Eosinophil Chemotaxis and Anterior Uveitis from Topical Dimoprit and Nordimoprit

Eosinophil Chemotaxis and Anterior Uveitis From Topical Dimoprit and Nordimoprit

Joel S. Mindel*tt Alan H. Friedmaat Tali Haimov,f Alex D. Kharlamb,*t and Jonathan M. Freilichj-

Topical application of the H2-histamine receptor agonist, dimaprit (S-[4-N,N-dimethylaminopropyl]isothiourea), produced eosinophil chemotaxis into the anterior segment of rabbit eyes only when an

H2-antagonist was co-administered. Nordimaprit (S-[4-N,N-dimethylaminoethyl]isothiourea), a structural homologue of dimaprit that lacked activity at histamine receptors, produced eosinophil chemotaxis

whether or not an H2-antagonist was co-administered. Onset of eosinophil chemotaxis began after 2 or more days of treatment, and was accompanied by corneal edema, opacifkation, and ocular inflammation. There was no concurrent eosinophilia in the peripheral blood or in the conjunctiva. The response occurred in pigmented and albino rabbit eyes, and was facilitated by prior co-administration of proparacaine eye drops. Another dimaprit homologue without activity at histamine receptors, homodimaprit (S-[4-N,N- dimethylaminobutyl]isothiourea), did not produce eosinophil chemotaxis when applied topically, nor did

the H2-agonists impromidine, histamine, or 4-methylhistamine, whether co-administered with an H2- antagonist or not. It was concluded that dimaprit and nordimaprit produced a selective eosinophil che-

motaxis unrelated to H,- and H2-histamine receptor activity. However, the H2-agonist activity of dimaprit appeared to inhibit this response unless neutralized by an H2-antagonist. Topical application of dimaprit with an H2-antagonist or nordimaprit alone may allow large numbers of non-degranulated eosinophils, free of other cell types, to be harvested from the aqueous humor. Invest Ophthalmol Vis Sci 27:1504- 1511,1986

Histamine is both an Hr and H2-receptor agonist. histamine produced random eosinophil migration Drugs that are more specific for each of these receptors (chemokinesis) and reduced true chemotaxis. Jones and have been synthesized. For example, dimaprit,1 im- Kay13 reported that an initial exposure to histamine 2 3 promidine, and 4-methylhistamine are primarily H2- was chemotactic, but that repeated histamine exposure agonists, while cimetidine4 and tiotidine5 are primarily prevented this effect. This apparent self-deactivation H2-antagonists. In addition, chemical homologues of might have been the result of histaminase release by these drugs have been made that lack significant ago- eosinophils.14 The limited evidence in the literature nist or antagonist activity at histamine receptors,6 e.g., supports the view that eosinophil movement, be it che- nordimaprit and homodimaprit (Fig. 1). Use of these motaxis or chemokinesis, is the result of stimulation I12 15 various agents has allowed identification of Hr and of H2-receptors.' Clark et al found that, with regard H2-receptors at a variety of sites. For example, H2-re- to migration stimuli, eosinophils behaved as a heter- ceptors have been identified in the uterus, heart, and ogeneous population. They speculated that this might on the ocular surface.7'8 be due to variations in the relative frequencies of H,- The chemotactic effect of histamine on eosinophils and H2-receptors, or to the existence of a third type of 9 10 is a subject of controversy. Clark et al and Turnbull histamine receptor, i.e., an H3-receptor. believed that histamine was selectively chemotactic for During preliminary studies on the intraocular pres- eosinophils. However, Litt" reported that histamine- sure effects from the topical application of a combi- induced eosinophil movement was a non-specific effect nation of an H2-antagonist, cimetidine, and an H2-ag- caused by its acidity. Wadee et al12 also found that onist, dimaprit, an acute inflammatory response was noted. The anterior segment of rabbit eyes developed From the ""Ophthalmology Section, Bronx Veterans Hospital, New a selective eosinophil infiltration. It was accompanied York, and the Departments of fOphthalmology and ^Pharmacology by miosis, corneal edema, and a variable amount of of the Mount Sinai School of Medicine, New York, New York. iris depigmentation. The present paper describes the Supported by the Medical Research Service of the Veterans Ad- investigations of this response. ministration, NEI Core Center Grant EY-01867, and the Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York. Materials and Methods Submitted for publication: January 29, 1986. Reprint requests: Joel S. Mindel, MD, PhD, Department of Oph- thalmology, Annenberg 22-14, Mount Sinai School of Medicine, One Dutch Belted rabbits, i.e., pigmented rabbits, were Gustave L. Levy Place, New York, NY 10029. used, except for one experiment with albino rabbits.

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Animals were maintained in compliance with the DIMAPRIT ARVO Resolution on the Use of Animals in Research. Cimetidine, dimaprit hydrochloride, nordimaprit

dihydrochloride, 4-methylhistamine dihydrochloride, NORDIMAPRIT histamine dihydrochloride, homodimaprit, and thiourea were 0.21 M unless so stated. Their pH's were

5.2, except for thiourea, which was 7.3. Solutions were 2V /3 HOMODIMAPRIT JC-S-CH2-CH2-CH2-CH2—N' made up every 2-3 days. Impromidine was supplied Hfr CH3 as a 3 mM, pH 4.5 solution by the manufacturer; it was used as such and also at dilutions of 0.3, 0.6, 1.2, CH3 CH2—CH2—NH2 and 1.8 mM. Tiotidine solubility was limited, and a 4-METHYLHISTAMINE Np=(? 0.01 M, pH 5.6 solution was used, as it was close to saturation. A proparacaine HC1, 0.5% commercial preparation provided local anesthesia and was used at CH3v IMPROMIDINE times to promote corneal penetration of the other drugs. When proparacaine was administered, it was

always the first eye drop given. When both an H2-an- CH3, CIMETIDINE J—CH2—S—CH2—CH2—NH—C—NHCH3 tagonist and an H2-agonist were administered, the for- N NCN mer was given before the latter. Drugs were administered as 50 n\ eye drops or injected subconjunctivally or intravitreally in 50 /il volumes. Eye drops were given TIOTIDINE ))—£-CH2-S-CH2-CH2-NH-C-NHCH3 by holding the lids away from the globe and opposed NCN for several seconds to prevent overflow. Topical ap- plications of different drugs were separated by at least Fig. 1. Structures of the histamine H2-receptor agonists, dimaprit, impromidine, and 4-methylhistamine; the H -receptor antagonists, 5 min. 2 cimetidine and tiotidine; and the H2-receptor inactive homologues The eosinophil uveitis was evaluated by observing of dimaprit, nordimaprit and homodimaprit. for miosis, iris depigmentation, and corneal clouding. Histologically, the response to all drug treatments was evaluated using light microscopy with hematoxylin- toxicity of impromidine, only one eye of each rabbit eosin stains and Luna eosinophil granule stain.16 Dif- was treated when this drug was used. If uveitis occurred, ferential white cell counts of Giemsa stained peripheral the eye drops were continued for an additional 48 hr blood smears were performed before and at the con- to intensify the reaction, and then the rabbit was killed. clusion of all drug treatments. Rabbits were killed in Each of three albino rabbits received proparacaine a CO gas tank or by intravenous pentobarbital. + cimetidine + dimaprit to the right eye and propar- 2 acaine + dimaprit to the left eye, three times a day until 48 hr after at least one eye developed an inflam- Intraocular Pressure matory response. The rabbit was then killed. Five rabbits had their intraocular pressures measured 4 times a day, while the right eye received: a) days 1- Table 1. Topical drugs producing anterior 7, dimaprit HC1, 0.1 M (2.5%), twice a day; b) days 7- segment eosinophil chemotaxis 14, dimaprit HC1, 0.21 M (5%), twice a day; and c) days 14-25, cimetidine and dimaprit HC1, both 0.21 Number Eyes Response Onset M and both twice a day. The left eyes received NaCl Treated Responding (Days) eye drops of matching pH and tonicity. Intraocular pressures were measured using proparacaine topical H2-antagonist activity Proparacaine + cimetidine anesthesia and a pneumatonometer. + nordimaprit 5 5 2-3

H2-antagonist and H2-agonist Eosinophil Chemotaxis activities Tiotidine + dimaprit 4 1 7 Eye drops were applied three times a day for 12 or Proparacaine + cimetidine + dimaprit 6 6 3-11 more consecutive days in the various combinations Proparacaine + tiotidine shown in Tables 1 and 2. One eye would receive a drug + dimaprit 4 4 3-4

or drug combination without proparacaine, and the No H2 activity contralateral eye would receive the same drug or drug Nordimaprit 5 5 5-10 combination with proparacaine. Due to the systemic Proparacaine + nordimaprit 5 5 2-3

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Table 2. Topical drugs not producing anterior ministered to the right eye. On the fifteenth day, the segment eosinophil chemotaxis animals were killed, and their eyes and blood smears were examined microscopically. Number Days Eyes Treated Comment Intravitreal drugs were injected once in both eyes of eight rabbits anesthetized with pentobarbital. Both eyes H2-agonist activity received the same drugs using 25-gauge needles inserted Dimaprit 4 12 Impromidine, 3 mM 4 1-2 All died through the pars plana: rabbit 1, cimetidine; rabbits 2 4-Methylhistamine 5 14 and 3, dimaprit HC1; rabbit 4, impromidine; rabbit 5, Proparacaine + dimaprit 4 21 tiotidine; rabbit 6, dimaprit + cimetidine in the same Proparacaine + impromidine, 3mM 8 1-2 All died 50 /il injection; and rabbits 7 and 8, dimaprit and ci- Proparacaine + histamine 5 14 metidine in two separate, but simultaneous, 50 /il in- Proparacaine + 4- jections to each eye. methylhistamine 5 14

H2-antagonist activity In Vitro Depigmentation Cimetidine 5 14 Tiotidine 4 14 Iris-ciliary bodies were removed from rabbits im- Proparacaine + cimetidine 5 21 Proparacaine + tiotidine 4 21 mediately after death. Each tissue was divided into four Proparacaine + cimetidine approximately equal-sized parts. One of the four parts + homodimaprit 5 14 was incubated in each of the following for 3 hr at 37°C:

H2-antagonist and H2-agonist dimaprit HC1, 5%; cimetidine, 5%; dimaprit, 2.5%-ci- activities metidine, 2.5%; or saline, 1.2%. Tissues were then ob- Cimetidine + dimaprit 4 12 Proparacaine + cimetidine served grossly and by light microscopy for disruption + impromidine, 3 mM 8 1-2 All died of the pigment-containing cells. Proparacaine + cimetidine + impromidine, 1.8 mM 4 14 Proparacaine + cimetidine Results + impromidine, 1.2 mM 4 14 2 of 4 died Proparacaine + cimetidine In the preliminary studies, dimaprit HC1, 2.5% and + impromidine, 0.6 mM 4 14 5.0%, had no effect on intraocular pressure in pig- Proparacaine + cimetidine mented rabbits. The combination of dimaprit HC1 5% + impromidine, 0.3 mM 2 14 Proparacaine + cimetidine and cimetidine 5.3% also had no effect on intraocular + histamine 5 14 pressure, but did produce miosis followed within 24 Proparacaine + cimetidine hr by a variable amount of iris depigmentation and + 4-methylhistamine 5 14 corneal clouding (Fig. 2). Light microscopy of hema- No H2-activity toxylin- and eosin-stained tissue slides revealed edema Thiourea 4 14 Proparacaine 3 21 and marked eosinophil infiltration of the cornea, iris, Proparacaine + homodimaprit 5 14 and ciliary body (Fig. 3). The aqueous humor was filled with eosinophils (Fig. 4). Luna-stained tissue slides confirmed that the cells were eosinophils (Fig. 5). The Time Course Study conjunctiva, although edematous and hyperemic, was not infiltrated with eosinophils. There was marked loss Ten rabbits were treated bilaterally with propara- of iris stromal pigment. The iris pigment epithelium caine + cimetidine + dimaprit eye drops three times was affected, but less so. The ciliary bodies were less a day. Two rabbits each were killed after 1, 2, and 4 depigmented than the irides, but also demonstrated days of treatment. The four remaining rabbits were some loss of pigment epithelium as well as disruption treated until eosinophil uveitis occurred in at least one of the non-pigmented epithelium. This in vivo pigment eye, and then the eye drops were discontinued. Four cell disruption could not be reproduced in vitro by and eight days later, two rabbits each were killed. The incubating the iris and ciliary body for 3 hr in solutions eyes of all rabbits were removed for histological ex- of dimaprit, cimetidine, or a cimetidine-dimaprit amination. combination. Pigmented rabbits were given eye drops of H2-ago- Subconjunctival and Intravitreal Drug Injection nists and antagonists, alone or in combination (Tables Five rabbits received daily subconjunctival injections 1, 2). Dimaprit alone or with proparacaine did not for 14 consecutive days. After applying one eye drop produce a response. Nor did other H2-agonists and an- of proparacaine to each eye, a temporal subconjunctival tagonists, alone or in various combinations. Only when injection of dimaprit was administered bilaterally, and an H2-antagonist (cimetidine or tiotidine) was admin- a nasal subconjunctival injection of cimetidine was ad- istered with the H2-agonist dimaprit was eosinophil

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Fig. 2. Eosinophil anterior uveitis. A, Miosis, iris depigmentation, and corneal clouding produced by proparacaine + cimetidine + dimaprit eye drops three times a day. B, ContraJateral eye which received proparacaine + cimetidine. Photographs were obtained 3 days after discontinuing the drops to allow sufficient corneal clearing for visualization of the iris of the responding eye.

uveitis produced. Prior administration or proparacaine proparacaine, or with an H2-antagonist. However, appeared to facilitate the development of eosinophil homodimaprit was without effect (Table 2). The ra- uveitis from an H2-antagonist + dimaprit combination, pidity of the onset of the response to nordimaprit was e.g., only one of four eyes developed eosinophil uveitis facilitated by the prior administration of proparacaine when proparacaine was not used, while nine of nine or an H2-antagonist. Nordimaprit, like dimaprit, pro- eyes developed eosinophil uveitis when proparacaine duced severe corneal opacification. There were several was used. differences in the reactions from these two drugs. First, Time course studies demonstrated that the marked the eosinophil-filled aqueous humor produced by the eosinophil infiltration of the anterior segment was not topical H2-antagonist + dimaprit treatments did not present until the miosis occurred that heralded the on- contain fibrinous material, while all combinations of set of the intraocular inflammation. At the time of the topical nordimaprit produced a marked fibrinoid re- onset of miosis, the inflammatory cells were approxi- sponse (Fig. 6). Second, nordimaprit did not cause iris mately half eosinophils and half neutrophils. Shortly depigmentation by gross inspection. However, on mi- thereafter, the response intensified and became almost croscopic examination, no intact iris stromal mela- purely eosinophilic. When treatment was discontinued nocytes could be identified in nordimaprit-treated eyes, after eliciting the full response, the eosinophils disap- although the iris pigment epithelial melanocytes were peared over the next few days. By the fourth day, few intact. Third, nordimaprit produced either a transient could be identified; grossly, the corneal clouding had miosis, lasting less than 24 hr, followed by a marked nearly resolved, and the iris remained depigmented. mydriasis, or directly produced a marked mydriasis. At no time did the peripheral blood smears demon- Subconjunctival dimaprit, with or without cimeti- strate eosinophilia. Differential cell counts for eosin- dine, failed to produce a marked eosinophil infiltration. ophils remained at 2-3% before, during, and after Beginning on the third to fifth day of treatment, there treatment. was a localized area of iris depigmentation superiorly Albino rabbits responded in a similar manner, except in both eyes of all five rabbits. However, this did not that iris depigmentation was not possible. The right progress, nor did corneal opacification or miosis occur. eyes, treated with proparacaine + cimetidine + di- Histologic examination revealed some mild fibrin de- maprit, developed eosinophil anterior uveitis in 3-8 position in the anterior chambers with an occasional days, while the left eyes, treated with proparacaine eosinophil present. + dimaprit, did not. There was no eosinophilia of the The single intravitreal injections of the ^-antago- peripheral blood. nists, cimetidine and tiotidine, alone or in combination Topical administration of the two dimaprit homo- with the H2-agonist, dimaprit, failed to produce an in- logues without H2-receptor activity produced differing flammatory reaction of any type. Eyes were examined, results. Nordimaprit produced an intense eosinophil histologically 4 days after drug administration, except uveitis (Table 1), whether administered alone, with for rabbits 3 and 8 which were examined 8 days after

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Fig. 3. Microscopic appearance of the edema and eosinophil infiltration produced by proparacaine + cimetidine + dimaprit eye drops in the comea (top, left) and iris (top, right). (He- matoxylin-eosin, XI60). Fig. 4. Microscopic appearance of eosinophil accumulation in aqueous humor in response to topical proparacaine + cimetidine + dimaprit. (second row, left) Adjacent to cornea, (second row, right) adjacent to iris. (Hematoxylin-eosin, X80). Fig. 5. Microscopic appearance of eosinophils in aqueous humor in response to topical proparacaine + cimetidine + dimaprit. The cells are intact and do not appear to have degranulated. (third row, left) Hema- toxylin-eosin stain, (third row, right) Luna stain, which gives eosinophil-granules a rust brown appearance. (X400). Fig. 6. (bottom) Microscopic appearance of the fibrinoid material and eosinophils in the aqueous humor in response to topical nordimaprit. (Hematoxylin-eosin, X160).

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treatment. When the H2-agonist, dimaprit, was injected Intravitreal dimaprit alone did produce a marked re- alone, it produced a marked posterior uveitis, vitritis, sponse in three of four eyes, but the many eosinophils and retinitis in three of four eyes within 48 hr. This were outnumbered by even larger accumulations of response consisted primarily of neutrophil cells with a neutrophils. The reasons for these differences between relative eosinophilia of approximately 15-30%. topical and injected dimaprit were not clear. The con- Impromidine, 3 mM was lethal within 48 hr when junctiva did not accumulate eosinophils during topical applied topically or injected intravitreally. Preceding or subconjunctival drug application. Thus, the eosin- each impromidine eye drop (an H2-agonist) with a ci- ophil response differed from a naturally occurring im- metidine eye drop (an H2-antagonist) did not prevent mediate hypersensitivity reaction, and suggested that or delay this lethality. The eyes were not inflamed, ei- corneal or intraocular drug metabolism played a role. ther by gross or histological examination. In the 12 hr Dimaprit was S-[4-N,N-dimethylaminopropyl]- preceding death, the rabbits appeared normal, but ex- isothiourea (Fig. 1). Nordimaprit had one less carbon hibited labored breathing. Autopsy revealed bilateral atom and lacked activity at histamine receptors. Yet, massive pulmonary edema and marked brain edema nordimaprit produced a marked eosinophil chemotaxis with herniation of the cerebellum. Lower concentra- under all conditions of topical administration; i.e., tions of impromidine, 0.3-1.8 mM, were not as toxic, alone, with H2-antagonists, and with proparacaine. and were successfully given for a 2-week period as eye Dimaprit's structural similarity to nordimaprit ap- drops (Table 2) with and without cimetidine. These peared to be the cause of its chemotactic effect, because eyes did not exhibit ocular inflammation or eosinophil other H2-agonists, e.g., impromidine, 4-methylhista- infiltration by gross inspection or on histologic ex- mine, and histamine, whether given alone, with an amination. H2-antagonist, or with proparacaine, were ineffective. Homodimaprit (S-[4-N,N-dimethylaminobutyl]iso- Discussion thiourea) and thiourea were also ineffective. The mechanism by which dimaprit and nordimaprit exerted The eosinophil chemotactic effect of topical dimaprit their actions was unknown. However, both of these appeared to be augmented by two factors. First was drugs have been shown to affect another white cell, the the presence of an H2-antagonist which, presumably, lymphocyte, in a manner suggesting that histamine re- neutralized dimaprit's H2-agonist activity. For example, ceptors were not involved. Dimaprit profoundly in- dimaprit and proparacaine + dimaprit did not elicit a hibited mitogen-induced lymphocyte activation,17"19 response, while tiotidine + dimaprit, proparacaine and its action was not diminished by H2-receptor an- + cimetidine + dimaprit, and proparacaine + tiotidine tagonists. Further, nordimaprit was even more effective + dimaprit produced a marked, and essentially pure, than dimaprit. Perhaps, in the present studies, dimaprit eosinophil inflammation. Second was the use of topical and nordimaprit produced eosinophil chemotaxis in- proparacaine which, presumably, enhanced corneal directly. N-methyl-p-methoxyphenethylamine form- penetration of the H2-antagonist + dimaprit. Thus, (1) aldehyde condensation product caused rabbit con- cimetidine + dimaprit was ineffective, while propara- junctival mast cells to degranulate and produced a lo- caine + cimetidine + dimaprit produced a marked re- calized eosinophilia.20 While many mast cells are found sponse, (2) only one of four eyes responded to tiotidine in the conjunctiva, a smaller number is found intra- + dimaprit while all four eyes responded to propara- ocular.21-22 Mast cells contain an eosinophil chemo- caine + tiotidine + dimaprit, and (3) the eye receiving tactic factor. If dimaprit or nordimaprit caused intra- proparacaine always responded before the contralateral ocular mast cells to degranulate, an eosinophil migra- eye that did not receive proparacaine. This last was tion could result. true for nordimaprit as well as H2-antagonist + di- The inflammatory responses evoked by topical nor- maprit combinations. dimaprit and H2-antagonist + dimaprit combinations Based on the effectiveness of proparacaine, it was were severe. The anterior and posterior chambers had assumed that subconjunctival and intravitreal injec- the histologic appearance of an eosinophil abscess, and tions, by bypassing the corneal epithelium barrier, eosinophils infiltrated the surrounding structures. Both would produce eosinophil chemotaxis more rapidly, albino and pigmented rabbits responded similarly. In and with less effort than using the same drugs as eye the past, rabbit neutrophils have been confused with drops. That this did not occur was unexpected. Two eosinophils, because the former have a cytoplasmic weeks of daily subconjunctival injections of dimaprit granule that stains red. Hence, these neutrophils have or cimetidine + dimaprit, each preceded by a topical been termed "pseudo-eosinophils."23 However, with drop of proparacaine anesthesia, failed to produce a this in mind, it was relatively easy to distinguish the significant response in any of the eyes treated. Intrav- larger eosinophils and their larger red granules. In ad- itreal cimetidine + dimaprit was also without effect. dition, the identity of the eosinophils was confirmed

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using Luna stain,16 which is specific for eosinophil eosinophils into the aqueous humor of rabbit eyes in granules. 2-4 days. These combinations may offer a new, and

While topical H2-antagonist + dimaprit combina- possibly superior, method for obtaining eosinophils. tions and nordimaprit produced similar inflammatory In summary, topical nordimaprit and dimaprit pro- responses with regard to cell type and corneal opaci- duced eosinophil chemotaxis into the eye. The eosin- fication, there were several differences. One was that ophils in the anterior and posterior chambers appeared miosis was a conspicuous component of the H2-antag- intact, i.e., they had not degranulated. The common onist + dimaprit reactions, while nordimaprit-treated structural characteristics of dimaprit and nordimaprit rabbits developed a marked mydriasis. The second dif- may lead to new insights into the mechanism of eosin- ference was that H2-antagonists + dimaprit caused ophil chemotaxis. In addition, these drugs may allow more disruption of iris pigment epithelium cells than large numbers of intact eosinophils to be harvested did nordimaprit. This pigment cell disruption did not from the aqueous humor for related studies. appear to be a direct result of drug-pigment cell inter- action, because incubating the iris and ciliary body with Key words: eosinophil, histamine, chemotaxis, dimaprit, nordimaprit dimaprit or cimetidine + dimaprit did not produce it. Third, nordimaprit elicted a marked fibrinoid response Acknowledgments in the aqueous humor. The reasons for these differences remain to be elucidated. Perhaps topical H2-antagonists Smith Kline and French Research Limited, Department + dimaprit released substances from eosinophils that of Pharmacology, Welwyn, Hertfordshire, United Kingdom, provided the cimetidine, dimaprit, nordimaprit, homodi- nordimaprit did not. For example, eosinophils can re- 14 maprit, 4-methylhistamine, and impromidine. Stuart Phar- lease anti-inflammatory agents, such as histaminase maceuticals, Wilmington, Delaware, provided the tiotidine. 24 and a substance that inhibits histamine release. Eo- Valerie L. Carter of NYU Medical School performed the sinophil granules also contain a basic polypeptide that intraocular pressure measurements. stimulates histamine release from basophils25'26 and enhances inflammation. This basic polypeptide com- References prises more than 50% of the granule protein in guinea 1. Durant GJ, Ganellin CR, and Parsons ME: Structure-activity 2728 pigs, and has a high arginine content. Dimaprit considerations, Part II. Agents and Actions 7:39, 1977. bears a closer structural relationship to arginine than 2. Durant GJ, Duncan WAM, Ganellin CR, Parsons ME, Blake- does nordimaprit. Histamine released from basophils more RC, and Rasmussen AC: Impromidine (SKF 92676) is a not only could elicit an inflammatory response itself, very potent and specific agonist for histamine H2 receptors. Na- ture 276:403, 1978. but it could react with H preceptor sites on eosinophils 3. Barker LA and Hough LB: Selectivity of 4-methylhistamine at

to cause them to release toxic superoxide anions as H,- and H2-receptors in the guinea-pig isolated ileum. Br J Phar- well.29 The resultant inflammation could produce the macol 80:65, 1983. miosis and pigment-cell disruption found following the 4. Brimblecomb RW, Duncan WAM, Durant GJ, Emmett JC, Ga- application of H -antagonist + dimaprit combinations. nellin CR, and Parsons ME: Cimetidine, a nonthiourea H2-re- 2 ceptor antagonist. J Int Med Res 3:86, 1975. Impromidine 3 mM was uniformly lethal within 48 5. Yellin TO, Buck SH, Gilman DJ, Jones DJ, and Wardleworth JM: ICI 125,211: a potent new antisecretory drug acting on his- hr. Prior administration of the H2-antagonist cimeti- tamine H2 receptors. Pharmacologist 21:266, 1979. dine was of no prophylactic value. Rabbits appeared 6. Ganellin CR: Chemistry and structure-activity relationships of to suffer cardiovascular collapse with marked pulmo- drugs acting at histamine receptors. In Pharmacology of Hista- nary and brain edema. Such a picture was consistent mine Receptors, Ganellin CR and Parsons ME, editors. Wright, with so-called histamine shock.30-31 PSG, 1982, pp. 101-102. In the past, studies of eosinophil chemotaxis and 7. Black JW, Duncan WAM, Durant CJ, Ganellin CR, and Parsons EM: Definition and antagonism of histamine H2 receptors. Na- degranulation have been hampered by the difficulty in ture 236:385, 1972.

obtaining large numbers of intact, i.e., non-degranu- 8. Abelson MB and Udell IJ: H2 receptors in the human ocular lated and non-fragmented, cells. One source has been surface. Arch Ophthalmol 99:302, 1981. heparinized blood, which must be washed, centrifuged, 9. Clark RAF, Gallin JI, and Kaplan AP: The selective eosinophil and passed through density gradients to achieve rela- chemotactic activity of histamine. J Exp Med 142:1462, 1975. 10. Turnbull LW, Evans DP, and Kay AB: Human eosinophils acidic tively small and variably pure yields, i.e., 64-98% of tetrapeptides (ECF-A) and histamine. Immunology 32:57, 1977. 32 33 the cells are eosinophils. ' A second source has been 11. Litt M: Studies in experimental eosinophilia: XI. Dependence peritoneal cavity washings from animals that have re- of eosinophilia, apparently induced by histamine, on acidity. ceived horse serum intraperitoneal injections for 4-6 Intl Archs Allergy Appl Immun 50:473, 1976. weeks.34 These eosinophils also require centrifugation 12. Wadee AA, Anderson R, and Sher R: In vitro effects of histamine 35 on eosinophil migration. Intl Archs Allergy Appl Immun 63: for purification. Topical proparacaine + nordimaprit 322, 1980. or proparacaine + tiotidine + dimaprit consistantly 13. Jones DG and Kay AB: Chemotactic activity of guinea pig eo- and selectively drew large numbers of apparently intact sinophils for the ECF-A acid tetrapeptides, histamine, histamine

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metabolites, and the effect of the H, and H2-receptor antagonists. 25. Zheutlin LM, Ackerman SJ, Gleich GJ, and Thomas LL: Stim- Int Archs Allergy Appl Immun 55:277, 1977. ulation of basophil and rat mast cell histamine release by eosin- 14. Zeiger RS and Colten HR: Histaminase release from human ophil granule-derived cationic proteins. J Immunol 133:2180, eosinophils. J Immunol 118:540, 1977. 1984. 15. Clark RAF, Sandier JA, Gallin JI, and Kaplan AP: Histamine 26. O'Donnell MC, Ackerman SJ, Gleich GJ, and Thomas LL: Ac- modulation of eosinophil migration. J Immunol 118:137, 1977. tivation of basophil and mast cell histamine release by eosinophil 16. Luna LG: Manual of Histologic Staining Methods of the Armed granule major basic protein. J Exp Med 157:1981, 1983. Forces Institute of Pathology. New York, McGraw Hill Book 27. Gleich GJ, Loegering DA, and Maldonado JE: Identification of Co, 1968, pp. 111-112. a major basic protein from guinea pig eosinophil granules. J Exp 17. Wickers MR, Arlington SA, Jones C, Martin D, and Melvin Med 137:1459, 1973.

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