575 TJI’S - October 2987 [Vol. 81

Tepler, I., Wallace, R. and Leder, P. 3698-3701 (1987) Cell 49,465-475 23 Steinman, G., Delorme, E. O., Law,

20 Ochuchi, N., Thor, A., Page, D. L., Hand, P. H., Halter, S. and Schlom,. J. w86j Cancer Wes. &5,2SlI-2Sb

21 Liderau. R.. Escot. C.. Thiilet. C.. Cham- pome, M. I?., B&et, M., Best, J. and Callahan, R. 0986) 1. Nutl Cancer Inst. 77,699-701

22 Greig, R. G., Koestler, T. I’., Trainer, D. L., Corwin, 5. I’., Miles, L., Kline, T., Sweet, R., Yokoyama, S. and Poste, G. (1985) Proe. Nuff Acud. Sci. USA 82,

Gelmann, E. P. and Lippman, M. E. (1987) Proc. Nutl Acad. Sci. USA 84,837- 841 Green, S., Walter, P., I&mar, V., Krost, A., Bioriezt, J. M., Argos, P. and Cht- bon, P. (1986) Nutare 320, Xi&l39 _ __.~

C. C. and Sager, R. (1987) Prac. Nutl Acad. Sci. USA 84,164-188 27

24 Kasid, A., Lippmti, M. E., Papageorge, A. G., Lowy, D. R. and Gehnann, E. P. (1985) Science 228,72S-728 28 Peres, R., Betshohz, C., Westermark, 8.

25 Albini, A., Graf, J. 0.. Kitten, T., Wein- and Heldin, C H. (1987) Cancer Res. 47, man, H. K., Martin, G. R, Veillette, A. 3425-3429 and Lippman, M. E. (1986) Proc. Nat1 29 Bronzert, D., Pantazis, P., Antonrades, Acud. Sci. USA 03,818243186 H. N., Rasid. A., Davidson, N., Dickson,

26 Dickson, R. B., Kasid, A., Huff, K. K., Bates, S., Knabbe, C.. Bronzert, D..

R.B.~d~ppm~M.E.~c.NatlAcud. Sci. USA (in press)

Ocular ~~~a~ion: a phmacobgical model

Intraocular inflammation such as uveitis, glaucoma and loss of integrity of the ocular surface are potentially blinding conditions. Also, vascularization of the cor- nea, or the retina, can lead to severe visual loss. It is therefore vital that several ocular models of infl~ation, which are essenti- ally painless, be developed not only to study mechanism, but also to find specific therapeutic agents that control the inflammation.

Ocular inflammation, like in- flammation in other areas of the body, is a complex process invol- ving the actions and interactions of several chemical mediators on ocular as well as non-ocular tis- sues and cells in the initiation, maintenance and termination of the inflammatory process. In order to understand the whole cas- cade of inflammatory responses (vasodilation, hyperemia, protein exudation, edema, chemokinesis and chemotaxis of leukocytes and other inflammatory cells) and the role of each chemical mediator in this process, it is essential to study the mechanism of inflammation in viva

The eye is an unique organ con- sisting of vascular tissues (con- junctiva, iris ciliary body, retina) and avascular tissues (lens, cornea and vitreous). Nutrients are pro- vided to the avascular tissues by the aqueous humor which is selectively secreted by the ciliary processes. Aqueous humor con- tains relatively little protein. The epithelial layer, the outermost layer of the cornea, is nourished by the tears and peripheral vascu- lar conjuctiva.

The four experimentdl ocular inflammatory models described below are generahy used to inves- tigate various aspects of the actions of steroidal and non- steroidal anti-inflammatory agents. Each model allows different characteristics of the test drug to be examined.

migration of adjacent conjunctival epithelium within 4-6 d. Migrated conjunctival epithelial cells resid- ing over the comeal surface become comeal epithelium with- in a couple of months. Anti- inflammatory corticosteroids in- hibit migration of conjunctival epithelium over the cornea while non-steroidal, aspirin-like drugs do not interfere with re- epitheli~i~tionl~.

Cornea1 wounding The cornea, although avascular,

is surrounded by vascular con- junctiva. The epithelial surface is continuous with conjunctival epithe~um. When the cornea1 sur- face is de-epithelialized by any of several methods, such as mech- anical scraping (scalpel blade or dental drill) or chemical cautery, a characteristic inflammatory reac- tion follows. This inflammatory reaction occurs even when only the central cornea is de-epithelial- ized. Polymorphonuclear leuco- cytes (PMNs) migrate from the peripheral conjunctiva across the conjunctival epithelial cell layer into the tear fluid. By the process of blinking, these PMNs are moved on to the comeal surface. PMNs also migrate from the conjunctiva through the cornea1 stroma to the site of injury but this is a much dower process, limited by migra- tion through stromal tissue. The peak of the tear fluid PMN res- ponse is reached within !5-6 h. However, PMN migration through the cornea1 stroma ma continue well beyond this time 1,x .

After initial total cornea1 de-

Repeated de-epithelialization of the cornea produces neovascular- ization (angiogenesis), i.e. migra- tion of conjunctival blood vessels over the avascular cornea. Corti- costeroids inhibit angiogenesis in the comeal wound model’. Thus, the comeal wound model can be used to investigate not only the underlying mechanism of inflam- mation, but also wound healing and angiogenesis in vim?.

Paracentesis

epithelialization using a scalpel blade or dental drill, the re- epithelialization of the injured cornea1 surface is completed by

Normally, the aqueous humor contains little protein. The com- position of the aqueous is rigidly maintained by selective secretory processes which are located in the iris-ci!iasy body. Paracentesis of the anterior chamber means the removal of aqueous humor from the anterior chamber of the eye. When aqueous humor is removed by means of a 30-gauge needle, the collapse of the anterior cham- ber traumatizes tissues of the anterior segment, especially the iris-ciliary body. After the first paracentesis, the anterior chamber is usually re-formed with aqueous humor within 30 min. However, the protein content of the aqueous humor is si~i~c~dy higher than normal because of the breakdown of blood-aqueous barrier, i.e. pro- tein exudation from the iris- ciliary body blood vessels into the aqueous humor. This response lasts up to 4-6 h following the first paracentesis. This type of infiam- mation is considered to be an acute type of inflammation and

@ 1987, Elewier PublicaWns, Cambridge 0165 - 6147/87/102.00

Cornea1 Wounding

Neovasculariza~ion

Corneol Surface

Fg. t. A schematicdiagram of various ocutarinflammatory models: (a) anterior portion of the eye showing wmeal epithet&m, wnjunctiva, anterior uvea, lens, and anterior chamber. EIore that wmeal ep~e~um is denuded strait; (b) 5-6 hours fo/~~~ wmeat deepitheliatization (6 mm centtatty) and thus cornea1 wounding, PMNs arrive on tha surface of the wrnea from aaacent conjunctival vasculature; (c) anterior portion of the eya showing e~e~cha~~ f&d with aqueous hu~r. Arrow indicates r~vat of aqueous humor by a 30.gauge needle: (d) after removal of aqueous humor - paracentasis- anterior chamber collapses (wrnea flattens) causing acute trauma to the @sues in the ante&r chamber; (e) 30 mih after the first removat of aqueous humor, anterior chamber is filted again with aqueous humor. However, because of the trauma, breakdown of the btwd-aqueous banter (reiaase of protein into the anterior chamber] awrS; (f) endotmin is injected into the poste&r portion (into vitreous) of the eye. (g) W-24 hours following endotoxin, PMNs and protein are released into anterior as well as postedor podion of the eye causing chronic in~mmad~ - Escherichia coli ~oto~in Ureitis; (h) repeated wmeat da-epithetiaiization results in invagination of Mood vessels on the (avasculattzed) cornea/ surface and naovascularization.

the unique feature of this model is that PM% are not found in the aqueous4.

Endotoxiu-induced uveitis ShigeBa or Escke~c~ia coli

endotoxin injected into the pos- terior cavity of the eye (intravit- real injection) produces chronic intraocular inflammation of the anterior as well as posterior por- tions of the eyes. In this type of i~mation, both the break- down of the blood-aqueous and blood-a-itmous barrier (increase in protein content of the aqueous and vitreous humor) and release

of PMN intcl the aqueous and vit- reous humor occur. Increase in protein content of the inflamed media otcrdrs within 6 h following intravitreal endotoxin injection. The peakof this response is reached at 24 h and lasts up to 72-96 h period. PMN release into the aqueous occurs within 14-24 h and lasts up to 72-96 h.

Bovine serum albumin (BSA)- induced uveitis BSA-induced uveitis is an im-

munogenic ocular infl~mation, Intravitreal injection of BSA pro- duces ocular inflammation within

TIPS - October 1987 IVol. 81

9-15 d and it completely subsides within a month. Following the recovery of the first in~ammato~ response, BSA is again inject- ed systemically. The sensitized eye develops inflammation within &6 h and this response reaches a peak at 24 h. Inflammation lasts up to 3-4 d. The predominant features of this model are conjuctival and iridial hyperemia, breakdown of the blood-aqueous barrier and PMN entry into the anterior cham- berss6.

Determination of intraocular inflammatory response

Inflammatory responses of the anterior chamber can be visual- ized in viva by split lamp examina- tion. Conjunctival and iridial hyperemia, a rise in the protein concentration (flare) and the entry of inflammatory cells can be eval- uated on a qualitative basis. Such clinical signs can be followed until inflammation has completely subsided. However, quantitative analyses require removal of aque- ous and vitreous humor to measure inflammatory media- tor(s) and protein content, and to count PMNs.

cl cl q

In the cornea1 wound model, the tear-PMN response is acute and can be studied for up to 5-6 h. In the paracentesis model, break- down of the blood-aqueous bar- rier occurs, but PMN infil~ation into the aqueous is absent. In the endotoxin- and BSA-induced uveitis model, both responses are predominant, but the BSA- induced uveitis model is im- munogenic. Thus, because of the different nature of these in- flammatory responses, the char- acteristics of mediator(s) of differ- ent inflammatmy responses can be studied. Also, by using ster- oidal and non-steroidail anti- inflammatory drugs in these models, the mechanism of drug action can be elucidated. Thus, the eye can be used: (1) to induce different types of inflammation to explore the involvement of various mediators’, and some or all ocular inflammatory responses can be studied by the application (topical, intracameral or intra- vitreal) of drugs and/or mediators to the normal eyesp9; (2) to investi- gate the mechanism of action of anti-inflammatory and as well as anti-infectious drugs3*6’8; and (3)

5 Bib, L. Z. 0977) Science 196, &65

6 Kulkarni, P. S., Bhattachrtjee, P., Eakins. K. E. and Sriniwsan, 8. D. (198lj curr. Eye Res. 1,49-17

7 Srinivasan. 8. D. and Kutkami, P.S. (19ao) fnvest OphfhniQmttl” Visual Sci. 19, 1087-1093

9 Kulkarni, P. 5. and Srinivasan, B. D. (1981) Erp. Eye Res. 33, 121-229

Phnrnracology~ E. S. Harktress Eie hifrcte, Columbia University, rS.30 West 168th Street, New York. NY 10032, U.5A.

Reference9 I Srinivasan, 8. D. (1982) ‘I’rurts. Am

OpfIfb~f~of. Sot. LXXX, 7594322

2 Srinivasan, B. D., Kulkami, P.S. and Eakins, Kc. E. !19B% in Adzwrtcs itr &es- tnglanditr nttd Thrombomne Rerearch (Vol. 71 (%mueisson, B., Ramwell, P, W. and Paolcttl, R., eds), pp. (161..851, _ _. Raven Press

3 Srinivasan, B. D. and Kuikami, P.S. 9 Bhattacherjee, P., Hammcmd, 8.. Sat- (1981) btT.JeSt. O~~i~~J~~Q~O~ b%Wd Sri. %,

mi4i91

mon, I., Stepney, ft. and Eakins, K. E. fW?!) Eur. f. F~~r~~~~~. 73,21-2%

4 Eakins, K. E. (1976) in prostQ~~#fld;~s: IO Sr~~~vasan, B. D. and Kulkami, P.S.

Pfrysiologi~Ql md Pkarmaco~o~ical and WJSlj Arch. Ophfbafnrol. 49, lot%-1089

Pafhologicaf Aspecfs (Karim, S. M. M., 11 Kulkarni, P. and Srinivasan, B. D. (1986) ed.), pp. 63-82. University Park Press I. Orrrl. Pharm-<of. 2, 171-175

AU antagonistdagonists are hkdy to be pdypharmic

their own

Dr Williams’ letter2 that classify- ing receptors* is not an easy task merits comment. I am glad we agre@ that the system far enzyme classification is not applicable to the classification of receptors. However, I do not agree that the classificatian of receptors presents such a formidable task, certainly not when compared with many other classifications, e.g. of species, languages, diseases, the physical status af patients, all of which can be regarded as success- ful taxonomies because they have gained use.

Receptors can be defined in objective, quantihtive, non-over- lapping terms with the use of pure chemicals, The chemicals, preferably antagonists, yield a grouping of characteristics, a set of rows and columns, with the ~ufo~at~on clustered according to the conjunction crf features that farm distinctive entitieP, ‘a con- silience of inductions’* as in other taxonomies, but here with repro- ducible number9 i.e. Kd values or EC& values. There are, as Dr Wil- liams mentioned, complications3 but not overwhelming ones or else we would net be able to talk and write of (Y, p, HI, etc, It needs to be noted that despite the complica- tions, so simple a technique as high affinity binding heralded receptors, Le. entities that were shown to be associated with functions3.

Dr Williams is correct in assert- ing that a classification system based on the I(a values of ‘poly- pharmic’ antagonist9 such as spiper- one cannot be readily extrapolated to processes in zliuo, i.e. lacking other evidence, the pharmaco- logical effects of spiperone cannot be attributed to blockade of a specific receptor, whether of dop- amine or of other substances. This stricture does not vitiate the use of polypharmic antagonists to class- ify receptors,

A receptor is defined by an array of & values of competitive antag- onists (like spi~rone) obtained by measuring the change in response to a specific agonist, e.g. 5-HT in vitro. Respect of certain cautions3 yields unequivocal results, testimony to which are lists of r<, values of antagonists for different receptors, If an antagon- ist, e.g. spiperone, has non- disting-lishable Kd values for more than one receptor of a single agonist, say 5HT, then that antag- onist would be of limited or no value in defining 5HT receptors. But its blockade of dopamine and histamine H2 receptor9 and other receptors, though of great ccm- sequence in understanding its pharmacological effects in V~PO, need not deter its use to classify receptors irr u&w. Obtaining & values in ~iz10, which is very difficult5, would not totally solve the problem of attributing drug

effects ia vi730 fo specific receptors. The ~oly~h~~~ effects present

problems, only when one neglects lo recognize (or to anticipate) that: the antagonist or agonist has affinities for more than one recep- tor, a neglect that could lead to false attribution. As Dr Williatns states, spiperone is best known as a dopamine antagonist, probably because that was its first observed effect. Additional work is needed to learn which of its pharmaco- iogical effects in viva rests on blockade of which receptor at what region of the brain, and what conflation and/or concatena- tion of effects results in its thera- peutic effect.

It must be assumed that all agonists and antagonists are ~oIypharmi~ until proved other- wise. It may be impossible to name a drug that in sufficient dose does not affect systems other than the one for which it is desig- nated. The presumption that a drug has an affinity for one recep- tor that somehow excludes affini- ties for other receptor9 seems to arise from the category it is placed in on discovery and frequently by its therapeutic use. Often the effect(s) in viva of the drug is then imputed to blockade of that one receptor and, consequently, that receptor is inferred to be associ- ated with the pharmacological effect. This series of events is best exemplified by the histamine HI antagonists because of their long history, Almost imm~iately these drugs became available, they were observed to have antimuscarinic effectsh’ powerful enough for some of them to be proscribed in patients with glaucoma or urinary retention’, To observation9 on their aut~mu~arini~ effects was adde,? clear evidence that many of them ijnve affinities for r@CeptOrS

of glutamate, aspartake, for the