Systematic variations in immune response-related gene transcript abundance suggest new questions about environmental influences on lacrimal gland immunoregulation.

PURPOSE: Retrospective analyses were undertaken to assess the hypothesis that environmental variables influenced immunophysiological status of lacrimal glands from untreated female rabbits that had been housed out-of-doors until they were acquired for use as controls for experimental studies. MATERIALS AND METHODS: Rabbits were euthanized within 5 days of arrival at University Vivaria. Glands were divided for histology and RNA extraction. Transcript abundances were determined with real time RT-PCR. Sections were stained for CD18 and rabbit thymic lymphocyte antigen. Environmental variables assessed were mean daily high temperature, low humidity, high temperature/low humidity ratio, and days with above average temperature/humidity ratio ("adverse days") during the prior 30 days. RESULTS: Spearman's analyses revealed numerous significant correlations. Numbers of T cells and abundances of mRNAs for CD8; CCL2, and CCL4; IL-1α and IL-1ß; the T(H)1 cytokine, IL-2; and the T(H)2- and B cell cytokines, IL-4, IL-6, IL-10, APRIL, and BAFF, all increased with adverse days, while IFN-γ mRNA abundance decreased. Glands from the group exposed to the most adverse days remained free of immunopathological lesions. Glands from the group exposed to the highest temperatures fell above the regression curves for IL-4, APRIL, and BAFF calculated for the other groups and had significantly higher abundances of mRNAs for prolactin, IL-18, CCL21, CCL28, CXCL8, and CXCL13. One of six glands from this group contained small immune cell aggregates; the others appeared normal. The only gland that presented with frank histopathology was from a group that had experienced benign conditions. CONCLUSIONS: Increasing adverse days correlated with increasing abundances of transcripts, including mRNAs for IL-2, IL-10, and CD8, outside the T(H)1/T(H)2 paradigm. The findings raise intriguing questions as to whether and how such changes might be associated with homeostatic phenomena.

Suppression of lymphocyte proliferation and regulation of dendritic cell phenotype by soluble mediators from rat lacrimal epithelial cells.

Lacrimal epithelial cells appear to constitutively secrete autoantigens to their underling stroma. The present experiments address the hypothesis that they also secrete soluble factors that regulate immune responses. Epithelial cells, spleen cells and lymphocytes were obtained from rabbits or rats and cultured in various configurations. Monocytes from rat bone marrow were matured to dendritic cells (DC) ex vivo. Proliferation was measured by [3H]-thymidine incorporation; surface MHC Class II and CD86 using flow cytometry; and mRNA relative abundances using real time RT-PCR. Microporous culture inserts containing rat lacrimal cells inhibited proliferation of rabbit lymphocytes co-cultured with autologous lacrimal cells and of rat lymphocytes co-cultured with TNF-alpha-stimulated DC. They inhibited CD86 and MHC Class II surface expression by maturating DC and reversed surface expression of CD86 but not MHC Class II by partially matured DC. Subsequent exposure of partially matured DC to mediators from rat lacrimal cells reversed the ability to stimulate lymphocyte proliferation. TGF-beta(1) and IL-10 mRNAs increased somewhat when rat lacrimal cells were isolated but decreased markedly in rabbit lacrimal cells. Antibodies to TGF-beta prevented soluble factors from rat lacrimal cells from inhibiting proliferation of rabbit lymphocytes co-cultured with rabbit lacrimal cells, but recombinant TGF-beta alone did not mimic the soluble factors. IL-10 immunopositivity was detected in epithelial cells of interlobular ducts and occasional interstitial cells in rabbit lacrimal gland. Rat lacrimal epithelial cells secrete TGF-beta and other factors that synergize to suppress lymphocyte proliferation and regulate DC maturation. Interlobular duct epithelial cells in rabbit lacrimal glands may express similar functions.

Autoimmune dacryoadenitis and keratoconjunctivitis induced in rabbits by subcutaneous injection of autologous lymphocytes activated ex vivo against lacrimal antigens.

Autologous peripheral blood lymphocytes (PBL), activated in a mixed cell reaction when co-cultured with purified rabbit lacrimal epithelial cells, are known to induce a Sjögren's-like autoimmune dacryoadenitis and keratoconjunctivitis when injected directly back into the donor animal's inferior lacrimal gland (LG). This study shows that autoreactive lymphocytes injected subcutaneously in a site away from the LG is capable of inducing an autoimmune disease in a rabbit. Induced disease (ID) develops more slowly, taking 4weeks as compared to 2weeks in the direct injection model. Initially, both clinical symptoms and histopathology are less pronounced than in the direct injection ID model, but later the immunocytochemistry shows the same CD4+/CD8+ ratio of 4:1 for both injection methods. The finding that lymphocytes activated against lacrimal antigens can travel or home from the injection site back to the inferior and superior LG, as well as the conjunctiva, suggests that these anatomical sites may have common epitopes that induce pathogenic CD4+ T cells that produce a Sjögren's-like syndrome.

Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist.

Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 microM carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca2+ and secrete beta-hexosaminidase in response to acute stimulation with 100 microM CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 microM CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal-lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of beta-hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)-binding proteins, Gq and G11, were decreased. Additional biochemical changes included decreased contents of 47 kDa Gs and Gi3, protein kinase Calpha and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K-ATPase from the plasma membranes to endomembrane compartments and decreased content of beta-hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor-signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.

Lacrimal gland epithelial cells stimulate proliferation in autologous lymphocyte preparations.

Autoimmune dacryoadenitis is a frequent cause of lacrimal insufficiency. In order to test hypotheses regarding mechanisms that can trigger this syndrome, we developed a method to obtain a preparation of rabbit lacrimal gland epithelial cells essentially free of immune-system cells. The method relies on controlled digestion to disperse lacrimal acini, and recovers acini by filtration through various sizes of nylon mesh. Purity and integrity of the preparation were established qualitatively using light and electron microscopy. Contamination by immune-system cells was quantitated by immunohistochemistry using anti-CD18, and -RTLA (rabbit thymic lymphocyte antigen) antibodies. The novel method produced preparations of highly-purified lacrimal gland epithelial cells (pLGEC) with expected morphological characteristics with less than 1.5% of the cells staining for CD18 or RTLA. The method also yielded preparations of lacrimal gland interstitial cells (LGIC) enriched for lymphocytes; in these preparations either CD18 or RTLA were detected on nearly 10% of the cells. pLGEC promoted proliferation in preparations of autologous splenic lymphocytes (SPL) that was blocked by anti-MHC class II but not anti-MHC class I antibodies. This observation, combined with the apparent requirement that pLGEC must contact the autologous lymphocyte preparation to promote proliferation, supports the hypothesis the proliferation arises from antigen-presentation via MHC class II by pLGEC.

Autologous lacrimal-lymphoid mixed-cell reactions induce dacryoadenitis in rabbits.

Autoimmune dacryoadenitis, such as occurs in Sjögren's syndrome, is a frequent cause of lacrimal insufficiency, which in turn can cause dry eye. Rabbits are used frequently to test ocular therapies. Our goal is to develop a rabbit model of autoimmune dacryoadenitis to identify and test candidate therapies. Our approach arises from the observations that lacrimal gland epithelial cells stimulate proliferation in cultured autologous lymphocyte preparations and that an anti-MHC II antibody blocks this proliferation. The purpose of this study was to determine if injecting this proliferating autologous mixed cell reaction could induce dacryoadenitis in rabbits. After establishing that irradiated lacrimal gland epithelial cells stimulate proliferation in autologous peripheral blood lymphocytes, irradiated cells from a single lacrimal gland were co-cultured with autologous lymphocytes and after 5 days the mixed cell reaction, or components of the reaction, were injected into the contralateral lacrimal gland of the donor rabbit. After 2 weeks, the injected glands were removed and lymphocytic infiltration quantitated using digital image analysis of immunostained histological sections. Injecting an autologous mixed cell reaction of co-cultured irradiated lacrimal gland epithelial cells and lymphocytes reliably induced abundant periductal foci of >200 lymphocytes expressing CD18 and/or a rabbit thymic lymphocyte antigen (RTLA). Injection of medium or autologous lymphocytes alone elicited little response; injections of lymphocytes cultured with lysates of lacrimal gland epithelial cells elicited variable, modest responses. These lysates did not stimulate proliferation in the mixed cell reaction and proliferation was not observed if a porous membrane separated co-cultured lacrimal gland cells and lymphocytes. The results demonstrate that injecting an autologous mixed cell reaction of lacrimal gland epithelial cells and lymphocytes reliably creates a model of autoimmune dacryoadenitis. The relative ineffectiveness of components of the reaction to do the same supports the hypothesis that lacrimal gland epithelial cells trigger or exacerbate lacrimal autoimmune disease by presentation of autoantigens via MHC II. This experimental system can aid efforts to further understand mechanisms of diseases, and to identify and test candidate therapies.

Prolactin and prolactin receptors in the lacrimal gland.

Light and electron microscopic immunocytochemistry, in situ hybridization and Dot Blot analysis revealed intracellular localization of prolactin-like molecules and prolactin mRNA in epithelial cells of the lacrimal glands of rabbits. There was also positive immunostaining for prolactin receptors on acinar cells and some interstitial cells. On Western blots of homogenates of whole lacrimal gland, isolated lacrimal acinar cells, isolated lacrimal interstitial cells and peripheral blood lymphocytes, prolactin antibody consistently labeled protein bands migrating at approximately 36 and 50 kD. These data confirm that lacrimal gland acinar cells produce endogenous prolactin-like molecules, but also express prolactin receptors. Since prolactin immunoreactivity has been detected in tear fluid and we found no accumulations of immunogold label in endocytic or transport vesicles, we hypothesize that the prolactin-like molecules in tear fluid originate primarily from synthesis within the acinar cells. We hypothesize further that prolactin from pituitary and other non-acinar cell origin has a modulating influence on acinar cell activity as well as immune function in the lacrimal gland, and that some of the prolactin-like molecules produced by the acinar cells contribute to these functions by autocrine/paracrine mechanisms.

Development of the vasculature of the anterior pituitary: ontogeny of basic fibroblast growth factor.

This study correlates the ontogeny of basic fibroblast growth factor (FGF) with the development of the vasculature of the anterior pituitary (AP) in two strains of rat, Sprague-Dawley (SD) and Fischer 344 (F344). Immunolocalization of FGF was followed from the first appearance of Rathke's pouch (RP) in 12-day (12d) fetuses, through each day of fetal development, and in 5, 20, and 50d postnatal female rats. In addition, the ontogeny of folliculo-stellate cells (FSC) is described, since previous studies suggested that these unique cells might function as phagocytes in the regulation of FGF. In both rat strains, vascularization of the AP commenced in 16d fetuses. In 15-20d fetuses, dense foci of immunopositivity for extracellular FGF were apparent at sites of capillary penetration adjacent to partially disrupted, immature gonadotropes. Localization of FGF was first detected in immature gonadotropes in 18d fetuses and persisted in the cytosol of a subpopulation of gonadotropes thereafter. In 15d fetuses, FGF was localized within the cytosol intimately associated with the peripheral-facing plasma membranes of all cells of the adenohypophysis, and persisted to variable degrees in later fetal stages. Localization of FGF within nuclei of AP parenchymal cells was evident only in 16-17d fetuses. Although the ontogeny of FGF and vascularization of the AP was very similar in both rat strains, the ontogeny of FSC differed markedly. In both strains, follicular lumens contained FGF during late fetal and early postnatal development. However, both electron microscopy and immunostaining for S-100 marker protein revealed that the postero-lateral edges of the AP of F344 rats often lacked FSC when compared to SD rats, a situation which could compromise regulation of FGF by FSC at the AP periphery in that strain, and thereby contribute to the neovascularization from systemic blood vessels known to occur in that strain during prolactinoma formation.

Is cellular disruption the mechanism of release of basic fibroblast growth factor from anterior pituitary gonadotropes?

Adult female Fischer 344 (F344) and Sprague-Dawley (SD) rats, intact and ovariectomized (10-30d), have been used for immunolocalization of basic fibroblast growth factor (FGF). Tissues were selected from three specific sites (postero-lateral, lateral wing, and anterior wedge) of the periphery of the anterior pituitary (AP) carefully maintaining the association between the gland and the highly vascular meningeal connective tissue which envelopes it. In both rat strains, most of the periphery of the AP was characterized by intact parenchymal cells delimited from the meningeal connective tissue by an intact basal lamina. However, foci also were evident in which parenchymal cells projected directly into the connective tissue without a basal lamina intervening. These zones, designated the Non-Delimited Peripheral Parenchyma (NDPP), were present minimally in control rats, but were more numerous in ovariectomized rats. Profiles of focally disrupted gonadotropes were evident within the NDPP of 20-30d ovariectomized rats juxtaposed against intact, granulated parenchymal cells. Partially disrupted gonadotropes also were evident within the peripheral parenchyma within approximately 100 mu of the edge, and occasionally the disruptions resulted in an association of neighboring gonadotropes as a syncytium. FGF was localized only within the cytosol of gonadotropes, i.e., cells immunopositive for LH-beta and FSH-beta subunits. Gonadotropes nearer to the edges of the AP, especially the postero-lateral edge, were the most intensely stained. Electron microscopy and immunostaining for S-100 protein, a marker for folliculo-stellate cells (FSC), demonstrated that in intact and ovariectomized SD rats FSC were present in all peripheral zones of the AP, whereas portions of the postero-lateral periphery of the AP of intact and ovariectomized F344 rats often lacked FSC. We propose that FGF may be released from the cytosol of gonadotropes by a mechanism of cellular disruption. FGF released at peripheral sites of the AP would be well-positioned to stimulate angiogenesis from systemic blood vessels within the meninges. Since FSC are known phagocytes within the AP, their consistent presence in the periphery of the AP of SD rats may help regulate the effects of the released FGF, and by contrast, their absence in F344 rats may intensify or prolong the effects of released FGF. Such differences may underlie the higher incidence of pituitary tumors in F344 rats.

Development of the vasculature of the pars distalis in a tumor-susceptible strain of rat (Fischer 344) differs from vascular development in a non-tumor-susceptible strain (Lewis).

The development of the vasculature of the pars distalis of two strains of rat, Fischer 344 (F344) and Lewis (LEW), was followed in 16-day (16d) and 20-day (20d) fetuses, and in 1-day (1d), 5d, 20d, 50d, and 6-month-old females. No differences in the two strains were apparent in 16d fetuses; and the capillaries that were present were immature, i.e., tall, non-fenestrated endothelial cells, and were surrounded by poorly delineated pericapillary spaces. Immature capillaries also were predominant in 20d fetuses of both strains. Agranular folliculo-stellate cells were identifiable, projecting endfeet to the parenchymal basal lamina in 20d F344 fetuses, but not in LEW fetuses. Postnatally, the capillaries of LEW rats became progressively more thin-walled and fenestrated, and were surrounded by a pericapillary space that was well delimited by basal laminae at 20d. In 50d and 6-month LEW rats, capillaries were intact and surrounded by well-defined pericapillary spaces. By comparison in F344 rats, the capillaries remained more immature even in 50d rats and older. In addition, in F344 rats focal disruptions in endothelial cells and disruptions in parenchymal and capillary basal laminae were present in all postnatal stages, and a dramatic accumulation of plasma was evident within the pericapillary spaces at 20d. Endfeet processes of folliculo-stellate cells were abundant at the parenchymal basal lamina of 1d and 5d F344 neonates, but only rarely were identified in LEW neonates. Some activation of folliculo-stellate cells, i.e., increased numbers of lysosomes and dilated endoplasmic reticulum, was present in 50d F344 rats. Connective-tissue cells within the pericapillary space also were numerous and activated in F344 rats. Discrete gaps in the parenchymal basal lamina were evident subjacent to the folliculo-stellate cell endfeet in F344 rats but not in LEW rats. The vascular bed of F344 rats differs in its development from that of LEW rats. Characteristic of the F344 strain is a persistence of more immature capillaries, an inherent vascular fragility, and an activated state of folliculo-stellate cells.

Spontaneous tumors in aging female mice are more prevalent in the lateral pituitary zones.

The localization of gross and microscopic spontaneous pituitary tumors was examined in aging female C57BL/6J mice. These tumors were lactotroph adenomas, by morphological and immunocytochemical criteria. Each lobe of the pars distalis was divided into three zones of approximately equal size and the number of tumors in each zone was counted. Twelve out of 30 tumors were located entirely within the most lateral zone. An additional 14 tumors occurred in both the most lateral and the interjacent zones. Thus, almost 90% of the observed tumors were localized in more lateral zones of the pars distalis (Chi-squared test, p less than 0.01). These findings support a hypothesis that lower portal blood dopamine levels reaching lateral portions of the pars distalis are a factor in the higher incidence of lactotroph adenomas in these zones.

Ultrastructural features of Adriamycin-induced skeletal and cardiac muscle toxicity.

In this study, the authors examined the effect of the anti-tumor agent Adriamycin, a known cardiotoxin, on mouse heart, diaphragm, and gastrocnemius muscle. Using an established model of Adriamycin cardiac toxicity, they found that 4 days after the intraperitoneal injection of 20 mg/kg of Adriamycin, characteristic heart lesions, including vacuolation of the sarcoplasmic reticulum, interstitial edema, and mitochondrial degeneration, were demonstrated in all treated animals. Furthermore, similar, but much more severe, myocyte damage was demonstrated in the diaphragm; muscle toxicity followed a decreasing gradient of injury from the peritoneal to the thoracic surface of the tissue. On the other hand, treatment with Adriamycin resulted in an increase in the size and number of lipid droplets in the red fibers of the gastrocnemius muscle without any other ultrastructural evidence of drug-induced damage to myocytes. An examination of the pharmacokinetics and metabolism of Adriamycin after intraperitoneal treatment revealed that relative drug levels in muscle (diaphragm much greater than heart much greater than gastrocnemius) paralleled the degree of ultrastructural damage observed. This study indicates that treatment with Adriamycin can produce significant injury to non-cardiac muscle in a fashion that strongly resembles the characteristic pattern of Adriamycin-related damage to the heart, and that the degree of myocyte damage is apparently dependent upon the Adriamycin concentration in the tissue.

Pituitary tumorigenesis in aging female C57BL/6J mice: a light and electron microscopic study.

Spontaneous pituitary tumors have been studied by light and electron microscopy in female C57BL/6J mice at 2 1/2, 11, 15, 22, 23, 24 and 30 months. Tumors were evident macroscopically in greater than 50% of mice 22 months or older, and greater than 80% on microscopic evaluation. Active and hypertrophied mammotrophs were the predominant cell type within the tumors at 22-30 months, often totally filling large portions of the tumor mass. Exocytosis of secretory granules was extensive from the mammotrophs, but much less abundant from other parenchymal cells. Somatotrophs and gonadotrophs were also present, and appeared active and often strikingly hypertrophied. The tumors were characterized by disruptions of parenchymal and capillary integrity which resulted in the formation of large vascular lakes lined solely by tumor cells, generally mammotrophs. Apparent metastasis of tumor cells into the vascular lakes was also observed. In 11- and 15-month mice small tumors or pretumor foci were evident in some mice on microscopic evaluation, although they were not visible macroscopically. Their degree of development was somewhat variable, but they had essentially the same features as more advanced tumors in older mice. pretumor foci were characterized by more moderate disruptions of parenchymal cell and capillary integrity; cellular hypertrophy, particularly of somatotrophs and gonadotrophs; and the presence of small vascular lakes. In 2 1/2-month mice tumors could not be localized macroscopically or microscopically, and the pituitary was composed of well-defined cell cords and an intact capillary bed. However, small focal zones of capillary and tissue disruption were apparent occasionally in 2 1/2-month mice. These findings indicate that the process of pituitary tumorigenesis in female C57BL/6J mice is initiated by midlife, with subsequent progressive development into large, mammotroph-dominated tumors.

Cations in the rat pars distalis ultrastructural localization.

Pituitary glands of female Sprague Dawley rats were fixed using the potassium pyroantimonate-osmium tetroxide technique by immersion or vascular perfusion. Both fixation procedures resulted in similar patterns of cation localization visualized as electron-dense precipitate within cells of the pars distalis. Nuclei were prominent sites of localization. Cytoplasmic precipitate occurred in association with the endoplasmic reticulum, typically within the cisternal spaces, and also was localized within the mitochondrial matrix and cristae, as well as Golgi membranes, small Golgi-associated vesicles, and multivesicular bodies. Immature secretory granules often contained precipitate between the core material of the granule and the enclosing smooth membrane. Frequently small antimonate-containing vesicles bordered the immature secretory granules. Precipitate was variable in secretory granules of more mature appearance although precipitate was apparent occasionally just within a granule's enclosing membrane. Granules closest to the plasma membrane often contained increased amounts of precipitate and small vesicles containing precipitate were observed fusing with them. Instances of granule release by emiocytosis often revealed a clustering of precipitate behind the core material away from the emiocytotic stoma, as well as at the stoma, and frequently an increased electron density of filamentous material radiating from the granules' enclosing membranes or from the adjacent plasma membrane. Exposure of section material to the chelating agents EGTA and EDTA indicate that calcium is the primary cation localized within the cytoplasm of these secretory cells. These findings are consistent with a role for calcium as a facilitator in the processes of transport and release of secretory granules.