Identification of steroidogenic enzyme mRNAs in the human lacrimal gland, meibomian gland, cornea, and conjunctiva

PURPOSE: Sex steroids exert a significant influence on the health and well-being of the ocular surface and adnexa. These hormones affect multiple aspects of the lacrimal and meibomian glands, conjunctiva, and cornea, and have been linked to the development of many ocular surface pathologies. We hypothesize that these hormone actions, as in other tissues, occur predominantly after the local synthesis of androgens and estrogens from adrenal precursors. To begin to test this hypothesis, we analyzed whether human ocular surface and adnexal tissues and cells contain the steroidogenic enzyme mRNAs necessary for the intracrine synthesis and metabolism of sex steroids. METHODS: Total RNA was isolated from human lacrimal and meibomian glands and immortalized corneal and conjunctival epithelial cells. Samples were reverse transcribed to cDNA and analyzed for the presence of enzyme mRNAs by real-time PCR. Positive and negative controls included human placental cDNA and the absence of template, respectively. RESULTS: Our results show that human lacrimal and meibomian glands and corneal and conjunctival epithelial cells contain the mRNAs for steroid sulfatase, 3beta-hydroxysteroid dehydrogenase (HSD)-Delta-Delta-isomerase type 1, 17beta-HSD types 1 and 3, aromatase, and glucuronosyltransferase. In contrast, only lacrimal and meibomian tissues appeared to contain detectable mRNA for sulfotransferase. CONCLUSIONS: If the corresponding mRNAs are translated, our results indicate that human ocular surface and adnexal tissues contain the enzymatic machinery necessary for the intracrine synthesis and metabolism of sex steroids.     

Immunofluorescence localization of cystatins in human lacrimal gland and in the exorbital lacrimal gland of the rat.

Cystatins are widely distributed natural inhibitors of cysteine proteinase. They occur both intra and extracellularly in various cells and tissue fluids including tears. Using an immunofluorescence technique with antibodies against rat cystatin S, an inhibitor of submandibular gland origin, cystatin-like immunoreactive material was demonstrated in the acinar cells of the exorbital lacrimal gland of the rat. Administration of the cholinergic agonist carbachol caused a depletion of cystatin from the acinar cells. This depletion was followed by a partial restitution in 6-8 h. Administration of the beta-adrenergic agonist isoproterenol for 4 days, which caused a marked hypertrophy of the submandibular gland, had no effect on the structure, weight, or cystatin content of the exorbital lacrimal gland. After such treatment, however, single large cells with intense staining for cystatin were encountered. Cystatin-like immunoreactive material was also demonstrated in human lacrimal gland using antibodies against human cystatin S. These data suggest the notion that tear cystatins are secreted by the lacrimal glands.     

糖尿病大鼠泪腺、结膜及角膜组织病理学观察

目的 探讨糖尿病造成的泪腺、角膜、结膜组织损伤。方法 雄性SD大鼠40只。随机分为糖尿病组和对照组，各20只。分别于成模后6、8、10、12周断颈处死大鼠，每组各5只。光镜下观察泪腺、角膜、结膜组织病理学改变。结果 糖尿病组随时间依次表现为泪腺细胞水肿、增生，腺泡及导管萎缩，纤维增生及淋巴细胞浸润，角膜上皮、基质层水肿，结膜杯状细胞减少。结论 糖尿病导致的泪腺、角膜、结膜等眼组织破坏是糖尿病性眼表病变的病理基础。     

Downward extension of the lacrimal gland under the bulbar conjunctiva after trabeculectomy

PURPOSE: To describe two cases in which the lacrimal gland was considered to extend downward under the bulbar conjuctiva following trabeculectomy. PATIENTS AND METHODS: We report two patients who complained of epiphora after trabeculectomy. A biopsy was performed on the tissue under the bulbar conjunctiva on the periphery of the bleb of case 1. The anatomical relationship between the holes and the avascular filtering bleb in case 2 was investigated using ultrasound biomicroscopy. RESULTS: In case 1, a biopsy showed the lacrimal gland tissue filtrated with mononuclear cells. In case 2, ultrasound biomicroscopy revealed no connection between the multiple leaking holes and localized filtering bleb. CONCLUSION: Following trabeculectomy at the superior temporal quadrant, we should consider downward extension of the lacrimal gland as a possible source of a patient's complaint of epiphora.     

正己烷染毒大鼠结膜及泪腺损伤的组织病理学和超微结构观察

目的研究正己烷染毒大鼠结膜和泪腺组织的损伤。方法32只雄性SD大鼠随机分成对照组和染毒组。采取静式吸入染毒方式，正己烷浓度为35．2g／m^3，染毒1、3、7、14d后处死大鼠，对照组为吸入空气的空白对照。观察结膜和泪腺组织的组织病理学和超微结构的改变。结果染毒7d和14d大鼠结膜上皮细胞微绒毛减少。部分基底细胞出现核固缩和核内空泡，线粒体水肿、缩小或消失。染毒14d大鼠结膜杯状细胞明显减少。正己烷染毒大鼠的泪腺细胞出现水肿和淋巴细胞浸润。结论正己烷染毒大鼠的结膜和泪腺组织出现明显的损害，将会影响正常泪膜稳定性的维持，导致眼表疾病的发生。     

T-lymphocyte subsets in the human lacrimal gland

This study examines the microscopical appearance, location, distribution, subdivision and density of T-lymphocytes in the human lacrimal gland. Fourteen glands, 7 from either sex, were removed and frozen shortly after the donors' death, and processed for immunoperoxidase staining, utilizing a biotinavidin system and one of the following monoclonal antibodies: Anti-Leu-1 and -Leu-4, which recognize T-lymphocytes, anti-Leu-2a, which binds to suppressor/cytotoxic T-cells, and anti-Leu-3a + 3b, which recognizes helper/inducer cells. The T-lymphocytes in the human lacrimal gland were small to medium sized, mainly located in the interacinar tissue, often adjacent to an acinus or close to a collecting duct. The median number of each T-cell subset per defined field (0.086 mm2) at x 500 magnification was as follows: Males: Leu-1: 3.1, Leu-2a: 4.9, Leu-3a + 3b: 2.8, Leu-4: 4.5. Females: Leu-1: 4.3, Leu-2a: 5.0, Leu-3a + 3b: 3.7 and Leu-4:5.8. The sex difference was not statistically significant. The helper/suppressor cell ratio in the human lacrimal gland was 0.57 for males and 0.74 for females.     

Immunopathology of ocular onchocerciasis 3. Th-2 helper T cells in the conjunctiva

Ocular onchocerciasis is the second most common infectious cause of blindness in the world. Th-2 helper T (Th-2) cells are thought to play a critical role in immediate hypersensitivity against allergens and extra-cellular parasitic infections. Th-2 cells secrete a high amount of IL-4, regulate IgE production, and recruit eosinophils and mast cells. Conjunctival biopsies from ten African patients with ocular onchocerciasis were evaluated for the presence of II-2, IL-4, mast cells and major basic protein (MBP), a marker for eosinophils. IL-4 mRNA was detected in seven of ten conjunctival specimens using in situ hybridization, yet IL-4 was detected in only one specimen using immunohistochemical staining. In contrast, IL-2 mRNA was detected in three of ten conjunctival specimens and IL-2 was detected in two specimens. There were greater numbers of mast cells and the presence of MBP in specimens with IL-4 mRNA. Furthermore, the three biopsies containing both IL-2 and IL-4 mRNA had greater numbers of CD4+cell infiltration and the patient with IL-4 protein in his conjunctiva also had the highest IgE in his aqueous humor. These findings suggest that Th-2 cells and their lymphokines are important for the localized host responsiveness to ocular onchocerciasis.     

Identification of T lymphocytes in ocular adnexal neoplasms by hybridoma monoclonal antibodies

We performed indirect immunofluorescence with the OKT series of hybridoma monoclonal antibodies in order to determine the total number of T cells and their subset distribution, that is, the percentages of helper (OKT3+T4+T8-) and suppressor/cytotoxic (OKT3+T4-T8+) T cells, in 28 ocular adnexal lymphoid neoplasms. OKT3+T4+ (helper) T cells vastly predominated in each of ten benign, polyclonal ocular lymphoid proliferations. The helper-suppressor T-cell ratio (T4-8) ranged from 2.5 to 8.2 (mean, 5.4) in these ten cases. In comparison, the mean T4-T8 ratio in 24 benign reactive lymph nodes was 3.4. These findings strongly suggested that the polyclonal ocular lymphoid proliferations represent a T-cell antigen-dependent response characterized by a proliferation of helper T cells, which in turn drive B cells to proliferate and to differentiate, eventually resulting in the formation of a clinically detectable tumor. The mean T4-T8 ratio was 2.3 in 18 ocular and in 16 nodal monoclonal B-cell proliferations, suggesting that the benign T cells in these proliferations represent a residual cell population rather than a distinctive subset originating in response to the B-cell neoplasm.     

Immunopathology of ocular onchocerciasis 3. Th-2 helper T cells in the conjunctiva

Ocular onchocerciasis is the second most common infectious cause of blindness in the world. Th-2 helper T (Th-2) cells are thought to play a critical role in immediate hypersensitivity against allergens and extra-cellular parasitic infections. Th-2 cells secrete a high amount of IL-4, regulate IgE production, and recruit eosinophils and mast cells. Conjunctival biopsies from ten African patients with ocular onchocerciasis were evaluated for the presence of II-2, IL-4, mast cells and major basic protein (MBP), a marker for eosinophils. IL-4 mRNA was detected in seven of ten conjunctival specimens using in situ hybridization, yet IL-4 was detected in only one specimen using immunohistochemical staining. In contrast, IL-2 mRNA was detected in three of ten conjunctival specimens and IL-2 was detected in two specimens. There were greater numbers of mast cells and the presence of MBP in specimens with IL-4 mRNA. Furthermore, the three biopsies containing both IL-2 and IL-4 mRNA had greater numbers of CD4+cell infiltration and the patient with IL-4 protein in his conjunctiva also had the highest IgE in his aqueous humor. These findings suggest that Th-2 cells and their lymphokines are important for the localized host responsiveness to ocular onchocerciasis.     

T cell populations in the lacrimal gland during aging

The present study examined the influence of age and gender on T cell populations in the lacrimal gland. Lacrimal (exorbital) glands were obtained from male and female rats at 19 days (pre-puberty), 9 weeks (adult) and 14 months (mid-life) of age and tissues were processed for T cell subset identification. In females, the density of total (W3/13+ and OX 19+), helper/inducer (W3/25+) and suppressor/cytotoxic (OX 8+) T cells underwent a significant increase in tissues from before, to after puberty. Following this rise, the density of all T cell populations decreased in glands from young adult to midlife females. This pattern of accumulation contrasted with the T cell profile presented by glands from males: T cell densities appeared unaffected from 19 days to 9 weeks of age, and then either declined (OX 19+, W3/25) or remained unchanged (W3/13+, OX 8+) in tissues of 14 month rats. An influence of gender on the distribution of T cells was also apparent if results were corrected for age-associated variations in lacrimal gland weight. Thus, the absolute number of all T cell populations rose dramatically in glands of both sexes from pre- to post-puberty. However, from 9 weeks to 14 months of age, the total content of W3/13+, OX 19+, W3/25+ and OX 8+ lymphocytes decreased 2-fold in glands of females, but did not vary in tissues of males. Of interest, the number of W3/25+ and OX 8+ cells was analagous in all age groups examined. Moreover, the combined total of W3/25+ and OX 8+ cells was greater than that of W3/13+ or OX 19+ cells at every age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective interactions of lymphocytes with neonatal and adult lacrimal gland tissues

The lacrimal gland is a functional part of the mucosal immune system and is populated by lymphoid cells that begin to appear early in neonatal development. To define the events controlling the accumulation of these cells, an in vitro adherence assay was used to investigate the interactions of lymphocyte populations with neonatal and adult lacrimal tissue. It was found that (1) lymphocytes adherence to neonatal lacrimal tissue is significantly enhanced over that seen in adults; (2) the increased binding is caused, in part, by adherence to nonacinar structures in neonatal tissue; (3) binding to both neonatal and adult lacrimal gland tissue is an active process that is observed only with viable lymphocytes; (4) lymphocyte adherence to neonatal and adult lacrimal gland tissues is differentially affected by metabolic and cytoskeletal inhibitors; and (5) attachment appears to require the presence of Ca++, a lymphocyte surface protein, and may involve target tissue carbohydrate recognition. These findings suggest that the initial accumulation of lymphocytes in the neonatal lacrimal gland results from a generalized enhanced binding capacity of the developing tissue and that the preferential binding of certain populations (thoracic duct and mesenteric lymph node lymphocytes) to acinar cells maintains the pool in the adult lacrimal gland.     

The T cell receptor in normal and inflamed human conjunctiva.

The majority of human peripheral blood and lymphoid tissue T cells express the TCR alpha/beta heterodimer, while the TCR gamma/delta is expressed on only a small subset of T lymphocytes. However, the majority of murine intraepithelial lymphocytes and most Thy-1+ murine dendritic epidermal cells express the TCR gamma/delta. Selective homing of avian TCR gamma/delta bearing lymphocytes to the intestinal epithelium also has been shown. These findings have suggested that these cells play a role against transformation and infection. More recently, a role in autoimmunity also has been proposed. We examined normal human conjunctiva and inflamed conjunctiva from patients with ocular cicatricial pemphigoid (OCP), an autoimmune disorder, and atopic keratoconjunctivitis (AKC). The majority of T cells in the epithelium and substantia propria of normal conjunctiva expressed the TCR alpha/beta. Tropism of TCR gamma/delta-expressing lymphocytes to normal human conjunctiva was not present. However, in OCP, there was a statistically significant increase in the absolute number of TCR gamma/delta cells/mm2 (epithelium, 33.9 +/- 10.5 [mean +/- standard error of the mean] vs. 159.9 +/- 51.5, P = less than 0.0008; substantia propria, 4.1 +/- 0.9 vs. 240.1 +/- 191.3, P less than 0.002) and TCR gamma/delta cells as a percentage of CD3+ cells (epithelium, 0.18 +/- 0.06 vs. 0.39 +/- 0.07, P = less than 0.02; substantia propria, 0.10 +/- 0.05 vs 0.33 +/- 0.08, P = less than 0.03). This was not the case for AKC. These findings suggest that TCR gamma/delta lymphocytes play a specific but undefined role in certain conjunctival inflammatory conditions and may be important in autoimmunity.     

Immunohistochemical localization of epidermal growth factor receptor and epithelial antigen in tumors of the human conjunctiva,eyelid, lacrimal gland,and orbit

Background: Increased numbers of epidermal growth factor (EGF) receptors are observed in squamous cell carcinomas of human lung, head, neck, and cervix. We studied the presence of EGF receptors and epithelial antigen in some ophthalmic lesions. Methods: Immunohistochemical staining for EGF receptors was assessed in tumors of human conjunctiva, eyelid, lacrimal gland, and orbit with monoclonal antibodies (EGF-R1 and clone 29.1). Reactivity of Ber-EP4, which reocgnizes epithelial antigen, was also examined. Results: Strong staining of EGF-R1 and clone 29.1 and weak to moderate staining of Ber-EP4 were demonstrated in conjunctival squamous cell carcinomas. Cell membranes of conjunctival papilloma were moderately or strongly stained with these antibodies. Ductal components in sebaceous gland adenoma of the eyelid and pleomorphic adenoma of the lacrimal gland were positively stained. The antibodies did not bind to reactive lymphoid hyperplasia of the orbit and Wegener's granulomatosis. Relatively good correlation for immunostaining reaction was observed among EGF-R1, clone 29.1, and Ber-EP4 in each tumor. Conlcusion: Immunostaining using EGFR1, clone 29.1, and Ber-EP4 may be useful in differentiating epithelial tumors from non-epithelial lesions. Strong immunostaining for EGF receptor may be the hallmark of epidermoid malignancy.