AL
AJP * February 1988
beled markers and the dye were administered intravenously before topical ocular treatment with the test solution. The eyes were enucleated immediately after sacrifice, the bulbar conjunctiva dissected free of the globe at the limbus and trimmed free of extraocular muscle. Tissue samples from each animal were counted in a scintillation counter (Beckman Gamma 8500), along with a l-ml blood sample from the same animal. Tissue samples were then dried in an oven at 50-55 C to constant weight. Results are expressed as the contralateral difference (left eye minus right eye) in milliliters of blood or extravascular albumin per gram dry weight tissue.
Statistical Analyses of Results Statistical analyses were performed by using analysis of variance, the Student t test, and a parallel line assay after the method of Finney."
Results The addition of a fixed 1000-ng dose of LTD4 to graded doses of LTB4 caused a significantly greater neutrophil response than did LTB4 alone at every
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dose selected. Indeed, an increase in cell number in response to LTB4 alone was seen only with the 100-ng and 1000-ng doses, but in the presence of LTD4, the l-ng and 1 0-ng doses evoked a clearly significant neutrophil infiltrate (Figure 1). Neutrophils showed no response whatsoever to LTD4 alone. The LTD4-induced potentiation of the neutrophil response to LTB4 was studied further by examining the 10-ng and 100-ng doses of LTB4 in combination with graded doses of LTD4 over the range of 10-1000 ng. Increasing the dose of LTD4 did not significantly affect the neutrophil response to LTB4, neutrophil numbers being directly related to the fixed dose of LTB4 used (Figure 2). Eosinophil responses evoked by LT combination treatments differed markedly from those of the neutrophil. When used by itself, LTD4 has proved to be more potent than LTB4 in causing a dose-dependent increase in conjunctival eosinophil numbers.3' In the present studies, the low doses of LTB4 (1 ng and 10 ng) did not evoke eosinophil infiltration, but appeared to potentiate the response to 1000 ng LTD4. The LTD4 response, however, was not augmented by the higher doses of LTB4 (100 ng and 100 ng); not even an additive effect was apparent with
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Figure 1-The effect of LTD4 on LTB4Anduced neutrophil infiltration into the guinea pig con-
junctiva. Graded doses of LTB4 were administered in the presence (k-) and absence (U---_) of 1000 ng LTD4. a-Cell counts obtained in the conjunctival epitheb-Counts obtained lium. in the conjunctival subepithelium. Cell counts are expressed as the mean contralateral difference (left eye minus right eye) per single HPF ± SEM. *P < 0.05. **P < 0.01. LTBJLTD4 responses relative to the response to LTB4 alone. n = 6-8
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IL Figure 2-Neutrophil infiltration into the guinea pig conjunctiva when graded doses of LTD4 were administered in the presence of 10 ng LTB4 (u-u)and 100 ng LTB4 (h -). The response to LTD4 alone was also examined for comaparative purposes (v-s). Cell counts obtained in the conjunctival epithelium. b-Counts obtained in the conjunctival subepithelium. Cell counts are expressed as the mean contralateral difference (left eye minus right eye) per single HPF ± SEM. *P < 0.05. **P < 0.01. LTBJ/LTD4 responses relative to the response to LTD4 alone. n = 6-8 animals.
357
LTB4/LTD4 SYNERGISM IN THE CONJUNCTIVA
Vol. 130 * No. 2
EPITHEUAL NEUTROPHILS
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those combination treatments (Figure 3). The synergistic activity between LTB4 and LTD4 in the modulation of eosinophil response was also evident upon comparison of the effects of graded doses of LTD4 in combination with fixed doses of LTB4 (Figure 4). Increasing doses of LTD4 over the range of 10-1000 ng in the presence of 10-ng or 100-ng fixed doses of LTB4 caused eosinophil numbers to increase. Eosinophil infiltration in response to the LT combinations that employed the 10-ng dose of LTB4 was significantly greater than that evoked by LTD4 alone, but the response to combinations containing the l00-ng dose of LTB4 was comparable to that elicited when LTD4 was used by itself. Only the epithelial counts are shown for eosinophils because of the minimal cell numbers present in the subepithelial regions, indicating that eosinophil emigration was directed toward the conjunctival epithelium. In contrast, neutrophil infiltrates were characteristically diffuse and not focused toward any specific region of the conjunctiva. Relatively higher neutrophil numbers were usually found in the subepithelial regions, as compared with the
epithelium. When the concentration of each component in the LTB4/LTD4 mixture was 100 ng or higher, eyes
1
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treated with these combinations exhibited some instances of conjunctival epithelial damage and reduction of goblet cell populations. Damaged areas occurred in patches, which were coincident with the presence of fragmented eosinophils or free eosinophil granules. Light-microscopic examination failed to produce evidence of neutrophil fragmentation or free neutrophil granules. Damage to tissue areas other than the conjunctival epithelium was not found (Figure 5). Tissues in other treatment groups appeared undamaged, even when high numbers of leukocytes were present. Moreover, there were no visual indications of leukocyte fragmentation or free cytoplasmic granules induced by treatment with the separate LT components. Both neutrophils and eosinophils appeared intact in tissues that were treated with LTB4 only (Figure 6) and LTD4 only (Figure 7), regardless of the dose used. Contralateral control eyes in all treatment groups appeared normal, with no significant leukocyte presence nor any signs of tissue damage or depletion of goblet cell populations (Figure 8). Treatment with LT combinations also led to a dramatic increase of leukocyte presence in the anterior chamber, cornea, and iris/ciliary body; leukocyte infiltrations into these tissues did not occur in response
358
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In order to ascertain if leukocyte infiltration was related to effects on conjunctival microvascular permeability, plasma extravasation was examined. Graded doses of LTB4, over the range of 10-100 ng, in the presence of 1000 ng LTD4 caused an increase in conjunctival microvascular permeability, but this increase was not related to the dose of LTB4 (Figure 1Oa). LTB4 by itself had no effect on microvascular permeability. When graded doses of LTD4 over the range of 10-1000 ng were administered concurrently with 100 ng LTB4, the increase in conjunctival microvascular permeability was similar to that evoked by LTD4 alone (Figure lOb). In comparison, BK elicited a dose-related increase in conjunctival extravascular albumin content that was much greater than any LTinduced response (Figure lOc). Extravascular albumin accumulation evoked by treatment with BK, a nonapeptide that is known to increase microvascular permeability, was measured for investigational pur-
1 000
LTB4 (ng) Figure 3-The effect of graded doses of LTB4, administered in the presence (h.and absence (u- --) of 1000 ng LTD4 on eosinophil infiltration into the guinea pig conjunctival epithelium. Subepithelial cell counts are not shown because of the minimal numbers of eosinophils observed in this region. Cell counts are expressed as the mean contralateral difference (left eye minus right eye) per single HPF ± SEM. *P < 0.05 indicates LTB4/LTD4 responses relative to the response to 1000 ng of LTD4 alone (arrow). n = 6-8 animals.
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centrations in LT combination treatments, while corneal penetration by eosinophils appeared dependent on the dose of LTD4 in the LT mixture. Neutrophils were clearly more numerous than eosinophils in all of the observed corneal infiltrates (Figure 9). Both types of leukocytes appeared intact and no damage to the cornea was evident in any treatment group in which a corneal leukocyte infiltrate was elicited. The patterns of leukocyte response were also notably different in the subretinal vascular region comprised by the ora serrata and choroid. The magnitude of eosinophil response in these areas qualitatively paralleled conjunctival eosinophil counts in all treatment groups, and the cells appeared normal. Neutrophils were never observed in these regions.
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Figure 5-The fomix region of conjunctival tissue treated with a 1000 ng LTBJ1 000 ng LTD4 combination. The subepithelial region is extensively infiltrated by neutrophils (N), which show no tendency toward focused emigration, whereas eosinophils (E) migrate to the conjunctival epithelium. Goblet cell (G) size and number are diminished in regions of conjunctival epithelium where fragmented eosinophils (e) and numerous free eosinophil granules (gr) are present, whereas areas containing intact eosinophils are undamaged. Neutrophils that have achieved the epithelium (large arrowheads) are intact. Biood vessles (V), which are filled with erythrocytes, are undamaged. (Luna's eosinophil stain, X160) Figure 6-The fomix region of conjunctival tissue treated with 1000 ng of LTB4. Intact neutrophils (N) are present in the epithelium and subepithelium, while the directional emigration of eosinophils (E) toward the conjunctival epithelium is clearly shown. Eosinophils are intact, and the epithelial layer is normal. Goblet cells (G) are also intact. Marginated neutrophils (large arrowheads) are evident in the microvasculature (V). (Luna's eosinophil stain, X160)
LTB4/LTD4 SYNERGISM IN THE CONJUNCTIVA
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because it did not cause neutrophil or eosinophil infiltration at any dose tested. Measurement of tissue blood content is obtained in the course of calculating extravascular albumin content and affords a convenient method of quantitating the hyperemic response in the tissue. Tissue blood content was therefore examined for investigation of whether the observed leukocyte infiltrations were associated with increased blood volume in conjunctival tissues. The increase in tissue blood content observed in response to graded doses of LTB4 in the presence of 1000 ng of LTD4 was not related to the dose of LTB4, and LTB4 alone did not cause an increase in tissue blood content (Figure 1 la). Graded doses of LTD4 in the presence of 100 ng LTB4 also gave rise to increased conjunctival blood content that was not clearly linked to the dose of LTD4, although LTD4 by itselfcaused a slight dose-related increase in blood content (Figure 1 lb). In contrast, BK evoked a very sharp dose-related increase in conjunctival blood content that was greater than any LT-induced increase (Figure 1 lc). The question remained whether the mechanism by which LTD4 exerted its potentiating effects on LTB4induced leukocyte infiltration could be explained by the increased microvascular permeability and the modest hyperemia evoked by LTD4. The ability of BK to cause increased plasma leakage and hyperemia in the conjunctiva without evoking leukocyte emigration provided an advantageous tool with which to explore this possibility. BK was, therefore, used in place of LTD4 as the microvascular permeability factor in combination with LTB4. Graded doses of LTB4 over the range of 1-1000 ng in the presence of 1000,ug BK did not cause significant enhancement of conjunctival leukocyte infiltration relative to that which was evoked by LTB4 alone (Figure 12). Indeed, in terms of eosinophil numbers, the BK/LTB4 combination involving the 1000 ng dose of LTB4 led to a slight, but significant, decrease (Figure 12c).
infiltration exhibit a clearly significant neutrophil emigration when LTD4 is present. LTD4 is more potent than LTB4 in causing eosinophil infiltration into the conjunctiva. However, low doses of LTB4 that do not themselves cause an eosinophil infiltrate appear to augment the response to LTD4. Higher concentrations of LTB4 do not exert an additive effect with LTD4; a net depression of the overall predicted response was apparent. The emigration of leukocytes into tissues is considered a complex process involving humoral and cellular responses. The modulation of neutrophil and eosinophil emigration observed for LTB4 and LTD4 interactions could arise from the direct effects of these LTs on the leukocytes, their influence on vascular events, or a combination of both. This issue was pursued experimentally. Leukocyte emigration and increased microvascular permeability are major pathobiologic features of the inflammatory process.'512 Moreover, it has been suggested that changes in microvascular permeability may modulate the rate and degree of cellular infiltration into an inflamed site.53" Thus, LTD4, being a microvascular permeability factor in the guinea pig
poses
Discussion We report here that LTD4 and LTB4 can interact in a synergistic fashion to modify in vivo leukocyte responses in the guinea pig conjunctiva. LTD4 potentiates the neutrophil response to LTB4, even though no neutrophil emigration is evoked by LTD4 itself. Low doses of LTB4 that do not evoke any leukocyte
conjunctiva,49,56,57 could exert an influence on leukocyte emigration. In order to ascertain whether LTD4 effects on LTB4-induced neutrophil emigration could be explained by changes in conjunctival microvascular permeability, we employed BK for its ability to cause plasma extravasation in our model without evoking leukocyte infiltration. Over the tested dose range of 10-1000 jg, BK causes a greater increase in conjunctival microvascular permeability than LTD449 but does not induce leukocyte infiltration into the guinea pig conjunctiva. Because BK did not enhance LTB4-induced neutrophil infiltration, it follows that increased microvascular permeability cannot account for the LTD4 influence on the neutrophil response to LTB4. Furthermore, BK causes a far greater increase in conjunctival blood content than do LT combinations, indicating that hyperemia is not a major contributory factor in the mediation of the observed cellular responses. Leukocyte emigration patterns do not appear, therefore, to be a passive consequence of increased blood content in the conjunctiva. Leukocyte adhesion to the inner wall of the vascular endothelium is reported to be a prerequisite for diapedesis into the interstitium.54-58 LTB4 is not only a potent leukocyte chemoattractant but also induces reversible neutrophil adhesion to the vascular endo-
Figure 7-The fomix region of conjunctival tissue treated with 1000 ng LTD4. Many intact eosinophils (E) have achieved the epithelium, which is undamaged. Goblet cells (G) are normal. Note the absence of neutrophils. Marginated eosinophils (arrowheads) can be seen in one blood vessel (V). (Luna's eosinophil stain, X1 60) Figure 8-Representative oontralateral control tissue. No significant neutrophil or eosinophil infiltrates are present. Goblet cells are large and abundant. The particular tissue sample which is represented in this photomicrograph is from the contralateral control eye of an animal which received 1000 ng LTB4 in the left eye. (Luna's eosinophil stain, X100)
362
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a-Plasma extravasation in Figure 10-The effects of LTB4/LTD4 combinations and of BK on conjunctival microvascular permeability in the guinea pig. -) and absence (.b-Plasma extravasation in response to response to graded doses of LTB4 administered in the presence { --i) of 1000 ng LTD4. c-Plasma extravasation in response to graded doses of BK. graded doses of LTD4 in the presence (@-*) and absence (u.- _.-) of 100 ng LTB4. Plasma extravasation is expressed as the mean contralateral difference (left eye minus right eye) in milliliters of extravascular albumin content per gram dry tissue weight ± SEM. *P < 0.05 indicates LTBJLTD4 responses relative to the response to LTB4 alone. n = 6-8 animals.
thelium in vivo,26'59 and influences on margination may partially contribute to synergistic interactions of LTB4 and LTD4. However, the differential nature of responses of eosinophils and neutrophils to the LT combinations suggest direct alterations of leukocyte activity by LTB4/LTD4 interactions. This postulate is also indirectly supported by the failure of LTB4 to affect LTD4-induced increases in conjunctival microvascular permeability, demonstrating that LTB4/ LTD4 synergism is not a generalized phenomenon associated with all pathophysiologic responses. The direct modulation of leukocyte function by multiple mediators resulting from the 5-lipoxygenation of arachidonic acid has been discussed in the literature. O'Flaherty and co-workers have proposed
that some arachidonate derivatives (eg, LTB4) serve as agonists, while others (eg, 5-HETE) might serve as potentiators of cell function.60 Goetzl has suggested that the constituents of SRS-A might regulate certain leukocyte functions through multiple independent effects.6' It is unlikely that the distinct leukocyte responses we report herein are due to competition for the same receptor site by these LTs. Neutrophils possess a subset of stereospecific cell surface receptors with a high affinity for LTB4.62-4 Binding to this receptor site is not affected by LTD4,61'65 implying a separate subset of receptors. Thus, LTB4 and LTD4 are almost certainly acting at independent surface receptor sites, and this could serve to greatly promote leukocyte activity. This supposition is made more at-
Figure 9a-Comea of eye treated with a 1000 ng LTB4/1 000 ng LTD4 combination. Leukocyte infiltrates are typically observed in the anterior portion of the corneal stroma, neutrophils (N) being more numerous than eosinophils (E). This pattem of infiltration results from all treatments that elicit corneal infiltration. There is no evidence of leukocyte degranulation or fragmentation, and the cornea eplium (ep) and endothelium (en) are normal. Leukocytes are rarely observed in the comeal epithelium. b-Representative contralateral control tissue. No neutrophils or eosinophils are present in any part of the cornea. (Luna's eosinophil stain, X100)
364
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Figure 11-The effects of LTB4/LTD4 combinations and of BK on conjunctival blood content in the guinea pig. a-Tissue blood content elicited by graded doses of LTB4 in the presence (*-d)and absence (_---_) of 1000 ng LTD4. b-Tissue blood content elicited by graded doses of LTD4 in the presence c-Tissue blood content elicited by graded doses of BK. Tissue blood content is expressed as the mean (e-*) and absence (v- --) of 100 ng LTB4. contralateral difference (left eye minus right eye) in milliliters per gram dry tissue weight ± SEM. *P < 0.05. **P < 0.01. LTB4/LTD4 responses to the responses for either LTB4 or LTD4 alone. n = 6-8 animals.
tractive when the regional patterns of neutrophil and eosinophil distribution that were observed in areas other than the conjunctiva are taken into consideration. Responses in the anterior chamber, cornea, and iris/ciliary body that were evoked by LT combinations could not be elicited by any dose ofthe separate LT constituents. The decreasing eosinophil counts in response to graded LTB4 doses administered concurrently with 1000 ng LTD4 are difficult to explain in this regard, but it is relevant to note that as neutrophil counts rise, the number of eosinophils tends to fall. This may indicate possible saturation of leukocyte attachment sites in the microvasculature. The regional differences in cell distribution that we observed may also be of relevance in understanding why leukocyte responses can vary according to tissue type. It is interesting in this regard to note the highly directional emigration of eosinophils, but not neutrophils, toward the conjunctival epithelium in response to LT treatments. The unfocused conjunctival neutrophil infiltrates observed in the present study are
similar to those found in ocular inflammatory states that involve neutrophilia.66 In addition, eosinophil fragmentation and free eosinophil granules are evoked specifically at the epithelium by high-dose LT combinations, but not by the separate LTs at any dose. There are also previous reports of eosinophil "targetting" of the conjunctival epithelium in response to topical LTs3' and histamine,47 and during immediate hypersensitivity.47 The latter two conditions induce eosinophil fragmentation confined to the epithelium, although the patterns of occurrence differ markedly.47 The pattern we describe for fragmentation and the presence of free cytoplasmatic granules induced by the LT combinations is similar to that of conjunctival immediate hypersensitivity. Perhaps these cellular consequences are caused by an eosinophil-specific chemoattractant that is produced and released by the conjunctival epithelium in response to treatment. It is noteworthy in this regard to emphasize that the conjunctival epithelium is contiguous with that ofthe cornea; yet no treatment evoked
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Figure 12-The effect of bradykinin on LTB4-induced leukocyte infiltration into the guinea pig conjunctiva. Graded doses of LTB4 were administered in the a and b-Neutrophil counts obtained in the epithelium and subepithelium, respecpresence (h- ^) and absence (s---_-) of 1000 ug bradykinin. tively. c-Eosinophil counts obtained in the epithelium. Subepithelial eosinophil counts are not shown because of the minimal number of eosinophils observed in this region. Cell counts are expressed as the mean contralateral difference (left eye minus right eye) per single HPF ± SEM. *P < 0.05 compares LTB4 responses in the presence and absence of bradykinin. n = 8-8 animals.
eosinophil "targetting" of corneal epithelium. Thus, synergy between LTB4 and LTD4 also exerts profound influences over a number of leukocyte functions that could result in subsequent pathologic consequences. Such a synergistic relationship has broad implications that may extend to a number of disease and trauma states. It is plausible that leukocyte responses during various pathologic conditions are more substantially mediated by the concomitant release of LTB4 and LTD4 than may have been previously appreciated. The LTs, as a class of compounds, can influence a range of cellular and vascular events in a manner that is consistent with a proinflammatory role. LTB4 has traditionally received separate consideration from the sulfidopeptide LTs because of its distinctly different structure and range of biologic activities. However, the ratio of peptide LTs and LTB4 released during a pathophysiologic response could vary according to tissue type. This ratio may be an important factor in determining the characteristics of the leukocytic response. Immediate hypersensitivity
reactions in conjunctival and airway tissues are characterized by extensive mucus secretions which contain high levels of intact and degraded eosinophils,21'6769 and elevated levels of peptide LTs are found in effusates from tissues undergoing these allergic reactions.1,14,15,70,71 Neutrophil accumulation is an important histologic event in psoriasis and ulcerative colitis. Lesions resulting from these diseases contain high levels of LTB4 as well as peptide LTs.7'-74 Leukocytes may also accumulate in tissue areas that have been traumatized by chemical, mechanical, or temperature effects. Corneal trauma results in altered arachidonic acid metabolism and increased levels of 5-lipoxygenase products,75'76 and neutrophils can enter the corneal stroma within hours of stimulation.77 Increased generation of sulfidopeptide LTs follows thermal or mechanical trauma in the rat.75 The results of our in vivo studies in the guinea pig conjunctiva indicate that LTB4 may synergize with LTD4, a peptide LT, to affect eosinophil and neutrophil movement and function in a manner that is independent of vascular events. Moreover, this interac-
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tion suggests that LTs possess other properties that may be of relevance across a broad spectrum of tissue-specific responses to insult. Our findings provide a further indication that specific LT antagonists may be beneficial in preventing or alleviating several pathologic events that characterize a number of clinically pertinent conditions.
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Acknowledgments We wish to thank Dr. J. G. Gleason (Smith Kline & French Medicinal Chemistry, Philadelphia) for kindly supplying LTB4 and LTD4.
