Dysfunctional Neural Regulation of Lacrimal Gland Secretion and its Role in the Pathogenesis of Dry Eye Syndromes

Dysfunctional Neural Regulation of Lacrimal Gland Secretion and its Role in the Pathogenesis of Dry Eye Syndromes DARLENE A. DARTT, PHD ABSTRACT Tears are a complex fluid consisting of three layers, each of which is secreted by a different set of tissues or glands. The aqueous portion of the tear film is produced predominantly by the lacrimal gland. Dry eye syndromes are diseases in which the amount and composition of tears are altered, which can lead to ocular surface damage. There are many causes for dry eye syndromes. One such cause is the alteration in the functions of nerves innervating the lacrimal gland and the ocular surface. The autoimmune disease Sjogren syndrome can deleteriously affect the innervation of the lacrimal gland. Damage to the sensory nerves in the ocular surface, specifically the cornea, as a result of refractive surgery and normal aging, prevents the normal reflex arc to the lacrimal gland. Both defects can result in decreased tear secretion and dry eye syndromes. This review will discuss the current information regarding neurally-stimulated protein, water, and electrolyte secretion from the lacrimal gland and delineate how nerve dysfunction resulting from a variety of causes decreases secretion from this gland. KEY WORDS aging, dry eye, epidermal growth factor, lacrimal gland innervation, neural dysfunction, neural regulation of lacrimal gland secretion, refractive surgery

Accepted for publication February 2004 From the Schepens Eye Research Institute, and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA Supported by NIH grant EY06117 Single copy reprint requests to: Darlene A. Dartt, PhD (address below) The author has no financial interest in any concept or product discussed in this article. Corresponding author: Darlene A. Dartt, PhD, Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, 20 Staniford St, Boston MA 02114 Tel: 617-912-0272; Fax: 617-912-0130; email: [email protected]. Abbreviations are printed in boldface where they first appear with their definitions. ©2004 Ethis Communications, Inc. All rights reserved.

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I. INTRODUCTION ry eye syndromes, a spectrum of diseases that alter the composition and/or amount of tears, deleteriously affect the ocular surface and can lead to ocular surface disease. The causes of dry eye syndromes are multifactorial, as the tears are an exceedingly complex fluid secreted by multiple tissues and glands, the latter being termed the ocular adnexa. An alteration in secretion by any of the ocular surface epithelia (cornea and conjunctiva), conjunctival goblet cells, lacrimal gland, or meibomian glands can lead to dry eye syndromes. Although there are numerous causes of dry eye syndromes, many of which are discussed in this issue, one major cause is a loss in the function of the nerves innervating the ocular surface epithelia and tear-producing glands resulting in a change in the cellular signaling pathways that they may activate. Botelho in 19641 introduced the concept that activation of sensory nerves in the cornea and conjunctiva stimulates tear secretion. This has recently been expanded by Stern et al2 and Mathers3 to the concept that the ocular surface (cornea, conjunctiva, accessory lacrimal glands, and meibomian glands) and the lacrimal gland are a functional unit. Mechanical, thermal, and chemical stimuli4 from the external environment activate sensory nerves in the cornea and conjunctiva. These sensory nerves form the afferent limb of a simple reflex arc and conduct the stimuli back to the central nervous system (Figure 1). The efferent limb of the reflex arc comprises the sympathetic and parasympathetic nerves that innervate the ocular surface epithelia and tear-producing glands. Activation of this reflex arc stimulates the epithelia and glands to secrete their respective components of tears onto the ocular surface, thereby protecting this surface from the original stimulus (Figure 1). All of the ocular surface epithelia and tear producing glands are innervated, and all, except perhaps the meibomian glands, respond by secreting their specific secretory products. The lacrimal gland, however, is a critical target of neural regulation. This gland responds by secreting lacrimal gland fluid containing proteins, electrolytes, and water onto the ocular surface. Proteins can be secreted by regulated or constitutive

D

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DYSFUNCTIONAL NEURAL REGULATION OF LACRIMAL GLAND SECRETION AND DRY EYE / Dartt OUTLINE I. Introduction II. Neural regulation of lacrimal gland secretion A. Overview B. Cholinergic agonist stimulation of lacrimal gland secretion C. α1-Adrenergic agonist stimulation of lacrimal gland secretion D. VIP stimulation of lacrimal gland secretion E. Nitric oxide stimulation of lacrimal gland secretion F. Interaction of epidermal growth factor neural stimulation of lacrimal gland secretion G. Summary III. Mechanisms by which neural dysfunction leads to dry eye syndromes A. Refractive surgery B. Sjogren syndrome C. Aging IV. Role of epidermal growth factor in dry eye syndromes V. Conclusion

pathways. Regulated protein secretion is acute and shortterm compared to the longer, chronic regulation of constitutively secreted secretory IgA (sIgA) by steroid hormones. Although the regulation of sIgA secretion is achieved predominantly by regulating synthesis of the protein rather than by regulating release, as occurs with nerves and exocytosis, neural agonists can modify sIgA secretion. Kelleher et al found a long-term inhibition of sIgA secretion with cholinergic agonists, whereas Schecter et al found a rapid stimulation.5,6 Neural and hormonal regulation of constitutive protein secretion as exemplified by sIgA will not be covered in this review. This review will focus on the cellular mechanisms used by nerves to stimulate regulated lacrimal gland protein secretion, as well as electrolyte and water secretion, and how an alteration of this process contributes to dry eye syndromes of the aqueous-deficiency type.7

tain the neuropeptides substance P, calcitonin gene-related peptide, and galanin, which can be released by neurogenic irritation, but do not participate in the reflex arc initiated by activation of corneal and conjunctival sensory nerves. Lacrimal gland sensory nerves can be activated by local irritation of the nerve endings that causes nerve conduction in the antidromic direction (opposite of the normal direction) and releases the sensory neuropeptides into the surrounding lacrimal gland tissue.12,13 The sensory neuropeptides cause limited lacrimal gland secretion.14 Parasympathetic nerves activated by the reflex arc initiated by activation of corneal and conjunctival sensory nerves release the neurotransmitters acetylcholine, a cholinergic agonist, and vasoactive intestinal peptide (VIP), which are both major stimuli of lacrimal gland secretion (Figure 1).15 Sympathetic nerves are activated by ocular surface sensory nerves, as are parasympathetic nerves, and release the neurotransmitters norepinephrine and neuropeptide Y. Norepinephrine, an adrenergic agonist, but not neuropeptide Y, is another major stimulus of lacrimal gland secretion.15 Acetycholine, VIP, and norepinephrine activate separate signaling pathways to stimulate lacrimal gland secretion. B. Cholinergic Agonist Stimulation of Lacrimal Gland Secretion

Stimulation of lacrimal gland secretion by cholinergic agonists was the earliest stimulus described and has been the focus of a considerable body of research. For a thorough review of the cellular mechanisms used by neural

II. NEURAL REGULATION OF LACRIMAL GLAND SECRETION A. Overview

Sensory, parasympathetic, and sympathetic nerves innervate the lacrimal gland. Parasympathetic nerves predominate and surround most acini with beaded varicosities that are the functional nerve endings.8-11 Sympathetic nerves are less abundant, and sensory nerves have the least dense innervation.8-10 Sensory nerves con-

Figure 1. Schematic representation of the neural regulation of the lacrimal gland in the normal state. Sensory nerves in the cornea and conjunctiva are activated to form the afferent branch of a reflex arc. This results in activation of the efferent branch of the arc that includes the parasympathetic and sympathetic nerves which innervate the lacrimal gland. Activation of these nerves causes the release of neurotransmitters, resulting in protein, water, and electrolyte secretion from the lacrimal gland. Similar reflex stimulation occurs in the ocular surface epithelia and other tear-producing glands to induce secretion. (Figure by Dr. Driss Zoukhri.)

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agonists to stimulate secretion, see Hodges and Dartt.16 Acetylcholine, the actual parasympathetic neurotransmitter, binds to muscarinic receptors (MAChR) of the M3 subtype located on the basolateral membranes of lacrimal gland acinar cells (Figure 2).17 This interaction stimulates the receptor, which, in turn, interacts with the G proteins, Gαq and Gα11.18,19 Gαq activates phospholipase Cβ (PLCβ) to break down phosphatidylinositolbisphosphate into 1,4,5inositol trisphosphate (1,4,5-IP 3 ) and diacylFigure 2. Schematic representation of the cholinergic pathway that stimulates protein secretion in the glycerol (DAG) (Figure lacrimal gland. The neurotransmitter acetylcholine (ACh) is released from parasympathetic nerves and binds to muscarinic (M3AChR) receptors to stimulate protein secretion. PLCβ—phospholipase Cβ; PKC— 2).20,21 The water soluble protein kinase C; IP3—1,4,5-inositol trisphosphate; IP3R - -inositol trisphosphate receptor; ER—endoplas1,4,5-IP3 diffuses from the mic reticulum; Ca2+/CaM dep protein kinase—calcium/calmodulin dependent protein kinase. basolateral membrane to bind with its receptors lateral membrane, where it is activated by DAG and phos(IP3R) on the endoplasmic reticulum. This interaction phorylates specific protein substrates found in the same causes the release of Ca2+ from the endoplasmic reticulum into the cytoplasm in the apical area near the secretory location as the PKCα.31 Phosphorylation of these substrates 22-24 2+ The decrease in the Ca content of the eneither directly or indirectly stimulates protein, electrolyte, granules. doplasmic reticulum causes the influx of extracellular Ca2+, and water secretion, as does Ca2+ (Figure 2). Two other 2+ which maintains an elevated intracellular [Ca ]. Two hyprotein kinase C isoforms, PKCε and -δ are also translopotheses have been suggested to explain the coupling of cated to cellular membranes by cholinergic stimulation, Ca2+ depletion of intracellular stores to Ca2+ influx. First, but the identity of the membranes is unknown.31 These 2+ a diffusible factor termed Ca influx factor (CIF) has been two protein kinase C isoforms also play a role in cholinproposed. Second, a conformational change in the IP3 reergic agonist-induced secretion, although they are less ceptor could cause the Ca2+ influx.25 In some cells, a role important than PKCα.32 PKCε and -δ also alter Ca2+ han2+ for 1,4,5-IP3 in Ca release may not be absolutely necesdling by the acinar cells. Activation of these two PKC sary.26 This increase in intracellular [Ca2+] either alone or isoforms inhibits the Ca2+ influx across the plasma mem2+ by activating Ca /calmodulin-dependent protein kinases brane that keeps the intracellular [Ca2+] elevated (Figure 27 stimulates secretion (Figure 2). Protein secretion is stimu2).33 This inhibitory action may serve to terminate stimulated by increasing the rate of fusion of secretory vesicle lation of secretion by cholinergic agonists. membranes with the apical plasma membrane, thus releasCholinergic agonists activate a second effector enzyme, ing the secretory proteins into the acinar lumen. Electrolyte phospholipase D (PLD), in addition to PLC.34 PLD hydrolyzes phosphatidylcholine, a phospholipid present in and water secretion is induced by increasing the activity of the cellular plasma membranes to produce phosphatidic potassium and chloride channels in the apical and basolateral acid. Phosphatidic acid can act as a signaling molecule or membranes of the acinar cells.28,29 For a more extensive description of the mechanism of electrolyte and water sebe degraded to DAG. Biochemical evidence suggests that cretion, see Hodges and Dartt16 or Walcott et al.30 there are two types of PLD in the lacrimal gland, one actiIn contrast to 1,4,5-IP3, the DAG produced upon the vated by cholinergic agonists and one activated by increashydrolysis of phosphatidylinositol-bisphosphate is lipid ing the intracellular [Ca2+] or activation of PKC.34 Several studies have established that the acinar microsoluble and remains in the plasma membrane. DAG actitubule array35,36 and the microtubule-based motor cytovates specific isoforms of protein kinase C (PKC) that are plasmic dynein37 facilitate the formation, maturation and translocated to the basolateral membrane upon stimula31,32 release of secretory vesicles at the apical plasma membrane tion. One of the most important isoforms is PKCα. Cholinergic agonist stimulation very rapidly, i.e., within of lacrimal gland acini stimulated by M3AChR. Prelimi30 seconds, causes the movement of PKCα to the basonary findings suggest that alterations in actin cytoskeleton 78

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can also affect the extent and fidelity of secretory protein targeting to the apical plasma membrane (personal communication, Sarah Hamm-Alvarez). For many years, regulation of actin filament disassembly has been noted as a key regulator of exocytosis in a related cell type, pancreatic acinar cells.38 Moreover, chronic exposure of cultured lacrimal gland acini to low doses of the cholinergic agonist, carbachol, elicits remarkable changes in the organization of actin cytoskeleton, particularly beneath the apical plasma membrane.39 No systematic investigations into the Figure 3. Schematic representation of the α1-adrenergic pathway that stimulates protein secretion in the lacrimal gland. The neurotransmitter norepinephrine (norepi) is released from sympathetic nerves long-term cytoskeletal and binds to α1-adrenergic receptors to stimulate protein secretion. PKC—protein kinase C. changes that may be caused by chronic changes in neutype of G protein responsible for the remaining response ronal innervation or modulation of the lacrimal gland have is still unknown. so far been conducted. However, altered signaling could Cholinergic agonists activate PLC and PLD as the efcertainly be expected to evoke cytoskeletal changes and, fector enzymes in the lacrimal gland. α1-Adrenergic agowith these, changes in other cellular functions that denists do not activate phospholipase C or D.34,41 It is known pend upon the cytoskeleton, including organization and that α1-adrenergic agonists increase the intracellular [Ca2+] function of the secretory pathway. by a small amount. There is controversy about the mechaC. α1-Adrenergic Agonist Stimulation of Lacrimal nism by which α1-adrenergic agonists work in the lacriGland Secretion mal gland. Patch clamp studies indicate the α1-adrenergic Norepinephrine released from sympathetic nerves can agonists activate PLC to increase 1,4,5-IP3 and Ca2+.42 The bind to both α1- and β-adrenergic receptors. In lacrimal biochemical studies from our laboratory do not support gland cells, when norepinephrine binds to α1-adrenergic this mechanism. Finally, Gromada et al43 propose that the receptors, it is a potent stimulus of secretion and activates increase in intracellular [Ca2+] is caused by the generation 2+ of cyclic ADP ribose that binds to ryanodine receptors on a specific signaling pathway involving Ca and PKC. In contrast, when norepinephrine binds to β-adrenergic rethe endoplasmic reticulum. Similar to IP3R, activation of ryanodine receptors releases Ca2+ from this store. Use of ceptors, it is a less effective stimulus of secretion and actidifferent types of preparations (single acinar cell compared vates a cAMP-dependent signaling pathway that will be to acini [groups of acinar cells]) could account for these discussed in the next section. different signaling mechanisms that are proposed to be Norepinephrine is not used to investigate the signalactivated by α1-adrenergic receptors. In addition, α1-adring pathways activated by stimulating α1-adrenergic receptors. Instead, specific α1-adrenergic agonists such as energic agonists activate PKC (Figure 3). Activation of phenylephrine and cirazoline are used. α1-Adrenergic agoPKCε by α1-adrenergic agonists stimulates protein secrenists bind to α1-adrenergic receptors on lacrimal gland tion. In contrast, activation of PKCα and -δ inhibits α1acinar cells. Although these receptors have been characadrenergic agonist-induced secretion. This inhibition could terized by molecular cloning, the subtype of α1-adrenerfunction to terminate α1-adrenergic agonist-induced segic receptor present in the lacrimal gland secretory cells cretion. The activated PKC isoforms function by phosphohas not yet been identified (Figure 3). Binding of α1-adrylating protein substrates that lead to increased fusion of renergic agonists to α1-adrenergic receptors couples this secretory granules with the apical membranes of secretory complex to the stimulation of Gαq subtype of G protein, cells for protein secretion and to the activation of ion chanas for cholinergic agonists and M3AChR. The stimulation nels and pumps for electrolyte and water secretion. of the Gαq G protein, however, does not account for the Unlike cholinergic agonists, stimulation of the lacrimal entire α1-adrenergic agonists induced response.40 The subgland by α1-adrenergic agonists does not translocate any of THE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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the PKC isoforms. In order to stimulate secretion, the activated, but not translocated, PKC isoforms would phosphorylate protein substrates in the same location as the α1-adrenergic receptors. It is interesting to note that when cholinergic agonists translocate PKCα and -ε to specific subcellular locations in lacrimal gland acinar cells, these isoforms stimulate secretion, whereas when α1adrenergic agonists activate these isoforms, they are not translocated and they inhibit secretion. This finding underscores the importance of PKC isoform location in determining the given function of an isoform, as the protein Figure 4. Schematic representation of the VIP-ergic pathway that stimulates protein secretion in the targets of the isoform and lacrimal gland. The neuropeptide vasoactive intestinal peptide (VIP) is released from parasympathetic nerves and binds to VIP receptors (VIPRI) to stimulate protein secretion. AC—adenylyl cyclase; cAMP— their co-localization with the cyclic AMP; PKA—protein kinase A. isoform are critical for the ultimate response. Identification is terminated by the enzyme cAMP phosphodiesterase tion of the protein targets of the individual PKC isoforms that converts cAMP to 5’ AMP. activated by cholinergic compared to α1-adrenergic agonists is a priority for future investigation. VIP also increases the intracellular [Ca2+] to the same extent as α1-adrenergic agonists, but to a lesser extent than D. VIP Stimulation of Lacrimal Gland Secretion cholinergic agonists. One hypothesis is that an increase in VIP is released from parasympathetic nerves along with the intracellular [Ca2+] is necessary for the stimulation of the cholinergic agonist acetylcholine. VIP, in contrast to secretion, although additional signaling components can cholinergic agonists, activates a cAMP-dependent signalcontribute to the secretory process.47 That all three agonists —cholinergic, α1-adrenergic, and VIP— increase the ing pathway (Figure 4). VIP binds to two types of VIP intracellular [Ca2+] is consistent with this hypothesis. receptors, VIPR1 and VIPR2. VIPR1 is the predominant In addition to VIP, other agonists increase cellular cAMP receptor, as it is located on the acinar and duct cells, levels and stimulate lacrimal gland secretion. These inwhereas VIPR2 is located on myoepithelial cells. Binding clude β-adrenergic agonists, α-melanocyte stimulating horof VIP to its receptors stimulates the Gαs subtype of G mone (α-MSH), and adrenocorticotropic hormone protein19 that, in turn, activates the effector enzyme adenylyl cyclase (AC) (Figure 4). Activation of AC pro(ACTH).48,49 In the lacrimal gland, there are to date three different neurally-mediated signaling pathways that stimuduces cAMP from ATP. Several types of AC are present in late lacrimal gland secretion, one activated by cholinergic the lacrimal gland, and their location has been deteragonists, one by α1-adrenergic agonists, and one by VIP. mined.44 One would expect that at least one subtype of AC would be located on the basolateral membranes, as All three agonists are released by the stimulation of the are the VIPR1s. None of the subtypes of AC, however, were efferent nerves innervating the lacrimal gland by activadetected in that location.44 AC could be translocated to tion of the afferent sensory nerves of the ocular surface. these membranes with VIP stimulation (as occurs with Two agonists (ACh and VIP) are released from parasympacholinergic stimulation and multiple components of celthetic nerves and one (norepinephrine) by sympathetic lular signaling45,46), or additional types of AC could be nerves. Thus, activation of nerves provides a major, critipresent but not yet identified in the lacrimal gland. cal mechanism to stimulate lacrimal gland secretion. An increase in the level of cAMP activates protein kiE. Nitric Oxide Stimulation of Lacrimal Gland nase A (PKA [Figure 4]). Similar to PKC and Ca2+/ Secretion calmodulin-dependent protein kinase, PKA phosphorylates Nitric oxide (NO) is produced as a gas by the enzyme specific protein targets that activate exocytosis (protein NO synthase (NOS).50 Of the three types of NOS identisecretion) or ion channels and pumps (electrolyte and fied (neuronal NOS, endothelial NOS, and inducible NOS), water) secretion. The cAMP-dependent signal for secre80

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only neuronal NOS and inducible NOS have been detected in the lacrimal gland. Neuronal NOS was found in lacrimal gland parasympathetic nerves,10,51 and inducible NOS was detected in cultured lacrimal gland acini.52 Elevation of NO can function as a stimulus of lacrimal gland secretion, as NO increased by NO donors or NOS substrates increases intracellular [Ca2+] and stimulates protein secretion.52,53 Evidence suggests that NO works by activating guanylyl cyclase and producing cGMP.52 Although not yet proven, there is evidence consistent with the hypothesis that activated Figure 5. Schematic representation of possible locations of EGF in the lacrimal gland and the target parasympathetic nerves tissue affected by the release of EGF from each location. MMP—matrix metalloproteinase. (Reprinted produce NO, as well as rewith permission from Dartt DA. Interaction of EGF family growth factors and neurotransmitters in reguleasing ACh and VIP. The lating lacrimal gland secretion. Exp Eye Res 2004;78:337-45, 2004.) NO would then activate lacrimal gland protein senot contain 6kD EGF and does not secrete it by exocytocretion by increasing the intracellular [Ca2+] and cGMP levels in the acinar cells. It is also possible that an agonist sis; rather, it contains membrane-bound precursor EGF produces NO, but there are none yet identified that eland other family members and releases them by evate cGMP in the lacrimal gland. ectodomain shedding, as do most other tissues.57 Only the salivary gland synthesizes the 6kD EGF and stores it F. Interaction of Epidermal Growth Factor with in secretory granules to be released upon the appropriate Neural Stimulation of Lacrimal Gland Secretion stimulus. Another dimension to neural stimulation of lacrimal The action of the released proEGF or other family memgland secretion is the effect of nerves on epidermal growth ber depends upon the location of the growth factor in the factor (EGF) and its family of growth factors, as well as lacrimal gland cell. If present on the basolateral membrane the effect of these growth factors on the signaling pathof acinar or ductal cells, the released growth factor could ways activated by neurotransmitters. Preliminary data have interact with EGF receptors on the basolateral membrane indicated that cholinergic agonists cause the release of EGF of the same or adjacent cells (Figure 5). If present on the from human lacrimal gland.54 EGF and its family memapical membrane, the released growth factor would enter bers are synthesized as membrane-bound proteins (prelacrimal gland fluid and be released onto the surface of the cursor growth factor) that are inserted into the cellular eye (Figure 5). There it could bind to EGF receptors present plasma membrane (Figure 5). Neural agonist stimulation on corneal or conjunctival epithelial cells and alter the funcor activation of PKC by phorbol esters can activate an ention of these cells. The lacrimal gland could thereby funczyme, usually a metalloproteinase, to cleave the extracelltion as a major source of EGF for the ocular surface. ular domain of precursor growth factor releasing proUpon the release of proEGF or other EGF family memgrowth factor into the extracellular space (Figure 5). This bers from the cellular membrane, each can interact with speprocess, known as ectodomain shedding, has been demoncific EGF receptors, also in the cellular membranes. There strated for the EGF family members transforming growth are four types of EGF receptors, known as erbB receptors, and factor (TGF)α and heparin binding (HB)-EGF.55 Recently, designated erbB-1 (the EGF receptor), erbB-2, erbB-3, and Le Gall et al56 found that pro-EGF can be released in the erbB-4 (Figure 6). EGF, TGFα, amphiregulin, and same way from a variety of cultured cells. These findings epiregulin bind to erbB-1.58 No known EGF family members bind to erbB-2, although the mucin MUC4, present suggest that cholinergic agonists can cause the release of in the lacrimal gland, does bind to it.59 The EGF family proEGF family growth factor from lacrimal gland, possimembers HB-EGF and betacellulin bind to erbB-1 and bly through activation of PKC. The lacrimal gland does THE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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erbB-4. The multiple heregulin isoforms and neu-differentiation factor, also members of the EGF family, bind to erbB-3 and erbB-4 (Figure 6). Binding of an EGF family member to an erbB receptor induces it to homoor hetero-dimerize with another erbB receptor and activates the intrinsic tyrosine kinase activity of the receptors (Figure 7). Activation of the receptor tyrosine kinases causes phosphorylation on specific tyrosine residues on the intracellular domain of the erbB receptors. Depending upon the residue phosphorylated, specific Figure 6. Schematic representation of the family of EGF receptors and EGF family memadapter proteins are recruited to the bers that bind them. EGF—epidermal growth factor; TGFα—transforming growth factor α; erbB receptor and activated by tyAR—amphiregulin; Epi—epireugulin; HB-EGF—heparin binding EGF; Btc—betacellulin; Her— rosine phosphorylation. If Shc and heregulin; Neu—neu differentiating factors. (Modified from Harris RC, Chung E, Coffey RJ..EGF receptor ligands. Exp Cell Res 284:2-13, 2003.) Grb2 are recruited, the p44/p42 mitogen-activated protein kinase to inhibition of cholinergic agonist stimulated secretion. (MAPK) signaling pathway is activated (Figure 8). If phosThe increased intracellular [Ca2+] and activated PKC pholipase Cγ is recruited, the Ca2+/PKC pathway is stimulated (Figure 7). If phosphatidylinositol 3-kinase is recaused by cholinergic agonist stimulation activates the noncruited, the Akt-dependent pathway is induced. Different receptor tyrosine kinases Src and Pyk2 (Figure 8).61 Src and Pyk2 then activate the p44/p42MAPK pathway. The cellular responses, both long-term and short-term, can be step in the EGF-dependent signaling pathway that is actistimulated, depending upon the signaling pathway induced vated is not known, except that it does not involve activaby receptor tyrosine phosphorylation. tion of ErbB-1 receptors and recruitment of Shc and Grb2. In the lacrimal gland, EGF stimulates protein secreThe activation potentially occurs at the Raf molecules. Action.60 It does so by inducing phospholipase Cγ activity to increase the intracellular [Ca2+] and activate PKC (Figure tivation of p44/p42MAPK inhibits cholinergic agonist 7). EGF-stimulated protein secretion is not stimulated by stimulation of secretion and could serve to terminate the activating PI-3K or Shc/Grb2. The potential exists for EGF secretory response. and cholinergic agonists to affect each other in a complex Activation of the α1-adrenergic signaling pathway, stimulated by sympathetic nerve activity, also interacts with scenario. Activation of parasympathetic nerves releases acethe EGF-dependent signaling pathway.61 α1-Adrenergic tylcholine. Acetylcholine binds to M3AChR to activate PLCβ to increase the intracellular [Ca2+] and activate PKC to stimulate protein secretion. If the preliminary data of Yoshino54 are confirmed, acetylcholine would also cause ectodomain shedding of an EGF family member. If the growth factor is EGF and it is released from the basolateral membrane, then EGF would bind to ErbB-1 receptors and stimulate protein secretion-inducing PLCγ to increase the intracellular [Ca2+] and activate PKC. Cholinergic agonists also interact with the EGFFigure 7. Schematic representation of the EGF pathway that stimulates protein secretion in the lacridependent signaling pathmal gland. EGF binds to its receptor to stimulate protein secretion. PLCγ —phospholipase Cγ ; PKC— way using an additional protein kinase C, P- phosphorylated tyrosine residue. mechanism, and this leads 82

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agonists do not activate Src and Pyk2 as do cholinergic agonists; instead α1-adrenergic agonists transactivate the EGFR, erbB-1 (Figure 8). The mechanism of this transactivation is unknown; it does, however, recruit Shc and Grb2 that ultimately activates p44/ p42MAPK. Activation of p44/p42MAPK inhibits α1adrenergic agonist stimulation of secretion and could serve to terminate the secretory response, as for cholinergic agonists. In other tissues, agonists that transactivate the EGFR do so by Figure 8. Schematic representation of the interaction of cholinergic and α1-adrenergic pathways with activation metalloproteinases the EGF signaling pathway. α1-Adrenergic agonists transactive the EGF receptor. The adapter proteins to release EGF family memShc and Grb2 are recruited to the phosphorylated receptors and are tyrosine phosphorylated. This bers that, in turn, bind to actiattracts SOS to Grb2. SOS catalyzes the exchange of GDP for GTP on Ras to activate it. Ras induces a cascade of serine-threonine kinases including Raf (MAPK kinase kinase), MEK (MAPK kinase), and vate the EGFR.56,62 It is not MAPK (p44/p42 mitogen activated protein kinase or extracellular regulated kinase (ERK)-1 and -2). known if this occurs in the Cholinergic agonists activate the non-receptor tyrosine kinases Pyk2 and Src that activates MAPK lacrimal gland. through an unknown mechanism perhaps at the level of Raf. Activation of MAPK inhibits agoniststimulated protein secretion. In other tissues, G protein-linked receptor agocells. The released EGF would bind to the EGFR. Whether nists, such as β-adrenergic agonists, that activate cAMP this mechanism occurs in the lacrimal gland is currently also transactivate the EGFR. The interaction of β-adrenerunder investigation in my laboratory. If EGF is released gic agonists or VIP with the EGFR in the lacrimal gland from the apical membrane, it would not activate ErbB-1 has not yet been investigated. receptors in lacrimal gland acinar or duct cells. Instead, G. Summary the EGF would enter lacrimal gland fluid and be released Activation of parasympathetic and sympathetic nerves onto the ocular surface to stimulate the corneal and conis a potent, critical stimulus of lacrimal gland protein, elecjunctival epithelia. Thus, nerves and growth factors interact trolyte, and water secretion. Neurotransmitters released in a complex web that is critical for the normal functioning of from these nerves activate Ca2+-, PKC-, and cAMP-depenlacrimal gland, as well as ocular surface, epithelia. dent signaling pathways that have been extensively invesIII. MECHANISMS BY WHICH NEURAL tigated and directly stimulate secretion. In addition, choDYSFUNCTION LEADS TO DRY EYE linergic and α1-adrenergic agonists activate the EGF-deSYNDROMES pendent signaling pathway that terminates the action of As reviewed in the previous section, nerves in coordicholinergic and α1-adrenergic agonists on secretion. Cholinergic agonists also release EGF or an EGF family memnation with EGF provide the predominant regulation of ber from the lacrimal gland. The released EGF would actilacrimal gland secretion of proteins, electrolytes, and wavate lacrimal gland secretion by a Ca2+- and PKC-depenter into the tear film. Loss of the sensory innervation to dent signaling pathway. If the released EGF is from the the cornea is well known to cause dry eye, largely by debasolateral membrane of acinar cells, this is inconsistent pressing the afferent limb of the reflex pathway that drives with the results of Ota et al,61 as cholinergic agonists do lacrimal gland secretion. This occurs for a variety of reanot transactivate the EGFR of acinar cells, a step indicasons, including diseases such as diabetes, herpes simplex tive of the release of EGF from the basolateral membrane keratitis, and neural keratitis; corneal transplantation; and of acinar. The effect described by Yoshino et al54 could be contact lens wear. Neural activation of lacrimal gland seconsistent with the results of Ota et al61 if EGF is released cretion can fail at multiple steps in the pathway, including from the apical membrane of acinar cells or if EGF is reactivation of sensory corneal nerves, activation of efferent leased from duct cells. Since α1-adrenergic agonists parasympathetic and sympathetic lacrimal gland nerves, transactivate the EGFR of lacrimal acinar cells it, is posrelease of neurotransmitters from nerves, induction of sigsible that α1-adrenergic agonists release EGF or an EGF naling pathways by neural agonists, stimulation of the sigfamily member from the basolateral membrane of acinar naling pathways themselves, exocytosis of secretory proTHE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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teins, and release of electrolytes and water (Figure 9). In the following sections of this review, several different states or diseases will be discussed in which differing defects in neural activation of lacrimal gland secretion occurs. A. Refractive Surgery

Three types of refractive surgery are performed today: photorefractive keratotomy (PRK), laser-assisted in situ keratomileusis (LASIK), and laser-assisted subepithelial keratomileusis (LASEK). All three types of surgery alter the corneal sensory nerves that drive tear, including lacrimal gland, secretion and result in dry eye.63 LASIK causes a greater decrease in tear production and the severity of dry eye than PRK.64 In one study, the incidence of dry eye symptomatic subjects post-LASIK was 95% after the first day, 85% after 1 week, and 60% after 1 month.65 No comparable study was performed for LASEK. The dry eye from refractive surgery results from damage to the corneal sensory nerves (Figure 9). In PRK, the damage is to the sensory nerve endings that terminate in the corneal epithelium that is removed by topical alcohol, mechanical scrape, or rotating brush during this procedure.66 In LASEK, dilute alcohol is used to separate the corneal epithelium from the stroma, allowing an epithelial flap to be raised and then repositioned after laser correction. The corneal epithelium is removed at the level of the basement membrane in both PRK and LASEK.67 As in PRK, the neural damage is to the nerve endings that terminate in the epithelium. In both PRK and LASIK, there is additional damage to the nerves in the stroma removed by the laser procedure and the greater the myoptic correction, the greater the dry eye symptoms.66 In LASIK, the corneal flap is made through the stroma, transecting the posterior corneal nerve trunks that enter the cornea at the 3 and 9 o’clock positions and provide the sensory innervation to the cornea.68 A superior hinge on the corneal flap severs both arms of the neuroplexus, whereas a nasal hinge transects only the nerves from the temporal side. Dry eye following LASIK surgery was greater in eyes with a superior hinge compared to a nasal hinge. The severed nerves grow back slowly over months and up to even a year.69,70 Evidence derived from confocal microscopy suggests that a population of these nerves never return to their original state.70,71 After PRK, corneal sensitivity was decreased at 1 week, with an additional decrease at 2 weeks, followed by a gradual recovery over 9 months to 1 year.72-75 After LASIK, corneal sensation was depressed to the lowest values for the first two weeks.76-79 In most individuals, dry eye following refractive surgery slowly improves with time. Signs and symptoms of dry eye are most severe immediately after surgery and improve over 3–6 months.80 This correlates with the immediate decrease in sensitivity of the central cornea measured by Cochet-Bonnet esthesiometry, which slowly returns to the original values over 3–6 months.80 It should be pointed out that Wilson81 and Wilson and Ambrosia82 suggest that in some patients, it is not an aqueous-deficient dry eye that results from LASIK surgery, but 84

a neurotrophic epitheliopathy, as Schirmer tests without anesthesia were never less than 12 mm of wetting. They suggest that a loss of trophic factors with severing of the corneal sensory nerve trunks causes the neurotrophic epitheliopathy. The results of Wilson and Ambrosia are consistent with a dry eye induced by an alteration in the composition of tears derived from the loss of neural stimulation of the lacrimal gland, other ocular adnexa, and the ocular surface epithelia. Alternatively, the increased volume of tears could reflect an inflammatory process that increased vascular permeability of the conjunctiva, allowing leakage of serum into tears, increasing their volume, but changing their protein, electrolyte, mucin, and lipid concentrations. Refractive surgery is creating a new type of dry eye patient who will require temporary treatment of dry eye. In addition, this surgery provides an iatrogenic experiment on an increasing number of individuals, demonstrating the crucial role of neural activity in maintaining a normal tear film and the loss of this activity in the pathogenesis of dry eye syndromes. The demonstration of the role of nerves in maintaining a normal tear film by the sequelae of refractive surgery adds to the existing literature that indicates a substantial role of neural stimulation in the production of tears. Even though the lacrimal gland is only one of several tissues that contribute fluid into the tear film in response to activated nerves, it is certainly a major component. Individuals undergoing refractive surgery provide an opportunity to study the role of sensory corneal nerves in the stimulation of tear production by the lacrimal gland and other tear-producing tissues in subjects that are free of the complications of ocular surface disease. These patients also provide an opportunity to evaluate potential treatments for dry eye syndromes. Similar caveats apply to experimental animals in which dry eye can be created by refractive surgery. This is a relatively simple type of dry eye that is temporary, free of complications of disease, and far less surgically invasive than trigeminal nerve dennervation. B. Sjogren Syndrome

Primary Sjogren syndrome is a chronic, systemic, inflammatory autoimmune disease in which lymphocytes infiltrate the salivary and lacrimal glands, resulting in dry mouth and dry eye. The pathogenesis of Sjogren syndrome is not well understood, although predominantly women are affected (For reviews, see 83-86). The hallmark of this disease is lymphocytic infiltration of the salivary and lacrimal glands by CD(4+) CD45RO(+) memory T cells forming foci of varying size.86 In addition, a small proportion of the infiltrating lymphocytes are B cells. The infiltrating cells destroy the surrounding exocrine gland tissue, including acinar cells, ductal cells, and nerves. Another sign of this disease is the presence of circulating antibodies. Anti-SS-A/Ro and anti-SS-B/La antibodies are diagnostic of the disease, but several other antibodies, including antifodrin and anti-M3 muscarinic receptor, have been detected. A variety of causes of Sjogren syndrome have been

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proposed, including a change in serum androgen levels,8789 inflammation of the lacrimal gland (see review by Stern

and Pflugfelder in this issue90), failure of local immunohomeostasis in the lacrimal gland and ocular surface,91 autonomic nervous system dysfunction,84,92 and functionblocking circulation antibodies.93-95 It is possible that some of these factors may be manifestations rather than causes of the disease, although there is not yet enough evidence to separate causes from manifestations. The role of autonomic nervous system dysfunction and anti-M3 muscarinic receptor antibodies are discussed below. Each of the causes of Sjogren syndrome may contribute to the disease, and treatments targeted at each of these components may provide effective relief. Combinations of therapies may provide even greater potential treatments, as the causes of Sjogren syndrome may be separate or overlapping. Thus, the study of each potential mechanism that contributes to the pathogensis of Sjogren syndrome is critical. As reviewed by Hocevar et al,84 autonomic nervous system dysfunction may be a feature of primary Sjogren syndrome that has been overlooked. Studies on the role of autonomic dysfunction have not always given consistent results. Andonopoulos et al,96 Kovacs et al,97 and Falkenbach et al98 found symptoms of autonomic dysfunction and abnormal function in at least one-half of patients. In contrast, Barendregt et al 99,100 and Niemela et al101 did not find evidence of autonomic involvement in patients with Sjogren syndrome. Tuominen et al102 found evidence of abnormal morphology in the subbasal nerve plexus of 40% of patients with Sjogren syndrome. A search of the literature reveals a substantial number of case reports of individuals with neuropathy associated with Sjogren syndrome and neuropathy associated with other connective tissue diseases. In one study, the prevalence rate of peripheral nervous system dysfunction in Sjogren syndrome was reported to be 20%.103 Most of the neuropathies described are sensory or autonomic dominant. Thus, the dry eye associated with Sjogren syndrome may, in part, be a consequence of a failure of the nerves innervating the ocular surface and ocular adnexa, and, in particular, the lacrimal gland, to function properly. Neural stimulation of lacrimal gland secretion involves activation of sensory nerves that, by a reflex arc, activates parasympathetic and sympathetic nerves that lead to the gland, stimulating the multiple signaling pathways that lead to secretion of lacrimal gland fluid into tears, as described in section II. The failure of nerves in Sjogren syndrome could occur at any of the multiple steps in the stimulus-secretion pathway and result in a decrease in lacrimal gland secretion and, hence, dry eye. Two different lines of evidence point to specific steps that could be dysfunctional in Sjogren syndrome: cytokine-mediated inhibition of neurotransmitter release from efferent nerves in the lacrimal gland, and function-blocking antibodies to M3AchR (Figure 9). Using the MRL/MpJ-Faslpr (MRL/lpr) murine model of Sjogren syndrome and its congenic control MRL/MpJ strain of mice, Zoukhri et al13,92 found an impairment of para-

sympathetic nerve activity in the lacrimal gland of the diseased MRL/lpr mice. Female MRL/lpr mice develop a progressive lymphocytic infiltration of the lacrimal gland with age that leads to decreased tear secretion. The progression of disease in the MRL/MpJ mice is much slower, allowing comparison of unaffected lacrimal glands of MRL/MpJ mice with affected glands of MRL/lpr mice at 8–12 weeks of age.92 The distribution of sensory, parasympathetic, and sympathetic nerves does not differ between age- and sexmatched MRL/lpr and MRL/MpJ mice from 4–16 weeks of age.13 The function of the nerves, however, is altered.92 As the MRL/lpr mice aged and lymphocytic foci appeared in the lacrimal glands, the ability of the parasympathetic nerves to release ACh or induce protein secretion in response to depolarizing concentrations of KCl was blocked.13,92 Concomitantly, addition of exogenous neurotransmitters, cholinergic and α1-adrenergic agonists, caused a significantly increased intracellular Ca2+ response and protein secretory response, as occurs in denervation supersensitivity.92,104 This finding was consistent with a functional “denervation” of the gland. Zoukhri et al discovered that interleukin (IL)-1α and -β, along with tumor necrosis factor (TNF)α produced by the invading lymphocytes and subsequently by lacrimal gland acini, blocked the release of neurotransmitters from lacrimal gland nerves.105 The results of Zoukhri and co-workers suggest that it is not the destruction of acini by lymphocytes that impairs lacrimal gland secretion in Sjogren syndrome, but that it is the decrease in neurally-mediated secretion in the remaining healthy acini that accounts for the loss of lacrimal gland function and the resulting dry eye (Figure 10). Based on this finding, treatments for dry eye might be developed that either prevent the inhibitory effects of cytokines on lacrimal nerves or bypass the nerves and stimulate the signaling pathways directly. A second type of nerve-related dysfunction has been substantiated in Sjogren syndrome. In 1998, Bacman et al106 demonstrated that IgG present in the sera of patients with primary Sjogren syndrome recognized and activated the M3AChR on lacrimal gland cell membranes isolated from rats (Figure 9). These antibodies bound to the receptor and increased IP3 production that was blocked by a non-subtype-specific muscarinic antagonist atropine and an inhibitor of M3AChR, 4-DAMP. These antibodies also activated nitric oxide synthase and increased cGMP production in rat lacrimal acinar cells.94,107 Unfortunately, others have not shown that cholinergic agonists produce NO and cGMP in the lacrimal gland of normal mice or rats. The effect of carbachol on the lacrimal gland from patients with Sjogren syndrome has not yet been tested. Bacman et al suggest that chronic interaction of these autoantibodies with lacrimal gland M3AChR increases cellular nitric oxide release leading to tissue damage.106 Beauregard et al did find that IL-1β can induce NO production in the lacrimal gland.108 Perhaps activation of M3AchR leads to production of IL-1β and this produces NO. Waterman et al also detected antibodies to M3AchR,

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but these antibodies inhibited the action of ACh on M3AchR.109 This inhibitory effect would lead to decreased lacrimal gland secretion and dry eye, as was detected in the labial salivary gland cells, which have an increased number of M3AChR in patients with Sjogren syndrome.110 Beroukas et al suggest that the increase in M3AChR results from the antagonistic effect of the antibodies.110 In response to the blockade of the M3AChR, the gland has increased synthesis of M3AChRs. In addition, these cells require a higher concentration of cholinergic agonists in order to achieve the same level of secretion seen in non-Sjogren patients.111 In another murine model of Sjogren syndrome, the NOD mouse, Figure 9. Schematic representation of the neural dysfunction of the lacrimal gland. Senantibodies to the M3AChR infused sory nerves in the cornea and conjunctiva are normally activated to form the afferent branch of a reflex arc. Dysfunction of sensory nerves can occur in many diseases or refractive into the mice decreased secretion by surgery, as described in the text. Activation of the efferent branch of the arc which inthe salivary gland, presumably by cludes the parasympathetic and sympathetic nerves that innervate the lacrimal gland will down regulating the M 3AChR rethen not be activated. Failure to activate parasympathetic and sympathetic nerves will lead to reduction of protein, water, and electrolyte secretion from the lacrimal gland resultsponse.112 It is not clear if the antiing in dry eye syndromes. (Figure by Dr. Driss Zoukhri.) body was function-blocking or caused down regulation due to chronic activation. Qian et al demonstrated that, in lacrimal glands, eye syndromes is not surprising, as previous research found that the tear film, tear-producing epithelial, and corneal chronic activation of the M3AChR did not lead to down regulation of this receptor, but rather to down regulation functions decrease with age. Mathers et al found that tear of post-receptor signaling molecules.39 functions, especially reflex tear capacity (Schirmer test withSubsequent studies found that the antibody in patients out anesthetic), tear volume, and tear osmolarity, declined with Sjogren syndrome was directed toward the second throughout life.114 Findings of Patel and Farrell115 were consistent with those of Mathers,114 as the former showed that extracellular loop of the M3AChR and that cytotoxic lymphocytes could potentially produce granzyme B, which tear film stability decreased with increasing age. cleaves molecules normally found in lacrimal acinar cells Multiple potential causes account for the decrease in (such as the M3AChR) into novel fragments that are imtear production with increasing age. A decrease in nerve munogenetic.94 A considerable body of information now function is one of them. Neural function could be altered suggests that M3AChR antibodies are generated in the sera at several levels in the stimulus-secretion process. First, of patients with Sjogren syndrome. These antibodies could the sensory nerves innervating the cornea and conjunccontribute to the development of dry eye, either by blocktiva could be defective. Second, the parasympathetic and ing the activation fluid of M3AChR and, hence, the prosympathetic nerves innervating the lacrimal gland could duction of lacrimal gland, or by chronic stimulation of the be altered. The potential changes in the nerves include a M3AChR that either induces down regulation of the redecrease in their number or a decrease in their activity ceptor or generates cytotoxic compounds. In either case, that would impair the release of neurotransmitters. Third, a therapy for dry eye could be developed that bypasses the signaling pathways in the acinar cells activated by the the activation of the receptor and activates the signaling neural agonists could be defective. pathway distal to the agonist-receptor interaction. Research on the human cornea has demonstrated that corneal sensitivity decreases with increasing age and that C. Aging the decrease is more pronounced around the 4th or 5th A recent epidemiological study on 39,876 females decade of life.116-118 These results suggest that the activity found that the number of individuals with dry eye synof the sensory nerves in the cornea decreases with aging. drome increases with age.113 Dry eye affects many women Thus, the driving force for lacrimal gland secretion is imin their 40s and 50s, but it is more prevalent among older paired with increasing age and could at least partially acwomen. Although the prevalence of dry eye is less in men count for the decrease in tear production and increase in than women, the same age-dependent increase in prevatear osmolarity that occurs with increasing age. lence occurs in men as well. The age-dependency of dry There are age-dependent changes in the lacrimal gland, 86

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as well as in the cornea, which could contribute to the decrease in tear production with age. In the human lacrimal gland, Damato et al119 and Obata et al120 found that with increasing age there occurred atrophy of secretory acini, secretory duct obstruction, ascending periductal fibrosis, obliteration of adjacent blood vessels along with an increased lymphocytic infiltration. In rat lacrimal glands, similar morphological changes occurred with aging when young adults (3–9 months old) were compared with elderly (24–28-month-old) animals.121-123 Additional Figure 10. Schematic representation of the neural regulation of the lacrimal gland in a diseased state. changes were observed in Sensory nerves in the cornea and conjunctiva are activated to form the afferent branch of a reflex arc. the aging rats. First, a deThis results in activation of the efferent branch of the arc that includes the parasympathetic and sympathetic nerves, which innervate the lacrimal gland. Activation of these nerves causes the release crease in the number and of neurotransmitters that is inhibited by cytokines released by lymphocytes present in the diseased intensity of lacrimal nerves lacrimal gland. This inhibits protein, water, and electrolyte secretion from the lacrimal gland resulting of all types—sensory, parain dry eye syndromes. (Figure by Dr. Driss Zoukhri.) sympathetic, and sympathetic— occurred in the elderly 24-month-old rats compathway; and 3) the signaling pathways activated by chopared to 3-, 5-, and 14-month-old rats.124 The activity of linergic and β-adrenergic agonists in the lacrimal gland these nerves was not determined. Second, the number of acinar cells (Figure 9). The resultant depression in lacrimast cells found throughout the lacrimal gland tissue was mal gland secretion could contribute substantially to the greater at 24 months than at 14 months.124 Finally, a dedecrease in reflex tear secretion and increase in tear osmocrease in protein secretion occurred in response to cholinlarity that occurs in humans with increased age. No studergic agonist stimulation, which reflects depressed activaies, however, have yet addressed the mechanism for the tion of the parasympathetic nerve pathway at an unknown deficiencies in neural activation of lacrimal gland secrepart of this pathway.123,125 No research on the effect of tion. The increase in lymphocytic infiltration and mast cell the other parasympathetic agonist, VIP, has been perinvasion of the lacrimal gland that was demonstrated with formed. A similar decrease occurred in response to β-adrincreased age could produce compounds that impair the energic agonist stimulation of protein secretion, but not function of the nerves and the signaling pathways, but to α1-adrenergic agonist stimulation, the two pathways these details have yet to be investigated. activated by sympathetic nerve stimulation.126 It should IV. ROLE OF EGF IN DRY EYE SYNDROMES be noted that the β-adrenergic secretory response is much Experimental evidence shows that tear EGF levels are smaller than the α1-adrenergic agonist response. Thus, there appears to be an age-dependent decrease in all types decreased in a variety of diseases, but especially in dry of lacrimal gland nerves and in function of the cholinergic eye, Sjogren syndrome dry eye, and Stevens-Johnson synand β-adrenergic signaling pathways leading to protein drome.127-129 The decreased level of EGF could contribute to the ocular surface disease. There is indirect evidence secretion, which could contribute to the decrease in tear that suggests that activation of corneal sensory nerves reguproduction. lates EGF levels in the lacrimal gland and in tears. First, The evidence in the literature suggests that with aging tear EGF levels correlate with reflex tear secretion, as meathere is a pervasive decrease in neural stimulation of lacrisured by the Schirmer I test.128,130 Second, wounding of mal gland secretion. The impairment has been found to the cornea increases the amount of EGF mRNA in lacrioccur at several levels including: 1) the corneal sensory mal glands in the rabbit,131 and the amount of EGF in nerves whose activation forms the afferent arm of the rerabbit tears increases immediately after wounding.132 Thus, flex pathway that stimulates lacrimal gland secretion; 2) activation of corneal nerves, at least in part, regulates EGF the lacrimal gland parasympathetic and sympathetic nerves levels in tears through an effect on the lacrimal gland, and whose activation forms the efferent arm of the stimulatory THE OCULAR SURFACE / APRIL 2004, VOL. 2, NO. 2 / www.theocularsurface.com

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this regulation may be impaired in a variety of types of dry eye syndromes. Clearly, additional research is necessary to substantiate the role of nerves in regulation of EGF release by the lacrimal gland, as well as the role of EGF in dry eye syndromes. In particular, no research has been performed on the functioning of EGF-mediated signaling pathways in the lacrimal gland in this disease. V. CONCLUSIONS Activation of sensory nerves in the ocular surface, as well as parasympathetic and sympathetic nerves in the lacrimal gland, is a major stimulus of lacrimal gland secretion. Maintenance of the appropriate amount and composition of lacrimal gland fluid is critical to a normal tear film and a healthy ocular surface. Dysfunction of the neural regulation of lacrimal gland secretion is an important cause of aqueous-deficient dry eye. This dysfunction can occur at many steps in the process by which the lacrimal gland is activated, including: 1) the afferent sensory nerves; 2) the efferent parasympathetic and sympathetic nerves; and 3) the signaling pathways by which nerves and growth factors stimulate secretion of proteins, electrolytes, and water. When different mechanisms in this process are altered, this results in diverse types of dry eye. In dry eye after refractive surgery, there is an impairment of the activation of sensory nerves. In Sjogren syndrome, a blockade in neurotransmitter release from efferent nerves or the activation of the M3AChR occurs. Finally, aging decreases the activation of sensory nerves as well as impairs neurotransmitter activation of the secretory process. Further study of these types of dry eye could identify potential treatments, treatments that would not only bypass the defect to restore secretion, but also have limited side effects. ACKNOWLEDGEMENTS The author would like to thank Ms. Robin Hodges for editorial assistance, Dr. Driss Zoukhri for artistic assistance, and Dr. Sarah Hamm-Alvarez for helpful discussions.

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