Ординатура / Офтальмология / Английские материалы / Dry Eye and Ocular Surface Disorders_Pflugfelder, Beuerman, Elliot Stern_2004

numerous secretory granules of the protein products to be secreted. Acinar secretions drain into intralobular ducts that converge into interlobular ducts and eventually merge to form the excretory ducts.

The acinar and ductal epithelia secrete water, electrolytes (Na+, Cl–, K+, Ca2+), protein, and mucus into the tear fluid. Two types of acini have been identified, the predominant serous acini that secrete fluid and proteins, and the mucous acini that stain positively for acid mucopolysaccharides. The serous acini vary in the complement of proteins that they secrete (31). Concentrations of proteins in tears can vary as well. For example, epidermal growth factor and IgA concentrations decrease following sensory stimulation, whereas concentrations of other lacrimal proteins such as lactoferrin and lysozyme remain constant in reflex tear fluid (32,33).

Lacrimal glands are components of the mucosal-associated lymphoid tissue (MALT). Lymphoid follicles with T and B lymphocytes and abundant IgAproducing plasma cells are scattered in the stroma surrounding secretory acini in the lacrimal glands (Fig. 4). Other secretory organs of the lacrimal functional unit include the meibomian glands (described in Chapter 12), the ocular surface epithelia, and the conjunctival goblet cells (described in Chapter 5).

The majority of tear secretion by the lacrimal epithelia occurs in response to neural stimulation (34). Parasympathetic, sympathetic, and sensory nerves innervate acini, ducts, and blood vessels of the lacrimal gland. Parasympathetic nerves release the neurotransmitters acetylcholine and VIP, whereas sympathetic nerves release norepinephrine, and sensory nerves release substance P and CGRP (35). Maintenance of the lacrimal gland secretory environment is also regulated by serum-derived factors, including sex hormones (e.g., androgen, estrogen, and progesterone), cortisol, insulin, thyroxin, and growth factors (36).

Parasympathetic cholinergic nerves are primarily responsible for signaling reflex tear secretion. Acetylcholine released from these nerve endings binds to M3 acetylcholine receptors on the basolateral cell membranes of secretory epithelia (35,37) and VIP binds to VIPergic receptors (38). Each receptor initiates a signal transduction cascade that results in increased levels of second messengers, fusion of secretory granules with the apical cell membrane, activation of cell membrane ion transporters, and insertion of ion pumps that together mediate secretion of electrolytes and osmotically entrained water. Specific signal transduction pathways have also been identified for sympathetic stimulation of tear secretion.

When acetylcholine binds to the extracytoplasmic domain of a M3 receptor in the lacrimal epithelial cell basolateral membrane, it activates the heterotrimeric GTP-binding proteins, Gq and G11, at the cytoplasmic surface (39). The α subunits, Gαq and Gα11, dissociate from the βγ subunits, release bound GDP, and bind GTP. Both GTP-bound α subunits are believed to mediate the same function, i.e., activation of the phosphatidylinositol-specific phospholipase C,

phospholipase Cβ (PLCβ). Phospholipase Cβ hydrolyzes phosphatidylinositol bisphosphate (PIP2) to generate the intracellular mediators, inositol 1,4,5-trisphos- phate (IP3) and diacylglycerol (DAG). IP3 activates IP3-regulated Ca2+ channels, presumably associated with the endoplasmic reticulum and Golgi complex, allowing Ca2+ that had been stored in those compartments to be released to the cytosol. The elevation of cytosolic Ca+ then activates a plasma membrane Ca2+ channel, ICRAC, to allow influx of additional Ca2+.

The other major parasympathetic neurotransmitter in the lacrimal gland, VIP, which also elicits protein secretion in the lacrimal gland (40), binds to type I and type II VIP receptors, which couple to Gs. The activated α subunit, GTP-Gαs, activates adenylyl cyclase, elevating cytosolic cAMP (38).

The major sympathetic neurotransmitter, norepinephrine, interacts with both α1- and β-adrenergic receptors. The β-adrenergic receptors, like VIP receptors, couple to Gs (39). α1-Adrenergic agonists elevate cytosolic Ca2+ (41). There are reports that α1-adrenergic receptors couple to Gq (42) in rabbit lacrimal gland, and that in rat lacrimal glands these receptors lead to stimulation of ADP-ribosylcy- clase to convert β-NAD to cADP-ribose, which activates ryanodine receptor Ca2+ channels to release Ca2+ from an intracellular pool distinct from the IP3-receptor regulated pool (43). The G proteins that couple α1 receptors to downstream mediators in rat lacrimal glands have not been delineated.

Lacrimal epithelial cells also respond to purinergic agonists, indicating that they possess P2Y1 receptors, but little is known about the purinergic innervation of the lacrimal glands (44).

It appears that parasympathetic and sensory nerves also may activate lacrimal epithelial secretion indirectly, by stimulating mast cells to release histamine (45). The lacrimal epithelial histamine receptors have not been characterized, but classically H1 receptors couple to Gq/G11, and H2 receptors couple both to Gs and Gq/G11 (46). Availability of histaminergic signaling pathways would raise the possibility that inflammation may initiate lacrimal gland secretion directly, i.e., without eliciting sympathetic or parasympathetic neurotransmission.

There are some interesting clues that cross-talk between the classical G pro- tein-coupled receptor (GPCR) signaling pathways and between GPCR and nonGPCR pathways may be physiologically significant. In addition to elevating cAMP, VIP stimulation also appears to cause a small but significant elevation of cytosolic Ca2+ (38). Neutralizing antibodies to Gs partially inhibit responses to the muscarinic cholinergic agonist, carbachol, suggesting that M3 receptors couple to Gs as well as to Gq/G11 (39). Epidermal growth factor stimulates protein secretion in rat lacrimal acinar cells (47), and M3 receptor stimulation causes transactivation of epidermal growth factor receptors (48). Carbachol stimulation also appears to activate MAP kinase, which, via pathways presently unknown, partially inhibits the secretory response (49). Interestingly, opioid receptor activation inhibits secretory responses, at least in some species, by stimulating Gi family proteins (50).

The intracellular signaling mediators, Ca2+ and diacylglycerol, elicit a set of downstream responses. Diacylglycerol is an activator of protein kinase C (PKC). Lacrimal acinar cells contain five different protein kinase C isoforms, PKCα, -β, -γ, -ε, and -λ/ι, all of which are activated by diacylglycerol and some of which also require elevation of cytosolic Ca2+. These appear to be differentially distributed among the various intracellular compartments and to play different roles in the secretory process (51).

VIII. ELECTROLYTE AND WATER SECRETION

Secretion of water by epithelial tissues is an osmotic phenomenon, driven by the active secretion of proteins, mucins, and electrolytes. Lacrimal epithelial cells contain aquaporin (AQP) water channels (52); AQP5 is present in the apical membrane, while AQP3 and AQP4 are present in the basolateral membranes (53). While the aquaporins are known to mediate the flux of water through cell membranes, aquaporin knockout mice do not exhibit a notable decrement in tear production.

Oncotic pressure associated with secretion of proteins and mucins likely contributes to the secretion of water, but it generally is believed that the greatest component of water secretion results from the epithelial cells’ ability to secrete electrolytes, primarily Na+, K+, and Cl–. Cumulative evidence from several laboratories suggests a cellular model for this process that involves five different transporters—Na+,K+-ATPase, Na+/H+ exchangers, Cl–/HCO3– exchangers, Na+K+Cl2– cotransporters, and K+ channels—arrayed in parallel on the basolateral plasma membrane, and at least two additional transporters—Cl– channels and K+ channels—arrayed in parallel on the apical plasma membrane (Fig. 5A) (54–58). Intracellular mediators and effectors generated by receptor activation lead to opening of the apical Cl– channels and both the apical and the basolateral K+ channels. Opening of the K+ channels hyperpolarizes the electrical potential difference across the cell membrane, thereby increasing the force driving Cl– out of the cell and into the forming lacrimal gland fluid. Intracellular mediators and effectors also activate Na+K+Cl2– cotransporters and Na+/H+ exchangers, accelerating Na+ influx and also accelerating Cl– influx, both directly and indirectly. The increased rate of H+ extrusion by activated Na+/H+ exchangers alkalinizes the cytosol. Cytoplasmic alkalinization increases the net influx of Cl– mediated by Cl–/HCO3– exchangers in the baslateral membrane kinetically, by increasing the cytosolic HCO3– concentration, and allosterically, by activating a pH-depend- ent regulatory site. Recruitment of additional Na+,K+-ATPase pump units to the plasma membrane from large intracellular stores helps the cell maintain ionic homeostasis.

It generally is assumed that Na+ ions, which are pumped back out of the cell into the surrounding interstitial space by Na+,K+-ATPase in the basolateral

membrane, are secreted through the epithelium via the paracellular pathway. The driving force is the lumen-negative transepithelial electrical potential difference generated by secreted Cl– ions (35).

IX. LACRIMAL PROTEINS

The population of large, proteinand mucin-containing secretory vesicles densely packed into the apical cytoplasm is perhaps the most striking structural feature of the lacrimal secretory epithelial cell (59). According to the classical merocrine secretory mechanism, the lacrimal secretory proteins are synthesized and mannosylated in the endoplasmic reticulum, and the carbohydrate groups are modified during subsequent transit through the Golgi complex. Secretory proteins are collected into transport vesicles in specialized domains of the trans-Golgi network, then, presumably, delivered to newly forming secretory vesicles. Both Ca2+ (60) and PKCα (61) activate exocytic fusion of preformed secretory vesicles with the apical plasma membrane, so that the content proteins are released into the forming lacrimal gland fluid.

A second apical secretory pathway recently has been discovered to emerge from the trans-Golgi network. In this pathway, secretory transport vesicles move directly to the apical plasma membrane in response to secretory stimulation (62) It is not yet known whether the same population of secretory transport vesicles, carrying the same spectrum of proteins, mediates direct secretion in the stimulated state and formation of secretory vesicles in the resting state. However, both processes appear to be driven by the microtubule minus-end-directed molecular motor, dynein.

After exocytic protein and mucin secretion, the lipids and proteins that had comprised the secretory vesicle membranes are endocytically retrieved and returned to the Golgi complex and trans-Golgi network, where they are collected into new secretory transport vesicles (63). The endocytic transport vesicles appear to be driven by the microtubule plus-end-directed molecular motor, kinesin. In-bound traffic, like the secretory traffic, is controlled directly by intracellular signaling mediators (64).

X.TRANSCYTOTIC SECRETION

Lacrimal gland fluid also contains macromolecular products that are synthesized by cells other than the secretory epithelial cells. The most notable of these is secretory IgA (sIgA), which is released by plasma cells in the space surrounding the epithelium. The mechanism of dimeric IgA secretion has been studied in MDCK cells transfected with polymeric immunoglobulin receptors (pIgR).

MDCK cells are a renal epithelial cell line that do not normally secrete IgA, but they offer the experimental advantage that the cells form polarized monolayers, with both surfaces accessible to manipulation; as such they may not necessarily recapitulate all the adaptations expressed by cells that are specialized for dimeric IgA transcytosis. The basic model is that newly synthesized polymeric immunoglobulin receptors emerge from the trans-Golgi network in transport vesicles targeted to the basolateral plasma membrane (65). At the basolateral membrane, polymeric immunoglobulin receptors bind dimeric IgA, and the complex is internalized to an early endosome. From the early endosome the complex is transferred to an apical or common endosome, where it is collected into budding transport vesicles targeted to the apical plasma membrane. The polymeric immunoglobulin receptor extracytoplasmic domain, which contains the dimeric IgA-binding region commonly referred to as secretory component, is cleaved from the membrane-spanning tail to release the secretory component-dimeric IgA complex, i.e., sIgA, into the forming lacrimal gland fluid.

A somewhat more nuanced model is suggested by observations that lacrimal acinar cells contain large intracellular pools of polymeric immunoglobulin receptors and, by analogy to an unusual pattern of traffic that receptors for epidermal growth factor, have been found to undergo in lacrimal acinar cells (66). According to this model, the bulk of the cell’s polymeric immunoglobulin receptors cycle constitutively between the intracellular pools and the basolateral plasma membrane. The polymeric immunoglobulin receptor pool turns over as the fraction that enter lysosomes and are degraded and the fraction that are hydrolyzed at the apical plasma membrane are replaced by newly synthesized polymeric immunoglobulin receptors emerging from the trans-Golgi network. Stimulation with carbachol dramatically increases secretory component secretion and presumably reduces polymeric immunoglobulin receptors’ traffic to the lysosomes (67). It is reasonable to assume that binding of dimeric IgA elicits a similar response, but possible dimeric IgA-polymeric immunoglobulin receptor signaling pathways in lacrimal secretory cells have not yet been investigated.

It has been known for some time that expression of polymeric immunoglobulin receptors is one of the lacrimal epithelial cell functions that exhibits a striking sexual dimorphism; tear secretory component and sIgA concentrations may be fivefold or more greater in males than in females (68). However, there is still no information about whether this dimorphism is related to males’ generally greater resistance to autoimmune lacrimal gland disease (69).

The general principles of the transcytotic secretion of dimeric IgA may apply to the secretion of other products as well. Lacrimal gland fluid contains serum albumin and IgG, and it also contains hepatocyte growth factor (HGF), which appears to be released by fibroblasts (70). Albumin and IgG secretion may occur via fluid phase

traffic through the series of compartments that comprise the transcytotic pathway. In contrast, HGF transcytosis may involve a receptor-mediated mechanism.

XI. INFLAMMATION AND SECRETION

It has been a puzzling observation that the lacrimal glands of Sjögren’s patients with severe salivary and lacrimal insufficiencies can contain large masses of secretory parenchyma (71). While it has been recognized that some mechanism(s) associated with lymphocytic infiltration must be responsible for maintaining a state of functional quiescence, until recently there has been little information about the nature of the signals involved. Secretomotor innervation appears largely intact, except in the immediate vicinity of lymphocytic foci. It seems well established that neurotransmission is impaired in the MRL/lpr mouse model of autoimmune lacrimal gland disease (72) and that the epithelial cells exhibit exaggerated responses to carbachol, as expected for denervation hypersensitivity (73). Moreover, impaired neurotransmission can be replicated ex vivo by treatment with IL-1 and TNF-α (74).

A different process may operate in human Sjögren’s syndrome. Immunohistochemical analyses of labial salivary gland biopsies from Sjögren’s syndrome patients indicate that plasma membrane muscarinic receptors are upregulated, as might be expected to occur in denervation hypersensitivity (75). Except within areas of lymphocytic infiltration, parasympathetic innervation appears intact in Sjögren’s syndrome patients’ labial salivary glands (76). Moreover, the patients’ saliva contains relatively high concentrations of VIP, suggesting that neurotransmission is not impaired (77). Ex-vivo studies indicate that epithelial cells from Sjögren’s syndrome patients’ labial salivary glands are unable to elevate cytosolic Ca2+ in response to stimulation with the cholinergic agonist, pilocarpine (78). Immunocytochemical studies indicate that expression of protein kinase Cα is decreased (79). Such observations suggest that signals associated with the presence of autoimmune infiltrates cause secretory epithelial cells to become functionally quiescent (80).

One possible clue to the nature of the quiescence-inducing signals is suggested by reports that Sjögren’s syndrome patients’ sera contain M3 receptor-acti- vating autoantibodies (81,82). If so, chronic autoantibody-mediated stimulation might induce functional quiescence by downregulating postreceptor signaling. Preliminary reports of studies with an ex-vivo model suggest that chronic stimulation with a half-maximal concentration of carbachol causes a wide spectrum of functional and biochemical changes, many of which mimic the changes documented in Sjögren’s patients’ labial salivary gland biopsies (83,84). Total muscarinic acetylcholine receptor ligand-binding activity per milligram of cellular protein is not altered, but receptors are redistributed from the endosomes to

the plasma membrane; protein kinase Cα immunoreactivity is downregulated; and the cells fail to elevate cytosolic Ca2+ or secrete protein in response to acute stimulation with 100 µM carbachol. Cellular expression of Gq and G11 is downregulated, suggesting that while the muscarinic receptors are present and their plasma membrane expression is upregulated, they are unable to transduce signals to downstream mediators and effectors. The cells exhibit several additional changes, which suggest that their ion and dimeric IgA secretory functions also are quiescent: Na,K-ATPase is internalized from the basolateral plasma membranes to endomembrane compartments, and total cellular content of polymeric immunoglobulin receptors is downregulated.

XII. SECRETORY EPITHELIAL CELLS MAY PROVOKE AND EXACERBATE AUTOIMMUNE INFLAMMATION

The specialized adaptations lacrimal epithelial cells have developed to fulfill their role in the lacrimal functional unit may cause them to participate actively in initiating and expanding the autoimmune responses that impair their function.

One of the early suggestions for the initiation of autoimmune responses to the Ro/SSA and La/SSB autoantigens was based on observations that their expression levels are upregulated and their subcellular localization shifts from the nucleus to the cytoplasm and plasma membranes in response to various perturbations, including viral infections, cytokine stimulation, and oxidative stress in vitro (85,86), and that an analogous redistribution of La/SSB occurs in Sjögren’s syndrome patients’ salivary glands (87). While this mechanism may indeed occur, and while it would increase exposure of the autoantigens to B-cell antigen receptors and autoantibodies, some additional process is required to account for the activation of CD4+ T cells, which dominate the Sjögren’s lymphocytic infiltrates.

More recent efforts to understand the pathways by which autoantigens come to be exposed and presented in the lacrimal and salivary glands have focused on the role of apoptosis. Apoptotic fragments have been shown to contain several known lacrimal autoantigens, including Ro/SSA, fodrin, and M3 receptors (88). Apoptosis appears to be a relatively rare event in the normal lacrimal gland, but it does occur at a detectable rate. Interestingly, ovariectomy causes a wave of lymphocyte apoptosis in the lacrimal gland, and this is followed by signs of increased epithelial cell necrosis (89). Both the lymphocyte apoptosis and the epithelial cell necrosis induced by ovariectomy can be prevented by administration of either dihydrotestosterone or estradiol (90). Presumably, apoptotic material and necrotic cell debris are cleared by macrophages, which may then process the autoantigens and present pathogenic epitopes.

An alternative model, in which intact, functioning lacrimal epithelial cells expose autoantigens and presented autoantigen eptitopes, has also been proposed. Compared to the absorptive epithelial cells that have been studied with the same analytical methods, lacrimal acinar cells contain unusually large intracellular pools of proteins that function classically in the plasma membranes. These include Na+,K+-ATPase, Na+/H+ exchangers, Cl–/HCO3– exchangers, muscarinic acetylcholine receptors, β-adrenergic receptors, epidermal growth factor receptors, and the heterotrimeric GTP-binding proteins, Gq, G11, Go, Gi3, and Gs (91,92). At least in ex-vivo preparations of acinar cells from rabbit lacrimal glands, recycling between the plasma membrane and the network of endomembrane compartments is remarkably rapid; half-times for internalization of extracellularly labeled membrane proteins are 30 s, and half-times for return to the plasma membrane are roughly 5 min (93).

This traffic may serve several goals. It may sustain a large flux of polymeric immunoglobulin receptors to the basolateral membrane to mediate dimeric IgA transcytosis, and a strategy of bulk membrane internalization and recycling may be energetically less costly than selective sorting of polymeric immunoglobulin receptors for endocytosis at the plasma membrane. It may permit the cell to fine-tune the number of Na+,K+-ATPase molecules in place in the plasma membrane to match changes in the rate of Na+ influx as Na+/H+ exchangers and Na+K+Cl2– co-transporters are activated and inactivated. It may permit rapid adjustments in cell surface area as cell volume changes in response to the osmotic losses associated with the effluxes of Cl– and K+ mediated by activated ion-selective channels. Finally, it may permit the continuous replacement and reactivation of inactivated neurotransmitter receptors to support secretory responses to sustained stimuli.

The apparent problem created by this functional specialization is that it also provides a pathway by which the cell may constitutively secrete potentially pathogenic autoantigens into the surrounding space. It is possible to induce autoimmune lacrimal gland disease by immunizing animals with several different lacrimal autoantigens (94–96). This result indicates that potentially pathogenic CD4+ T cells specific for lacrimal autoantigen epitopes must routinely escape clonal deletion (97,98). It also indicates that the epitopes recognized by autoreactive lymphocytes are constitutively available and are associated with antigen presenting cells in the milieu of the lacrimal glands. Therefore, it appears that lacrimal autoimmunity is a normal state. If so, it must normally be prevented from progressing to a disease state by immunoregulatory mechanisms.

According to the paradigm of normal lacrimal autoimmunity, autoimmune disease may result when any of a number of perturbations disrupt local immunoregulation, e.g., increased expression of pro-inflammatory cytokines, decreased expression of anti-inflammatory cytokines, and increased exposure of autoantigens. The endomembrane network is fairly complex. As mapped in

recent studies (62,66,92), the early basolateral endosome communicates with a late endosome, a recycling endosome, and the Golgi complex and the trans-Golgi network. The trans-Golgi network communicates with the recycling endosome and the late endosome, as well as with the regulated and recruitable apical secretory pathways. The late endosome communicates with the early endosome as well as with the prelysosome. The endomembrane network is not only complex, it also is very dynamic, and its organization appears to undergo significant changes in response to alterations of the local signaling environment. Moreover, the changes that have been observed experimentally are consistent with increased secretion of autoantigens as well as with increased plasma membrane exposure, although it is not yet known whether these are sufficient to disrupt local immunoregulation (99).

Once local immunoregulation has been disrupted, or once an infection or trauma has provoked an inflammatory response, it is possible that autoimmune responses may be initiated and spiral out of control. The presence of cytokines and inflammatory mediators in the local milieu appears to induce lacrimal epithelial cells to begin expressing major histocompatibility complex (MHC) Class II molecules (100). Preparations of MHC Class II+ epithelial cells are able to stimulate autologous lymphocytes to proliferate ex vivo, suggesting that they are able to function as antigen-presenting cells (100,101). This function raises the possibility that when MHC Class II expression has been induced, it leads to epitope spreading that evades the available immunoregulatory mechanisms.

Interruption of the afferent or efferent pathways results in paralysis of tear secretion. Immediately after interruption of lacrimal innervation, the acinar cells continue to produce tear proteins that are stored in secretory vesicles. These vesicles initially fill the acinar cells, but over time, the acinar cells atrophy in the absence of innervation.

XIII. TWO STATES OF THE FUNCTIONAL UNIT

To achieve ocular surface protection in a wide range of situations, the components of the functional unit may be activated differentially depending on environmental conditions and pathology (Fig. 6). Sensory innervation and hence the lacrimal functional unit can operate at two states of activation. The first state is under normal conditions (without trauma or pathology) where a constant, low level of input to the lacrimal functional unit arrives from sensory nerves on the ocular surface. In this first state of activation, sensory input is subthreshold, and the individual is not usually aware that the nerves are sensing the environment. The cornea sensory nerves act in concert with efferent sympathetic and parasympathetic innervation to modulate secretory activity by the lacrimal and meibomian glands, and by the conjunctival goblet cells. Tear flow is moderate without excess.