Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Before considering tentative theoretical paradigms that might explain how the androgens might confer protection against dry eye disease, it is necessary to first review the influences other reproductive hormones exert on the lacrimal glands.

Estradiol and Progesterone

Whereas estradiol and testosterone often have opposing actions, Azzarolo and coworkers found that estradiol – like DHT – prevents ovariectomy-induced apoptosis of lacrimal gland plasmacytes. Estradiol does not appear to influence pIgR expression in rat lacrimal epithelial cells. However, tear lactoperoxidase levels vary during the estrus cycle in rats and the menstrual cycle in humans – correlating with the changes in estradiol and progesterone levels. Microarray analyses of mouse lacrimal gland extracts indicate that estradiol and progesterone influence the expression of numerous gene transcripts. They increase expression of the chemokines CCL2 and CXCL15 and decrease expression of FoxP3 – a central transcription factor in regulatory lymphocyte function; these influences might seem consistent with the greater risk for inflammatory lacrimal gland disease in females. However, estradiol and progesterone also decrease expression of CD86, IL-12, and the chemokines CCL6, CCL12, and CCL28 – influences which might be expected to diminish inflammatory responses.

Prolactin

Prolactin is produced by the pituitary gland; as its name implies, its first discovered function was support of lactogenesis and lactation. However, prolactin has also been found to function as an autocrine/intracrine and paracrine mediator in a number of physiological systems. In the immune system, it acts as a mitogenic cytokine for Tcells and B cells, and as a differentiation factor for T cells – inducing them to express the prototypical TH1 cytokine, IFN-g. Administration of prolactin to hypophysectomized male rats increased Na, K-ATPase catalytic activity in lacrimal gland. However, a number of reports indicate that serum prolactin levels are elevated in women with Sjo¨gren’s syndrome and other autoimmune diseases. A study of reproductive hormone influences on lacrimal function revealed that increasing serum prolactin levels within the normal range of values for nonpregnant, nonlactating women were strongly correlated with decreasing lacrimal function – independently of menopausal status and use of estrogen replacement therapy.

Exocrine Products and Autocrine/Intracrine

and Paracrine Mediators

While they respond to prolactin as a classic hormone, lacrimal gland epithelial cells also express prolactin, and

they secrete it both as an exocrine secretory product and as a paracrine mediator. In the lacrimal glands of normal, nonpregnant female rabbits, immunopositivities for prolactin, as well as for transforming growth factor-beta (TGF-b), EGF, fibroblast growth factor (FGF)-2, are localized preferentially – but not exclusively – in ductal epithelial cells. In both acinar and ductal cells, the cytokine and growth factor immunopositivities are concentrated in the apical cytoplasm. Prolactin is localized in the regulated exocrine secretory vesicles; the mechanisms by which epithelial cells of the rabbit lacrimal gland secrete the other cytokines and growth factors have not been elucidated. Like prolactin and other secretory vesiclecontent proteins, TGF-b is released to the fluid forming within the lumen of the acinus-duct system in response to stimulation with cholinergic agonists.

As illustrated in Figure 1, lacrimal epithelial cells use their apical recycling endosome and early basolateral endosome as a transcytotic secretory apparatus. It is this apparatus which secretes sIgA and some free SC into the lumena of the acinus-duct system. They also use the early and recycling endosomes as a paracrine secretory apparatus that delivers products to the underlying stromal space. Both transcytotic secretion and paracrine secretion occur constitutively; although they can be accelerated by stimulation with cholinergic agonists, the steady-state pools of secreted products in the endosomes are quite small compared to the pools of products stored in regulated exocrine secretory vesicles. Experiments with ex vivo acinar cell models showed that increasing the concentration of prolactin in the ambient medium induces increased transcription of prolactin messenger RNA (mRNA). Increasing epithelial cell prolactin expression or increasing the prolactin concentration in the ambient medium decreased the amount of secretory proteins stored in apical secretory vesicles, and it induced the cells to express a novel population of regulated secretory vesicles that accumulated in the basal cytoplasm and released their contents at the basolateral plasma membrane in response to acute cholinergic stimulation.

Immunogold localization studies demonstrated that when acinar cells endocytose prolactin from their ambient medium, they traffic it dually to the endosomes that comprise their constitutive transcytotic-paracrine apparatus and to the secretory vesicles of the novel, induced paracrine apparatus. Thus, when acinar epithelial cells internalize prolactin secreted from the pituitary or from ductal epithelial cells, they may recycle it as a paracrine mediator.

Serum prolactin levels do not differ greatly between normal men and normal, nonpregnant women. However, serum prolactin levels increase markedly during pregnancy, and, by the time a pregnancy reaches term, mean serum prolactin levels are 10to 20-fold greater than the levels in nonpregnant, nonlactating females. Thus, the physiological hyperprolactinemia of pregnancy has

seemed to offer a natural model in which to study prolactin’s influences on the lacrimal glands.

Influences of Prolactin, Estradiol, and Progesterone during Pregnancy

The lacrimal glands of nonpregnant, sexually mature female rabbits normally contain small aggregates of

lymphocytes and plasmacytes, localized in the stromal spaces surrounding and spanning between venules and interlobular ducts. It might be noted that these are the same sites where the ectopic lymphoid tissues characteristic of Sjo¨gren’s dacryoadenitis develop. The immunoarchitecture undergoes a remarkable change during pregnancy, and it remains in the altered state throughout lactation and for some weeks following weaning. By the time a pregnancy reaches term, the aggregates have largely dissipated, and

lymphocytes and plasmacytes are primarily located in the thin stromal spaces surrounding acini.

The immunoarchitectural change is associated with several notable cytophysiological changes and functional changes. The basal rate of lacrimal gland fluid production decreases, while the rate of fluid production under cholinergic stimulation increases. Immunopositivities for TGF-b and prolactin increase substantially, and their localizations shift from the apical cytoplasm to the basal cytoplasm. It now appears that these redistributions occur because the novel paracrine secretory apparatus induced by the increased serum prolactin level captures prolactin and TGF-b away from the regulated exocrine secretory pathway. Accordingly, the level of prolactin excreted in lacrimal gland fluid decreases, and TGF-b becomes scarcely detectable.

Experiments that have not yet been published indicate that when ovariectomized rabbits are implanted with sustained-release pellets establishing pregnancy-like serum levels of estradiol and progesterone, the patterns of lymphocyte organization and of TGF-b and prolactin expression and localization change to resemble the patterns characteristic of pregnancy. Thus, estradiol, progesterone, or the two steroid hormones in concert act on ductal epithelial cells to increase their expression TGF-b. They may increase ductal epithelial cell prolactin expression either directly, or indirectly, that is, by increasing pituitary prolactin secretion (see Box 1). The increased level of prolactin then directs both mediators away from the regulated exocrine protein-secretion apparatus and directs prolactin into the novel paracrine apparatus.

As discussed below, the changes that occur in the lacrimal glands during pregnancy are analogous to those which occur over roughly the same time in the mammary glands. While the lacrimal glands are accessory organ of the visual system, the mammary glands are accessory organs of the reproductive system. Both glands also are effector organs of the mucosal immune system. Other organs of the male and female reproductive systems also play parallel roles as adaptive mucosal immune system effector organs, and their immunophysiological functions are, likewise, influenced by the reproductive hormones.

Reproductive Hormone Influences on Other Mucosal Immune System Tissues

Neither the testes nor the ovaries are normally populated by IgA+-plasmacytes. However, in males, IgA+ cells are abundant in the urethral glands and prostate; they are also present in the seminal vesicles in some species, but not others. Orchiectomizing male rats has little effect on the amount of IgA in the prostate and seminal

vesicles; subsequent administration of DHT causes a slight increase of the IgA content of the prostate, but not the seminal vesicle. There is little pIgR expression in the testes, vas deferens, or epididymis, but a significant level of expression in the seminal vesicles and a 20-fold greater level in the prostate. Orchiectomy decreases pIgR expression, and subsequent administration of DHT increases it threefold in the seminal vesicles and fourfold in the prostate. Interestingly, estradiol has no effect on pIgR expression in the seminal vesicles but doubles it in the prostate.

In females, IgA+ immune cells are abundant in the lamina propria of the fallopian tubes. They are sparse in the endometrium. Since endometrial gland epithelial cells contain IgM+ and IgA+, as well as J chain, it may be that the uterine lining secretes Igs derived primarily from the serum, rather than from local plasmacytes. In contrast, IgA+ cells are abundant within the epithelia and lamina propria of the endocervix, although somewhat less abundant in the ectocervix and vagina. Epithelial expression of pIgR roughly parallels the abundance of IgA+ cells. The level is significant in the fallopian tubes and endocervix. While there seems to be no clear evidence that pIgR is expressed in the ectocervix and vagina, the level of sIgA in cervical mucus increases just prior to ovulation and remains elevated throughout the luteal phase, and the uterine fluid contains a high level of sIgA throughout pregnancy.

As the alveolar epithelium of the mammary glands develops during pregnancy, ductal epithelial cells are induced to express pIgR, and the glands’ stromal spaces become populated by dIgA+-plasmacytes. During lactation, the mammary epithelium secretes sIgA as well as lactoperoxidase and other innate mucosal immune effector molecules. Like the other changes occurring during lactogenesis, the induction of mucosal immune effector functions is controlled by interacting influences of estradiol, progesterone, and prolactin. Most studies of hormonal influences on the mammary glands have been motivated by interest in normal lactogenesis, lactation, and postweaning involution, and in mammary carcinoma – rather than focused on the mammary glands’ mucosal immune functions. This work has shown that the systemic hormones orchestrate lactogenesis and lactation, in part, by regulating the expression of autocrine/intracrine and paracrine mediators. The ductal network of the mammary gland develops during puberty, largely under the influence of estradiol. Prior to pregnancy, TGF-b – which is expressed both by ductal epithelial cells and periductal mesenchymal cells – exerts pro-apoptotic and antiproliferative influences that prevent development of the alveoli – and, during pregnancy, alveolar development depends on the concerted influences of prolactin and progesterone. Increasing progesterone levels increase the abundance of TGF-b, but

they also increase expression of EGF, FGF-2, and TGFalpha (TGF-a) – which abrogate TGF-b’s anti proliferative and pro-apoptotic influences. Notably, the expression of prolactin by mammary epithelial cells also increases at this time. Despite its evident synergy with prolactin in promoting lactogenesis, the elevated level of progesterone inhibits lactation. Recent evidence suggests it does so by increasing expression of Wnt-5b, which is thought to maintain the undifferentiated state by promoting nuclear translocation of b-catenin, and by increasing expression of insulin-like growth factor-binding protein (IGFBP-5) – which suppresses insulin-like growth factor signaling. These inhibitory influences are removed and lactation becomes possible at parturition, when production of progesterone is abruptly suppressed.

Certain of the estradiol-, progesterone-, and prolactininduced mediators that determine development of the mammary epithelium also determine expression of the mammary glands’ mucosal immune functions. As reviewed elsewhere in the encyclopedia, TGF-b typically acts as a differentiation factor for immature dIgA+-expressing plasmablasts, inducing them to mature into dIgA-secret- ing plasmacytes as they arrive at mucosal immune effector sites. Normal plasmacytes – like other bone-marrow- derived cells – express an intrinsic apoptotic program, and their ongoing survival requires that this program be abrogated by survival signals from the local milieu. As noted above, prolactin plays a role in inducing ductal epithelial cells to express pIgR. Its known mitogenic influences on T cells and B cells suggest that it may also act as one of the factors which support the mature plasmacytes’ survival. Thus, prolactin may contribute to a counterpoise against plasmacyte’s apoptotic program as well as against the pro-apoptotic and antiproliferative influences TGF-b would otherwise exert on both the plasmacytes and the alveolar epithelium. Evidently, this counterpoise is maintained as the levels of TGF-b, prolactin, and other estrogenand progesterone-dependent factors increase during pregnancy, and it supports expansion of the population of plasmacytes that will produce dIgA for secretion in the milk.

Similar interactions between the sex steroids and prolactin may account for the reproductive hormones’ influences on the other mucosal effector organs of the female and male reproductive systems, and the data available so far indicate that they do so for the lacrimal glands, as well.

Counterpoises between Contradictory Signals

Figure 2 summarizes the spatial and temporal actions of the differentiation and survival factors, as they are

organized in the lacrimal glands. The notions that plasmacytes express an intrinsic apoptotic program which must constantly be abrogated and that the steady-state pools of paracrine secretory products in lacrimal epithelial cells are small may help explain why they begin undergoing apoptosis so abruptly after ovariectomy. The pools of paracrine secretory products would deplete rapidly after the signals that support their ongoing expression are removed. Unpublished findings that prolactin immunoreactivity is present in the nuclei of plasmacytes seem to confirm that prolactin is one of the lacrimal epithelial paracrine mediators the influence plasmacytes. This may only be part of the explanation; however, and it is possible that testosterone, estradiol, and, perhaps, progesterone as well also might interact with prolactin and other survival factors to generate synergistic signals that maintain plasmacyte survival.

While prolactin may be one of several factors that provide mitogenic signals abrogating TGF-b’s pro-apoptotic and antiproliferative influences, it appears that TGF-b may provide a counterpoise to prolactin’s lymphoproliferative and proinflammatory influences. As reviewed elsewhere in the encyclopedia, there is evidence that the transcytotic apparatus mucosal epithelial cells use to internalize dIgA and release sIgA into the fluid they produce inevitably secretes a significant burden of autoantigens to the underlying stromal spaces. Thus, newly matured dendritic cells that emigrate from the lacrimal glands to the draining lymph nodes carry with them lacrimal epithelial autoantigens. They process the autoantigens to generate epitopes that their surface MHC class-II molecules will present to CD4+ T cells, and they also release the autoantigens for sampling by IgM+-B-cell antigen receptors. There is now evidence that TGF-b induces immature dendritic cells to mature into immunosuppressive antigen-presenting cells that prevent proliferation of autoreactive lymphocytes within the lacrimal glands and draining lymph node. Moreover, it is possible that dendritic cells that have matured within the lacrimal glands might also function as tolerogenic antigen-presenting cells – inducing the generation of TH3 or TR1 regulatory cells.

Microarray studies have clarified that the reproductive hormones influence the expression of other cytokines and growth factors apart from TGF-b and prolactin in the lacrimal glands. Further work will be needed to determine the extents to which the various mediators are expressed by infiltrating immune cells, acinar and ductal epithelial cells, and mesenchymal cells. Other factors in addition to the reproductive hormones are likely to influence epithelial expression of TGF-b and of prolactin and the plasmacyte survival factors. Nevertheless, the concept that the reproductive hormones orchestrate counterpoises between contradictory signals, summarized in Figure 3, may lead to detailed paradigms that explain why Sjo¨gren’s dacryoadenitis and the common histopathological syndrome

are both so much more prevalent among women, and why the onset of clinical dry eye disease is associated with events of the reproductive cycle and life cycle. There are several physiological states during which prolactin-mediated influences might become excessive with respect to the available counterpoises (see Box 1). Given the burden of epithelial autoantigens constitutively present in the stromal space of the lacrimal glands, one might predict that such states favor the accumulation of autoreactive T cells and B cells. In a direct experimental test of the hypothesis, an adenovirus vector was used to transiently increase prolactin expression in lacrimal glands of mature female rabbits. As has been reported in preliminary form, increased abundance of prolactin transcripts was accompanied by increased

abundances of mRNAs for IFN-g and TGF-a, as well as accumulation of large lymphocytic infiltrates and apparent formation of germinal centers. Moreover, the lymphocytic foci persisted for weeks after the prolactin mRNA levels returned to normal. Of interest also is a recent report that, after having a primary relative with an autoimmune disease, carrying a pregnancy to term is the second greatest risk factor for Sjo¨gren’s syndrome appears to accord with this prediction. Both findings suggest that autoimmune activation can be suppressed after systemic hormone levels have returned to normal, but that autoreactive memory cells may persist and become activated as the age-related loss of reproductive steroids changes the immunoregulatory signaling milieu within the lacrimal glands.

Immunoregulation epithelial survival plasmacyte survival plasmablast differentiation

Figure 3 The capacity of the lacrimal gland to perform its exocrine functions – that is, secretion of electrolytes, water, and proteins – and its mucosal immune functions – that is, maintenance of a population of mature, dIgA-secreting plasmablasts and transcytotic delivery of sIgA into the fluid being produced, while avoiding autoimmune inflammatory processes – depends on a system of counterpoises between contradictory influences. The reproductive hormones influence expression of many of the paracrine mediators that exert those influences.

See also: Adaptive Immune System and the Eye: Mucosal Immunity.

Further Reading

Ariga, H., Edwards, J., and Sullivan, D. A. (1989). Androgen control of autoimmune expression in lacrimal glands of MRL/Mp-lpr/lpr mice.

Clinical Immunology and Immunopathology 53: 499–508. Azzarolo, A. M., Eihausen, H., and Schechter, J. (2003). Estrogen

prevention of lacrimal gland cell death and lymphocytic infiltration.

Experimental Eye Research 77: 347–354.

Azzarolo, A. M., Mircheff, A. K., Kaswan, R. L., et al. (1997). Androgen support of lacrimal gland function. Endocrine 6: 39–45.

Azzarolo, A. M., Wood, R. L., Mircheff, A. K., et al. (1999). Androgen influence on lacrimal gland apoptosis, necrosis and lymphocytic infiltration. Investigative Ophthalmology and Visual Science 40: 523–526.

Bailey, J. P., Nieport, K. M., Herbst, M. P., et al. (2004). Prolactin and transforming growth factor-b signaling exert opposing effects on mammary gland morphogenesis, involution, and the Aky-forkhead pathway. Molecular Endocrinology 19: 1171–1184.

Ding, C., Chang, N., Fong, Y. C., et al. (2006). Interacting influences of pregnancy and corneal injury on rabbit lacrimal gland immunoarchitecture and function. Investigative Ophthalmology and Visual Science 47: 1368–1375.

Frey, W. H., Nelson, J. D., Frick, M. L., and Elde, R. P. (1986). Prolactin immunoreactivity in human tears and lacrimal gland: Possible implications for tear production. In: Holly, F. J. (ed.) The Preocular Tear Film in Health, Disease, and Contact Lens Wear, pp. 798–807. Lubbock, TX: Dry Eye Institute.

Kolek, O., Gajowska, B., Godlewski, M. M., and Motyl, T. (2003). Antiproliferative and apoptotic effect of TGF-b1 in bovine mammary epithelial BME-UV1 cells. Comparative Biochemistry and Physiology C 134: 417–430.

Mathers, W. D., Stovall, D., Lane, J. A., Zimmerman, M. B., and Johnson, S. (1998). Menopause and tear function: The influence of prolactin and sex hormones on human tear production. Cornea 17: 353–358.

Mircheff, A. K., Wang, Y., de Saint Jean, M., et al. (2005). Mucosal immunity and self-tolerance in the ocular surface system. Ocular Surface 4: 182–193.

Priori, R., Medda, E., Conti, F., et al. (2007). Risk factors for Sjo¨gren’s syndrome. Clinical and Experimental Rheumatology 25: 378–384.

Richards, S. M., Liu, M., Jensen, R. V., et al. (2005). Androgen regulation of gene expression in the mouse lacrimal gland. Journal of Steroid Biochemistry and Molecular Biology 96: 401–413.

Rosfjord, E. C. and Dickson, R. B. (1999). Growth factors, apoptosis, and survival of mammary epithelial cells. Journal of Mammary Gland Biology and Neoplasia 4: 229–237.

Rudolph, M. C., McManaman, J. L., Hunter, L., Phang, T., and Neville, M. C. (2003). Functional development of the mammary gland: Use of expression profiling and trajectory clustering to reveal changes in gene expression during pregnancy, lactation, and involution. Journal of Mammary Gland Biology and Neoplasia 8: 287–307.

Schechter, J., Carey, J., Wallace, M., and Wood, R. (2000). Distributions of growth factors and immune cells are altered in the lacrimal gland during pregnancy and lactation. Experimental Eye Research 71: 129–142.

Sullivan, D. A., Kelleher, R. S., Vaerman, J. -P., and Hann, L. E. (1990). Androgen regulation of secretory component synthesis by

lacrimal gland acinar cells in vitro. Journal of Immunology 145: 4238–4244.

Suzuki, T., Schirra, F., Richards, S. M., et al. (2006). Estrogen’s and progesterone’s impact on gene expression in the mouse lacrimal gland. Investigative Ophthalmology and Visual Science 47: 158–168.

Wang, Y., Chiu, C. T., Nakamura, T., et al. (2007). Traffic of endogenous, over-expressed, and endocytosed prolactin in rabbit lacrimal acinar cells. Experimental Eye Research 85: 749–761.

Lacrimal Gland Signaling: Neural

D Zoukhri, Tufts University, Boston, MA, USA

ã 2010 Elsevier Ltd. All rights reserved.

Glossary

Acinar cells – Highly polarized epithelial cells that form an acinus and whose primary function is to secrete proteins, electrolytes, and water. Exocytosis – The process in which molecules (such as secretory proteins) in a membrane-enclosed vesicle (secretory vesicle or granule) fuse with the plasma membrane and are then released outside the cell.

Muscarinic receptors – A subtype of receptors for the neurotransmitter acetylcholine that is more responsive to muscarine than nicotine. Neurotransmitters – Chemicals released by neurons to modulate the function of a target cell. Preocular tear film – A complex and highly structured moist film which covers the bulbar and palpebral conjunctiva, and the cornea. It is composed of water, electrolytes, proteins, mucins, and lipids.

Signal transduction – The biochemical events that conduct the signal of an external stimulus from the cell exterior, through the cell membrane, and into the cytoplasm.

Anatomy of the Lacrimal Gland

The lacrimal gland is a compound tubuloalveolar serous gland composed primarily of acinar, ductal, and myoepithelial cells (Figure 1). Acinar cells account for over 80% of the cell type present in the lacrimal gland and form the site for synthesis, storage, and secretion of proteins. Several of these proteins have antibacterial or growth factor properties and are crucial to the health of the ocular surface. Acinar cells are highly polarized cells with tight junctions surrounding each acinar cell on the luminal side and thus separating the plasma membrane into apical (luminal) and basolateral (serosal) components. The basal portion of the cell contains a large nucleus, rough endoplasmic reticulum, mitochondria, and Golgi apparatus, while the apical portion is filled with secretory granules.

Like the acinar cells, the duct cells are also polarized with the nuclei located basolaterally, whereas the rough endoplasmic reticulum and mitochondria are more apical. The primary function of the ductal cells is to modify the

primary fluid secreted by the acinar cells by absorbing or secreting water and electrolytes. The duct cells secrete a KCl-rich solution so that the final secreted lacrimal gland fluid is rich in K+. It has been estimated that as much as 30% of the volume of the final lacrimal gland fluid is secreted by the duct cells.

The myoepithelial cells lie scattered between the acinar and ducts cells and the basement membrane and are interconnected by gap junctions and desmosomes. These cells are highly branched and contain multiple processes which surround the basal area of the acinar cells (Figure 1). The myoepithelial cells are thought to contract because they contain muscle contractile proteins (a-smooth muscle actin, myosin, and tropomyosin). The contraction of these cells would help expel the fluid out of the acini and the ducts onto the ocular surface. In support of a functional role of lacrimal gland myoepithelial cells, receptors and intracellular signaling molecules for parasympathetic neurotransmitters have been described.

The lacrimal gland contains other cells: plasma cells, B and T cells, dendritic cells, macrophages, and mast cells. Immunoglobulin A (IgA)-positive plasma cells account for the majority of the mononuclear cells in the lacrimal gland. These cells synthesize and secrete IgA, which then is transported into acinar and ductal cells and secreted by these epithelial cells as secretory IgA.

Neural Control of Lacrimal Gland

Secretion

To ensure adequate production of the aqueous component of the preocular tear film, lacrimal gland secretion is under tight neural control. To this end, the lacrimal gland is densely innervated by the parasympathetic and sympathetic nervous system (Figure 2). Although scarce, sensory nerves are also present in the lacrimal gland (Figure 2). Nerves are located in close proximity to acinar, ductal, and myoepithelial cells, as well as blood vessels, and hence can control a wide variety of lacrimal gland functions. While each individual cell may not be innervated, gap junctions electrically and chemically connect cells within an acinus so that even noninnervated cells can respond to the neural stimulus.

In the lacrimal gland, parasympathetic nerves contain the neurotransmitters acetylcholine and vasoactive intestinal peptide (VIP). Sympathetic nerves contain the neurotransmitters norepinephrine and neuropeptide Y (NPY).

Sensory nerves contain the neurotransmitters substance P and calcitonin gene-related peptide (CGRP). Acetylcholine and VIP are potent stimuli of lacrimal gland protein and electrolyte/water secretion. Norepinephrine is also a potent stimulus of protein secretion, but a weak stimulus of electrolyte/water secretion. In contrast, NPYand CGRP are weak stimuli of protein secretion, while substance P does not appear to stimulate either protein or electrolyte/water secretion.

The stimulation of lacrimal gland secretion occurs through a neural reflex arc originating from the ocular surface (Figure 3). Neural reflexes are initiated by stimulation of the afferent sensory nerves of the cornea and conjunctiva or by activation of the optic nerve in response to intense light. Efferent parasympathetic and sympathetic nerves of the lacrimal gland are then activated to release their neurotransmitters (Figure 3). The neurotransmitters interact with and activate specific receptors located on the basolateral membranes of acinar and duct cells, which then

Figure 1 Schematic of the lacrimal gland and photomicrographs showing the three major cell types that it is composed of. The acinar cells, which account for 80% of the cell type present in the lacrimal gland, and ductal cells were stained with hematoxylin and eosin. The myoepithelial cells were identified immunohistochemically (brown stain) using an antibody against a-smooth muscle actin.

initiates a cascade of intracellular events known as signal transduction. Activation of these signal transduction pathways induces fusion of the preformed secretory granules with the apical membrane to release secretory proteins into the lumen (Figure 3). To trigger electrolyte and water secretion, ion channels and pumps, located in the apical and basolateral membranes, are also activated.

Signal Transduction Pathways Activated

in the Lacrimal Gland

Signal transduction proceeds in three steps: (1) initiation of the signal by interaction of the ligand (neurotransmitter, neuropeptide, or hormone) with its receptor; (2) amplification of the signal through the interaction of the receptor/G protein/effector enzyme leading to the generation of second-messenger molecules; and (3) termination of the signal through the action of protein phosphatases and membrane pumps to bring the amount of phosphorylated proteins and ions, respectively, back to resting levels (Figure 4).

Cholinergic Agonist-Activated Signal

Transduction Pathways

Acetylcholine, released from parasympathetic nerves, activates muscarinic receptors on the basolateral membrane of lacrimal gland cells. Of the five receptor subtypes (M1–5) identified, only the M3 or glandular subtype is present in the lacrimal gland. These receptors are coupled to the activation of phospholipases C and D (PLC and PLD, respectively) and the activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK, also known as extracellular signal-regulated kinase (ERK)) pathway (Figure 5).

PLC-coupled signaling pathway

Lacrimal gland M3 receptors are coupled, via the G- protein Gaq, to the effector enzyme PLC. Activated PLC hydrolyzes the plasma membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to generate two

Parasympathetic Sympathetic

+

+

+

+

Acinus

Secretion

Brain

Lacrimal gland

second-messenger molecules, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG; Figure 5).

IP3, a water-soluble molecule, diffuses to the endoplasmic reticulum where Ca2+ is stored in an inactive, bound form. It also interacts with specific receptors located on the endoplasmic reticulum to release Ca2+ into the cytosol. Depletion of these Ca2+ stores leads to an increase in the influx of extracellular Ca2+ across the plasma membrane. The IP3 receptor is a homotetramer of 310 kDa each and constitutes one of the largest of all known ion channels. Binding sites for IP3 are located within the N-terminal

domain, whereas the C-terminal regions form the intrinsic Ca2+ channel. Multiple isoforms of IP3 receptor have been cloned. They share significant similarity to each other and are encoded by at least four genes.

The activation of lacrimal gland cholinergic M3 receptors triggers a biphasic Ca2+ response: a rapid (usually referred to as peak) increase in [Ca2+]i due to IP3-induced release of Ca2+ from intracellular stores, followed by a slow and sustained (usually referred to as plateau) increase in [Ca2+]i due to influx of Ca2+ from the extracellular milieu. Both Ca2+ responses are necessary for cholinergic