Eye (1989) 3, 167-171

Cellular Response to Inflammation at the Limbus

GILBERT SMOLIN

San Francisco

Summary The role in ocular (limbal) inflammation of the ric.h vascular and lymphatic supply is discussed in detail. The distribution of lymphocytes, mast cells, Langerhans cells, macrophages and other cellular elements are discussed as well as their individual and collective response to inflammation whether due to infectious or non-infectious disease states. Among the non-infectious causes of Iimbal cellular inflammation are the four types of hypersensitivity response.

Inflammation results in the dilation of blood serum and blood elements, namely, C-reac­ vessels, the leakage of fluid into the extra­ tive protein and immunoglobulins, mac­ vascular spaces, and the migration of leuko­ rophages, neutrophilic polymorphonuclear cytes from the blood stream into these spaces. leukocytes, and lymphocytes, into the extra­ Specific chemical substances are frequently vascular spaces. released during inflammation. These sub­ Lymphatics are also present in the limbus. stances tend to heighten or perpetuate the The bulbar conjunctival lymphatics begin at response. the limbus in a series of arcades. There are The vascular component of inflammationis two interconnected plexuses (superficial and the most important because the external eye deep) that drain toward the palpebral com­ (i.e. limbus) is so richly supplied with vessels, missures, where they join with the lymphatics many of them grossly visible in tissues such as of the lids. Antigenic material and lympho­ the conjunctiva. cytes pass through these channels, ultimately The muscular branches of the ophthalmic reaching the draining lymph nodes and/or artery with their anterior ciliary branches central reticuloendothelial organs, where form the rich blood supply of the limbus.! The immunologic responses are initiated. arterial channels are composed of arterioles The conjunctiva has a rich supply of immu­ and end in a complex capillary network at the noglobulins A, G and M (IgA, IgG, and IgM), limbus. The conjunctival vessels form a super­ probably derived from the rich vascular ficialmarginal plexus which branches and as it supply and abundant plasma cells. progresses toward the limbus forms the termi­ The vasculature may also be the source of nal peripheral corneal arcades and recurrent the complement, properdin, and properdin vessels which run posteriorly to supply factor that are present in the tear film.2.3 3-6 mm of the perilimbal area. Immune com­ The sequence of vascular events in inflam­ plexes deposit in the extravascular spaces mation consists of preliminary vasoconstric­ around these vessels. tion, then arteriolar dilation, oedema Ocular inflammation first dilates the blood formation, blood cell aggregation, sticking of vessels and then allows for the leakage of blood cells to the vascular endothelium, and

Correspondence to: Gilbert Smolin MD, Francis I Proctor Foundation, University of California Medical Center, San Francisco, California 94143, USA. 168 GILBERT SMOLIN eventually, stasis. The minute vessels are not genase pathway to hydroxyeicosatetraenoic subject to neuronal regulation, but they are acid (HETE) or leukotrienes. instead affected by chemical substances that The mediators released by the mast cell are liberated from cellular stores or formed de may be preformed, secondarily formed (gen­ novo (i.e. leukotrienes, histamine, and erated by the interaction of primary mediators prostaglandins (PG». The larger vessels are and nearby cells and tissues), or granule subject to neurogenic mechanisms of vasomo­ matrix derived (which are preformed but do tor activity. not readily dissociate from the granule after Most inflammatory reactions are biphasic, discharge). The preformed mediators are released into the tissue immediately. These that is, there is an immediate response that include histamine, eosinophil chemotactic reaches its maximum eight to ten minutes factors of anaphylaxis, neutrophil chemotac­ after the application of a noxious stimulus. tic factor, superoxide anions, and This is followed by a levelling-off period of kininogenase. The kininogenase results in the variable duration, during which homeostasis formation of kinins, which can alter vascular can be achieved. This, in turn, is followed by a permeability dramatically. late phase of vessel dilation and increased vas­ Several of the secondary mediators are cular permeability that unfolds over a period formed immediately (PG) or within minutes of hours and culminates in the infiltration of (leukotrienes) and, unlike histamine, may last the tissue with blood elements (i.e. leuko­ for hours. The secondary mediators have cytes, C-reactive protein, complement). effects that enhance inflammation by The first phase is mediated by histamine or (1) increasing cyclic GMP (PF Gz and Fza), histamine-like substances and the second (2) causing chemotaxis of eosinophils and phase by the kinins and prostaglandins. neutrophils (HETE, PG 1z, and leuko­ The submucosal tissue of the conjunctiva triene B4), contains mast cells. In response to an allergen, (3) causing platelet aggregation (thrombox­ injury, or other appropriate stimuli, these ane Az), cells release histamine, as well as a variety of (4) increasing mucus secretion (PG Fza, Eb Iz, other substances that can cause blood vessel Dz and Az, HETE, and leukotrienes C dilation and increased vascular permeability and D) noted above by interacting with the HI recep­ (5) augmenting mediator release (PG Fza and tors present on the ocular surface. Stimulation HETE) of the HI receptor may also result in pruritis, (6) increasing vascular permeability (PG Ez PG generation, and an increase in cyclic and leukotrienes C, D, and E), and guanosine monophosphate (GMP). The (7) causing vasodilation (PG Ez and leuko­ latter causes enhanced release of the phar­ trienes C, D, and E).5 macologic mediators from the mast cells or circulating basophils. Hz receptor stimulation The inflammatory reaction is regulated by increases mucus secretion (a feature common substances that neutralise the effect of the to atopic ocular disease) and cyclic adenosine above mediators (i.e. peroxidases, monophosphate (AMP) production, which in arylsulfatases, and certain prostaglandins) turn decreases the release of pharmacologic and that elevate cyclic AMP, which in turn mediators from the mast cells and basophils. stabilises the mast cell membrane (certain Stimulation of either H receptor can result in prostaglandins, histamine). PGE may act as a vasodilation. negative feedback inhibitor of T-cell pro­ The activation and degranulation of mast liferation, lymphokine production, mac­ cells require Ca +. The serine esterase in the rophage activity, and natural killer cell cell wall is eventually broken down to cytotoxicity. 6 lysophosphatidyl choline and arachidonic Of the various stimuli that can produce acid. The arachidonic acid is subsequently inflammationin the limbus those that activate broken down by the cyclooxygenase pathway complement probably have the most far­ to PF and thromboxanes or by the lipoxy- reaching effects. This complex substance, CELLULAR RESPONSE TO INFLAMMATION AT THE LIMBUS 169

present in the external ocular tissue, can distinct areas. The epithelium over these become bound to antigen-antibody com­ CALT sites shows elongated microvilli with plexes, to simple aggregates of foreign pro­ few microplicae. The underlying lymphoid teins, and to certain toxic substances. The nodules are multiple and packed with lympho­ union of complement with these substances cytes. Antigens are preferentially processed triggers enzymatic reactions that actually at these sites. cause the formation of holes in cellular mem­ In addition to the active role of the com­ branes and the release of chemotactants ponents noted above in causing ocular inflam­ (attracting neutrophils) and anaphylatoxins. mation, the limbal tissue may be an innocent The integrity of the vascular endothelium may bystander for the deposition of immune com­ become permanently altered (which attract plexes. The anatomy of the vasculature at the eosinophils, basophils, and neutrophils). The corneal limbus may account for the deposition polymorphonuclear leukocytes attracted to of these complexes, which in turn results in this area can release a variety of proteolytic the binding of complement, the attraction of enzymes that cause tissue necrosis. polymorphonuclear leukocytes, and sub­ When damaged, the conjunctival epi­ sequent inflammation. thelium can release a thymocyte-activating The cellular response of the limbus can vary factor (ETAF), which can attract poly­ quantitatively and qualitatively depending morphonuclear leukocytes, fibroblasts, and upon the stimulus. Infectious and noninfec­ lymphocytes. This substance has interleukin­ tious processes may influence this response. I-like properties.s In general, nongranulomatous bacterial, fun­ Another interesting cell type found in the gal and parasitic infections attract neu­ limbal epithelium are the Langerhans cells or trophils; granulomatous bacterial infections dendritic cells (D cells). They play a major attract epithelioid cells, lymphocytes, mac­ role in the processing of antigen presented via rophages and possibly giant cells; and viral the epithelial surface and they carry unique infections a mononuclear response (unless histocompatibility antigens (Ia) that are of necrosis is extensive enough to attract neu­ importance in stimulating T and B lympho­ trophils). Noninfectious, nonimmune cytes.9 These cells bind antigens and carry aetiologies of limbal disease include toxic and them by way of the lymphatics to the draining degenerative processes. Immunologic lymph nodes, which leads to host sensitisa­ responses at the limbus can be divided in the tion.to They also stimulate helper T cells and B four classical hypersensitivity types. In the cells to participate in humoral immune type 1-anaphylactic response the com­ responses.ll The stimulated T and B lympho­ monest ocular manifestations occur with the cytes in the regional lymph nodes then migr­ hay fever type of anaphylactic reaction. There ate via the blood stream to the ocular adnexa. is rapid vascular congestion and conjunctival The T cells tend to home to submucosal sites chemosis. The conjunctiva looks milky or pale in the conjunctiva, whereas B cells home to pink. All these signs are the sequelae of the the lacrimal gland and accessory lacrimal release of the vasoactive amines. gland epithelia.12 The B cells can produce In the vernal or atopic type of keratocon­ immunoglobulins at these sites, particularly junctivitis a mononuclear cell infiltrate can IgA.13 occur at the limbus. The presence of lympho­ In different diseases, different populations cytes and macrophages suggest that this type of T cells can be seen (i.e. helper cells, sup­ of reaction has a slightly different pressor cells) in the conjunctival submucosal pathogenesis. tissueY A papillary limbal response in vernal' or Foreign substances can be processed locally atopy is the result of marked hyperplasia of by the musocal immune defence system. The connective tissue and of inflammatoryinfiltra­ tissue associated with this system is called the tion (with eosinophils, mast cells, basophils, mucosal-associated lymphoid tissue (MALT). lymphocytes, and macrophages pre­ In the adnexa, the conjunctival-associated domination). lymphoid system (CALT) is in histologically Limbal nodules may be single or multiple. 170 GILBERT SMOLIN

They appear as small, semitransparent, Herbert's peripheral pits of trachoma, which smooth, gelatinous elevations, usually in the are larger and permanent. interpalpebral fissure. Their corneal edge is Type II-Cytotoxic Response is the one sharp but their conjunctival edge blends grad­ that participates in the immune protection of ually with the normal tissue. Their colour may the host from pyogenic bacteria, transplanted vary from grayish white to pink depending tissue, and tumour cells. The action of the upon the degree of vascularisation. These lim­ antigen on the foreign cell, combined with bal masses may continue to grow and coalesce antibody (IgG or IgM), causes the death of until they completely surround the cornea. the affected cell by bringing about its contact Occasionally they extend centrally-with with phagocytes or killer cells. Phagocytosis is from minimal to extensive corneal invasion. 17 accomplished by a reduction in surface Thickening, broadening, and opacification charge; opsonic adherence directly through a at the upper limbus may extend onto the cor­ portion of the antibody, the phagocytes nea as a semitransparent hood. Peripheral adhering to antibody without the assistance of vascularisation of this area may extend into complement; or immune adherence through the cornea as a micropannus, rarely as a gross bound C3b, the phagocytes adhering to anti­ pannus. The pannus eventually clears, leaving body with the assistance of complement. The ghost vessels and a few gray opacities. Occa­ activation of complement occasionally occurs sionally the gelatinous limbal nodules extend and results in cell death. centrally to cover the entire cornea, and in a Some examples of these cytotoxic reactions few cases the corneal nodules have not been are organ transplantation reaction (in part), connected to the limbus. many drug reactions, Mooren's ulcer, and The limbal white points (Horner's points or mucous membrane pemphigoid. Trantas' dots) present in vernal or atopy are In Mooren's ulceration plasma cells are small, grayish white to whitish yellow dots abundant at the limbus.18 appearing singly or in large numbers, usually In cicatricial pemphigoid, as well as in bull­ at the upper limbus. They are sometimes seen ous pemphigoid of the skin, the basement on the bulbar conjunctiva and semilunar membrane of the epithelium is under attack folds, however, and very rarely on the tarsal by autoimmune processes. By the use of conjunctiva. They are present in about 69 per immunofluorescenttechniques, complement, cent of the cases of mixed vernal keratocon­ IgG, IgM, and IgA have all been found to be junctivitis (VKC) in 41 per cent of limbal localised in the basement membrane zone. VKC, and in 21 per cent of palpebral VKC. Neutrophils are attracted to these sites. They usually last for as long as a week. Path­ In the Type III-Immune Complex ologically they are small cysts filled with Response the injured cells or tissue are inno­ eosinophils, granules, and epithelial cells cent bystanders and do not possess the anti­ undergoing rapid degeneration. They are the genic determinants that can combine with the tip of the iceberg whose bulk lies in the deeper antibody that caused the injury. The antibody layers of the epithelium. reacts with its antigen, and the resulting In the limbal form of VKC, cysts and min­ immune complex binds complement, attracts ute marginal 'pits' also occur. The cysts are PMNs and then directly or indirectly injures ovoid, closely packed in some areas, contain tissues that are in the vicinity. The result can clear, colourless fluid, and appear to be ele­ be catarrhal ulceration; or peripheral corneal vated. They are true cysts formed by the infiltratesin a variety of conditions, e.g. rheu­ apposition of two epithelial layers between matoid arthritis, polyarteritis nodosa. The papillae and they contain mucin as well as distribution of the injury to the tissue is deter­ epithelial and inflammatory cells. The margi­ mined by the sites where the immune complex nal 'pits' appear as transparent, round to oval, form or are deposited (i.e. limbus). glassy spots in the opaque limbus. They are The mechanism of immune complex­ not depressed and represent areas in which induced injury is complicated. The size of the the limbal infiltration has almost returned to complex is important: Large complexes are normal. They are not to be confused with the easily phagocytised by the reticuloendothelial CELLULAR RESPONSE TO INFLAMMATION AT THE LIMBUS 171 system and do not localise in tissues; small electrophoresis. Br J Ophthalmol1975, 59: 279- complexes persist in the circulation but may 83. 3 Smolin G and O'Connor GR: 'Ocular Immunology', be too small to localise in tissues. It is the Boston, Little, Brown and Co, 1987, p.76. 4 11: intermediate sizes that localise in tissues, but Abelson MB and Udell H2-receptors in the the vascular permeability must increase human ocular surface. Arch Ophthalmol1981, 99: 302-5. before they can localise in the extravascular 5 Marom Z and Casale TB: Mast cells and their media­ areas, e.g. the limbus. The vasoactive amines tors. Ann Allergy1983, 50: 367-70. may play a crucial role in increasing this per­ 6 Goodwin JS and Ceuppers J: Regulation of the immune response by prostaglandins. J Clin meability, and platelets, complement, Immunol1983, 3: 295-9. anaphylatoxins, and mast cell or basophils 7 Gamble CN, Aronson SB, Brescia FB: Experi­ combined with IgE may also be sources of mental uveitis. Arch Ophthalmol1970, 84: 321- these vasoactive amines. 30. ( 8 Grabner G, Luger TA, Smolin G, Openheim JJ: Previous damage nonimmunologic or Corneal epithelial cell-derived thymocyte activity immunologic) to the blood vessels can also factor. Invest Ophthalmol Vis Sci 1982, 23: 757- increase their permeability and pave the way 63. 9 Gillette TE, Chandler JW, Greiner JV: Langerhans for the subsequent deposition of immune of the ocular surface. Ophthalmology 1982, 89: complexes in the surrounding tissues (the 700-10. Auer reaction). 10 Chandler JW and Gillette TE: Immunologic defense The Type IV-Cell-Mediated Immune mechanisms of the ocular surface. Ophthal­ 89: (CMI) response is due entirely to the activity mology1982, 700-10. 11 Silberberg-Sinakin E, Baer RL, Thorbecke GJ: of thymus-dependent lymphocytes. Langerhans cells: A review of their nature with The lymphocyte responds primarily to fixed emphasis on their immunologic functions. Prog rather than soluble antigens, participating in Allergy1978, 24: 268-94. reactions to many bacteria, viruses, fungi, 12 Chodirker WB and Thomasi TB Jr: Gamma globulins: Quantitative relationships in human tissue allografts, and neoplasms. There are serum and nonvascular fluids. Science 1963, 142: two types of this delayed hypersensitivity 1080-1. response. The two types are the classic, tuber­ 13 Jackson EE, Lally ET, Nakamura MC, Montgomery culin type in which T cells and macrophages PC: Migration of IgA bearing lymphocytes into 63: participate; and the cutaneous, basophilic salivary glands. Cell Immunol1981, 203-9. 14 Bhan AK, Fujikawa L and Foster CS: T-cell subsets type previously called the Jones-Mote and Langerhans cells in normal and diseased con­ reaction. junctiva. Am J Ophthalmol1982, 94: 205-12. The CMI response plays a role in protecting 15 Chandler JW and Axelrod AJ: Conjuncitiva-associ­ against infections and tumours and in destroy­ ated lymphoid tissue. In O'Connor GR (ed.), 'Immunologic Disease of the Mucous Mem­ ing transplanted tissue. branes'. New York: Masson, 1980, pp.63-70. 16 Bienenstock J, Johnson N, Perey DTE: Bronchial lymphoid tissue 1. Morphologic characteristics. References Lab Invest1973, 28: 686-92. 1 Wolff E: 'Anatomy of the Eye and Orbit'. Phila­ 17 Hogan MJ: Atopic keratoconjunctivitis. Am J delphia: Saunders WB, 1961, pp.379-381. Ophthalmol1953, 36: 937. 2 Bluestone R: Lacrimal immunoglobulins and comp- 18 Brown SL: Mooren's ulcer. Br J Ophthalmol1975, lement quantified by counter-immuno- 59: 670.