Lacrimal gland-directed B cell responses

Although it is accepted that IgA plasma cells predominate in the lacrimal gland, the factors leading to this prevalence are not known. A series of 4-day LPS-driven co-culture experiments performed with dissociated lacrimal gland and lymphoid cell populations was employed to study the direct effect of lacrimal gland cells on B cell differentiation. Lacrimal gland cells, when co-cultured with spleen or mesenteric lymph node cells, were found to suppress differentiation of cells to IgA, IgG, and IgM production. Furthermore, suppression of IgG and IgM responses occurred after co-culture of lacrimal gland cells with Peyer's patch cells. However, these Peyer's patch co-cultures led to a stimulation of the IgA response, a condition that was abrogated by removal of Peyer's patch T cells before co-culturing. Pretreatment of lacrimal gland cells with mitomycin C eliminated the suppression and stimulation previously observed. These results demonstrate the effects of lacrimal gland, both directly and indirectly through T cells, on B cell differentiation. These findings explain in part the preferential accumulation of IgA-plasma cells within the gland.     

Lacrimal gland involvement in zygomaticofrontal fracture sites

Injury to the lacrimal gland following fracture of the zygomatic complex is a rare occurrence. Recently, we have observed three cases of herniation of the lacrimal gland between the diastased frontal and zygomatic bone ends. We present these cases, postulate a mechanism for this phenomenon, and advocate open reduction and internal fixation in the primary management of severe distraction.     

Age-related changes in the rat lacrimal gland: Impressive morphology and enigmatic nature

The rat lacrimal apparatus includes several glands; among them, the exorbital lacrimal gland plays the central role. Its parenchyma and stroma undergo prominent morphologic changes with age. The parenchymal transformation includes metaplasia of some of its acini and their turning into Harderian gland-like structures (harderization), accumulation of gland ducts (“ductularization”), and morphologic dysplasia—cytomegaly, karyomegaly, and cell and nuclear polymorphism in the other part of acini. All these transformations are hormone-dependent and sex-specific: they more often appear in males. On the final stages of agerelated transformations, the lacrimal gland tissue is morphologically similar to a neoplasm and has neoplastic morphology but no other features of a tumor. Therefore, the rat lacrimal gland is an interesting object to study tissue and cell atypia. In the rat glandular stroma, lymphocytic infiltration and fibrosis appear with age; these changes are similar to processes taking place in human lacrimal apparatus involved in the pathogenesis of senile dry eye syndrome. The spontaneous changes in the rat lacrimal gland, predominantly in male rats, can be used as a model of the human lacrimal apparatus disorders.     

Calorie restriction: A new therapeutic intervention for age-related dry eye disease in rats

A decrease in lacrimal gland secretory function is closely related to aging and leads to an increased prevalence of dry eye syndrome. Since calorie restriction (CR) is considered to prevent functional decline of various organs due to aging, we hypothesized that CR could prevent age-related lacrimal dysfunction. Six-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR (−35%) groups. After 6 months of CR, tear function was examined under conscious state. After euthanasia, lacrimal glands were subjected to histological examination, tear protein secretion stimulation test with Carbachol, and assessment of oxidative stress with 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) antibodies. CR significantly improved tear volume and tended to increase tear protein secretion volume after stimulation with Carbachol compared to AL. The acinar unit density was significantly higher in the CR rats compared to AL rats. Lacrimal glands in the CR rats showed a lesser degree of interstitial fibrosis. CR reduced the concentration of 8-OHdG and the extent of staining with HNE in the lacrimal gland, compared to AL. Furthermore, our electron microscopic observations showed that mitochondrial structure of the lacrimal gland obtained from the middle-aged CR rats was preserved in comparison to the AL rats. Collectively, these results demonstrate for the first time that CR may attenuate oxidative stress related damage in the lacrimal gland with preservation of lacrimal gland functions. Although molecular mechanism(s) by which CR maintains lacrimal gland function remains to be resolved, CR might provide a novel therapeutic strategy for treating dry eye syndrome.     

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).     

The rat lacrimal gland expresses the alpha isoform of PKC. Further evidence for the PMA-activated and phospholipid-independent protein kinase activity.

The molecular heterogeneity of protein kinase C (PKC) is now widely documented. In our first report, we characterized the rat lacrimal gland PKC along with a phorbol 12-myristate 13-acetate (PMA)-activated and phospholipid-independent protein kinase activity [Mauduit P., Zoukhri D. and Rossignol B. (1989) Fedn Eur. biochem. Socs Lett. 252, 5-11. In this work, we show that when the rat lacrimal gland cytosolic fraction is chromatographed on hydroxyapatite, only one peak of PKC activity can be detected. Comparison with a rat brain cytosolic fraction indicated that it is PKC-alpha which is expressed in the rat lacrimal gland. This result was confirmed by the use of polyclonal antibodies raised against rat brain PKC-alpha, beta and gamma isoforms. We also provide evidence that free arachidonic acid activates PKC, as does PMA, in a calcium and phospholipid-free system.     

Characterization of lacrimal gland carbonic anhydrase VI.

We have previously demonstrated by immunohistochemistry the presence of secreted carbonic anhydrase (CA VI) in the acinar cells of the rat lacrimal glands. In this study we purified the sheep lacrimal gland CA VI to homogeneity and demonstrated by Western analysis that it has the same apparent subunit molecular weight (45 kD) as the enzyme isolated from saliva. RT-PCR analysis showed that CA VI mRNA from the lacrimal gland was identical to that of the parotid gland CA VI mRNA. An RIA specific for sheep CA VI showed the lacrimal gland tissue concentration of the enzyme to be 4.20 +/- 2.60 ng/mg protein, or about 1/7000 of the level found in the parotid gland. Immunohistochemistry (IHC) and in situ hybridization (ISH) showed that lacrimal acinar cells expressed both immunoreactivity and mRNA for CA VI. Moreover, CA VI immunoreactivity was occasionally observed in the lumen of the ducts. Unlike the parotid gland, in which all acinar cells expressed CA VI immunoreactivity and mRNA, only some of the acinar cells of the lacrimal gland showed expression. These results indicate that the lacrimal gland synthesizes and secretes a very small amount of salivary CA VI. In tear fluid, CA VI is presumed to have a role in the maintenance of acid/base balance on the surface of the eye, akin to its role in the oral cavity.     

Leucine aminopeptidase (LAP) activity and sexual dimorphism in rat exorbital lacrimal gland

Leucine amonopeptidase (LAP) activity was histochemically studied in the rat exorbital lacrimal gland. The enzyme is present in the secreting cells of both male and female prepuberal rats, in vivo and in monolayer cultures, while in the adult rat it is demonstrable only in the female. Furthermore, LAP is influenced by the sex hormones, disappearing in the female gland after testosterone treatment and appearing in the adult male gland after administration of estradiol or cyproterone acetate. These results show that the behaviour of LAP can be considered as another character of sexual dimorphism of the rat exorbital gland. Furthermore, our findings, showing an inverse relationship between LAP activity and the PAS positivity of the secretion products, suggest the hypothesis that the presence of this exopeptidase could induce qualitative modifications of one or more secreted glycoprotein.     

Alligator tears: a reevaluation of the lacrimal apparatus of the crocodilians

The Harderian gland is a poorly understood anterior ocular gland that occurs in most terrestrial vertebrates. Numerous extraorbital functions have been ascribed to the Harderian gland, principally based on its association with the nasolacrimal duct. Few studies have centered on archosaurs and the majority of those available focused solely on the Harderian gland of birds. Little is known about the lacrimal apparatus of the crocodilians. We examined the lacrimal apparatus of several specimens of Alligator mississippiensis anatomically, histologically, and histochemically and studied the embryogenesis of this system. The nasolacrimal duct possesses a distal secretory area, which is more convoluted than that of typical mammals or lepidosaurs. The alligator Harderian gland possesses a unique combination of characteristics found in lepidosaurs, birds, and mammals. Like that of both mammals and lepidosaurs, it is a large, tuboloacinar gland that appears to secrete both mucoprotein and lipids. However, the presence of blood vessels and immune cells is reminiscent of that of the avian Harderian gland. The immunogenesis of the alligator Harderian gland appears to be tied to the development of the vascular system. The presence of a distinct palpebral gland in the anterior aspect of the ventral eyelid is a feature unique to alligators. Based on position, this gland does not appear to be homologous to the anterior lacrimal gland of lepidosaurs. Lymphatic aggregations were also found in the palpebral gland. The presence of interstitial immune cells in the orbital glands of alligators suggests that the alligator lacrimal apparatus, like that of birds, may play a role in the head-associated lymphatic tissue system.     

Immunological characterization of soluble peroxidases from rat tissues including preputial gland

A highly active soluble peroxidase has been identified in the preputial gland of rats and characterized immunologically along with other soluble peroxidases of a number of rat tissues such as submaxillary gland, exorbital lacrimal gland and also of the uterine fluid of the estrogen treated rats. All these peroxidases have the native molecular weight around 73K as determined by gel filtration on Sephadex G-150. An antiserum raised against the pure bovine lactoperoxidase interacts with all these soluble peroxidases and immunoprecipitates the enzyme activity in a similar fashion when titrated against varied concentration of the antiserum. Following electrophoretic transfer to nitrocellulose by Western blotting, the antiserum crossreacts with the preputial, submaxillary and lacrimal gland protein of molecular weight around 73K and with the uterine fluid protein of molecular weight of 80K. An additional crossreacting protein of molecular weight of 80K is also evident in the lacrimal gland. All these enzyme preparations, however, contain another immunoreactive protein of molecular weight of about 64K. While 73–80K molecular weight interacting proteins may represent different forms of peroxidase, presumably with varied carbohydrate moieties, 64K molecular weight protein may be a precursor of the peroxidase which after posttranslational modification such as heme conjugation and glycosylation leads to formation of native enzyme. Rat harderian gland, unlike bovine origin, does not contain any detectable peroxidase activity. The immunoblot does not show the presence of any immunoreactive protein around 73K except the 64K molecular weight protein indicating that this gland can not synthesize the native peroxidase from this precursor probably due to some block in posttranslational modification.     

Lymphocyte migration into the lacrimal gland is random

The murine lacrimal gland demonstrates a high proportion of both IgA-producing cells and IgA-bearing cells, an observation consistent with tissues of the secretory immune system. This selective accumulation of IgA cells in the lacrimal gland was studied by observing the accumulation of various populations of fluorescein isothiocyanate-labeled lymphocytes in the gland. Labeled spleen cells were found to accumulate equally as well as mesenteric lymph node cells. T cells and T-cell-depleted populations of lymphocytes from spleen also showed migration into the gland. Surface staining of labeled cells found in the gland after 24 hr revealed that IgA-, IgG-, and IgM-bearing cells were all present in high proportions. These experiments demonstrate a random migration of lymphocytes into the lacrimal gland and suggest that the accumulation of IgA-bearing cells in the gland is regulated by the nonvascular microenvironment within the gland.     

Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction

Preferential outgrowth of the bud cells forms the basis of branching morphogenesis. Here, we show that lacrimal gland development requires specific modification of heparan sulfates by Ndst genes at the tip of the lacrimal gland bud. Systemic and conditional knockout experiments demonstrate the tissue specific requirement of Ndst1 and Ndst2 in the lacrimal gland epithelial, but not mesenchymal, cells, and the functional importance of Ndst1 in Fgf10-Fgfr2b, but not of Fgf1-Fgfr2b, complex formation. Consistent with this, Fgf10-induced ectopic lacrimal gland budding in explant cultures is dependent upon Ndst gene dose, and epithelial deletion of Fgfr2 abolishes lacrimal gland budding, its specific modification of heparan sulfate and its phosphorylation of Shp2 (Ptpn11 - Mouse Genome Informatics). Finally, we show that genetic ablation of Ndst1, Fgfr2 or Shp2 disrupts ERK signaling in lacrimal gland budding. Given the evolutionarily conserved roles of these genes, the localized activation of the Ndst-Fgfr-Shp2 genetic cascade is probably a general regulatory mechanism of FGF signaling in branching morphogenesis.     

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.     

Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.     

Dacryoadenopexy as a recognized factor in upper lid blepharoplasty

The recognition of a herniated lacrimal gland is done by simple examination; it is not usually taught to plastic surgeons. Fullness in the superotemporal aspect of an upper eyelid in younger patients often represents a ptotic gland. Suspension of the glands improves the surgical result of upper-lid blepharoplasty without compromising lacrimal outflow.     

Bevacizumab Eye Drop Treatment Stimulates Tear Secretion in Rats Through Changes in VEGF and NGF Lacrimal Gland Levels

VEGF and NGF are known to modulate corneal healing, neovascularisation and tear secretion. While a VEGF-NGF cross talk has been recently shown to modulate corneal healing in rats, it is not known whether it also plays a role in the regulation of lacrimal function. In this study we aim to investigate the effects of anti-VEGF eye drop treatment on lacrimal gland function and on the local expression of VEGF and NGF in rats. Tear function was measured in 3 months old rats by modified Schirmer test at baseline and after 3 weeks of topical anti-VEGF eye drop treatment. Whole lacrimal glands from rats were removed after treatment and analysed by ELISA for VEGF and NGF levels. To investigate if the effects of anti-VEGF were mediated by changes in the NGF-pathway, we repeated the experiments in RCS rats, a strain with NGF-pathway impairment associated with decreased tear flow. After topical treatment with anti-VEGF eye drops, an increase in tear secretion was observed in both wild-type and RCS rats. A significant decrease of VEGF levels was also observed in lacrimal glands of both RCS and SD rats, accompanied by a significant increase in NGF levels. Inhibition of VEGF at the ocular surface in rats results in changes of tear function and lacrimal gland levels of VEGF and NGF. Further studies on the VEGF/NGF cross-talk at the ocular surface may expand our knowledge on the pathogenesis of several diseases characterized by tear dysfunction.     

The presence of carbonic anhydrase in orbital glands. An enzyme-histochemical study in cynomolgus monkeys and rabbits

The distribution of carbonic anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbit. In the lacrimal gland of the cynomolgus monkey, a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.