Antigenic profile of the human lacrimal gland.

The antigenic profile of 13 normal formalin-fixed, paraffin-embedded human main and accessory lacrimal glands, biopsied from patients aged 11 to 78 years, was studied using a panel of 27 polyclonal and monoclonal antibodies. Secretory cells of lacrimal acini reacted with antibodies to S-100 protein and simple epithelium-type cytokeratins CK 7, CK 8, CK 18, and CK 19. Their luminal membranes were labeled with antibodies to carcinoembryonic antigen, epithelial membrane antigen, and epithelial glycoproteins recognized by Ber-EP4. Myoepithelial cells were often immunopositive for S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), and alpha-smooth muscle actin. More rarely, they reacted with antibodies recognizing CK 5, CK 13, and CK 14, which consistently labeled the basal cells of lacrimal ducts. Unlike myoepithelial cells, basal ductal cells were immunopositive for CK 7, CK 8, CK 18, and CK 19. In main excretory ducts, dendritic melanocyte-like cells co-expressing vimentin and S-100 protein intermingled with ductal epithelial cells. The luminal cells of lacrimal ducts basically paralleled secretory cells in their antigenic profile, although they lacked Ber-EP4 and were immunopositive for CK 4. Antibodies to neuron-specific enolase and synaptophysin reacted with nerve fibers among negatively reacting secretory acini. This antigenic profile closely parallels that of salivary glands and provides a basis for studies of lacrimal gland pathology.     

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.     

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.     

Characterization of immortalized rabbit lacrimal gland epithelial cells

Summary To establish an immortalized lacrimal gland epithelial cell line, the orbital lacrimal glands of normal New Zealand White rabbits were multiply injected with an immortalizing amphotropic retroviral vector (LXSN16E6E7) containing the E6 and E7 genes of human papillomavirus type 16. Lacrimal glands were removed after 2 d and acinar epithelial cells were isolated and cultured on Matrigel-coated 60 mm² plates containing DMEM-F12 supplemented with 5% Nu-serum V. Transformed cells were selected in G418 sulfate for 7 d and passaged. Morphology of the immortalized cells was similar to that described for normal acinar cells both in vivo and in vitro, with rough endoplasmic reticulum and secretory granules. These characteristics remained unchanged and the cells continued to exhibit typical polygonal epithelioid structure. The cells have been maintained in culture for 14 mo. and have gone through 58 passages without loss of proliferation or epithelial cell characteristics. Immunohistochemistry and Western blots showed positive reactivity to secretory component, transferrin, and transferrin receptor, which are typical proteins found in the lacrimal gland. Functional analysis by stimulation with a cholinergic agonist, carbachol (100 μM), resulted in a significant release of protein. This is the first report of an immortalized rabbit lacrimal epithelial cell. These cells will provide a valuable tool for the molecular analysis of lacrimal gland epithelial cell functions.     

Exploring the human lacrimal gland using organoids and single-cell sequencing

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Intracellular pH regulation in the mouse lacrimal gland acinar cells

Summary Intracellular pH (pH _i ) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pH _i was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pH _i only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH ₄ ⁺ and bicarbonate buffer solution gassed with 5% CO₂/95% O₂ was 26 and 46mm/pH, respectively Stimulation with 1 m acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pH _i . In the presence of amiloride (0.1mm) or the absence of Na⁺, application of ACh caused a significant decrease in pH _i and removal of amiloride or replacement with Na⁺-containing saline, respectively, rapidly increased the pH _i . Pretreatment with DIDS (0.2mm) did not change the pH _i of the nonstimulated conditions; however, it significantly enhanced the increase in pH _i induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na⁺ removal from the bath solution. We suggest that both Na⁺/H⁺ and HCO ₃ ^– /Cl^– exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.     

Establishing human lacrimal gland cultures with secretory function

Purpose

Dry eye syndrome is a multifactorial chronic disabling disease mainly caused by the functional disruptions in the lacrimal gland. The treatment involves palliation like ocular surface lubrication and rehydration. Cell therapy involving replacement of the gland is a promising alternative for providing long-term relief to patients. This study aimed to establish functionally competent lacrimal gland cultures in–vitro and explore the presence of stem cells in the native gland and the established in-vitro cultures.

Methods

Fresh human lacrimal gland from patients undergoing exenteration was harvested for cultures after IRB approval. The freshly isolated cells were evaluated by flow cytometry for expression of stem cell markers ABCG2, high ALDH1 levels and c-kit. Cultures were established on Matrigel, collagen and HAM and the cultured cells evaluated for the presence of stem cell markers and differentiating markers of epithelial (E-cadherin, EpCAM), mesenchymal (Vimentin, CD90) and myofibroblastic (α-SMA, S-100) origin by flow cytometry and immunocytochemistry. The conditioned media was tested for secretory proteins (scIgA, lactoferrin, lysozyme) post carbachol (100 µM) stimulation by ELISA.

Results

Native human lacrimal gland expressed ABCG2 (mean±SEM: 3.1±0.61%), high ALDH1 (3.8±1.26%) and c-kit (6.7±2.0%). Lacrimal gland cultures formed a monolayer, in order of preference on Matrigel, collagen and HAM within 15–20 days, containing a heterogeneous population of stem-like and differentiated cells. The epithelial cells formed ‘spherules’ with duct like connections, suggestive of ductal origin. The levels of scIgA (47.43 to 61.56 ng/ml), lysozyme (24.36 to 144.74 ng/ml) and lactoferrin (32.45 to 40.31 ng/ml) in the conditioned media were significantly higher than the negative controls (p<0.05 for all comparisons).

Conclusion

The study reports the novel finding of establishing functionally competent human lacrimal gland cultures in-vitro. It also provides preliminary data on the presence of stem cells and duct-like cells in the fresh and in-vitro cultured human lacrimal gland. These significant findings could pave way for cell therapy in future.     

Carbonic anhydrase isozyme VI in rat lacrimal gland

Using monoclonal antibody specific to rat carbonic anhydrase isozyme VI (CA VI), the isozyme was localized in the lacrimal gland. A minority of acini (less than 10% of the total) contained a few immunoreactive acinar cells. Enzyme histochemistry indicated that the CA VI-positive cells were the only cells possessing CA in the lacrimal acini. In the acinar cells, the reaction product for CA VI was distributed in the secretory granules and cytosol between secretory granules. Except for mitochondrial enzyme (CA V) activity, the intracellular distribution of enzyme activity was similar to that of CA VI immunoreactivity, suggesting that rat lacrimal acinar cells contain only CA VI and CA V. CA VI in the secretory granules was discharged into the acinar lumen and is considered to carry out its function on the surface of the conjunctiva and cornea. The cytosolic CA VI may function in situ and be involved in electrolyte and water secretion by the acinar cells. Polyclonal antibody to rat erythrocyte CA (CA I and CA II) stained only the interlobular ducts. In contrast, all the ductal elements exhibited CA enzyme activity. This discrepancy between immunohistochemistry and enzyme histochemistry suggests the presence of CA isozyme(s) other than CA I, CA II and CA VI in the lacrimal duct.     

Calcium dependence of exocytosis in lacrimal gland acinar cells

Simultaneous measurements of membranecapacitance and intracellular calcium concentration were used toexamine the calcium dependence of exocytosis in single acinar cellsfrom mouse lacrimal gland and to establish the quantitative relationbetween calcium concentration and rate of exocytosis. Application ofadrenergic or muscarinic agonists elevated intracellular calcium andevoked exocytosis, as indicated by an increase in membrane capacitance of single cells. The capacitance response to agonist stimulation waseliminated by internal dialysis with the calcium buffer EGTA, whichdemonstrated that the increase in intracellular calcium was necessaryfor agonist-evoked exocytosis. When internal calcium was elevated byapplication of the calcium ionophore ionomycin, exocytosis was evokedin the absence of agonist stimulation. Thus an increase inintracellular calcium was necessary and sufficient for exocytosis insingle acinar cells. The rate of change of membrane capacitanceincreased as approximately the third power of the calciumconcentration, which is similar to the dependence of exocytosis rate oncalcium concentration in other secretory cells.