Choroidal Mast Cells Research Articles

Choroidal Mast Cells and Pathophysiology of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) remains a leading cause of vision loss in elderly patients. Its etiology and progression are, however, deeply intertwined with various cellular and molecular interactions within the retina and choroid. Among the key cellular players least studied are choroidal mast cells, with important roles in immune and allergic responses. Here, we will review what is known regarding the pathophysiology of AMD and expand on the recently proposed intricate roles of choroidal mast cells and their activation in outer retinal degeneration and AMD pathogenesis. We will focus on choroidal mast cell activation, the release of their bioactive mediators, and potential impact on ocular oxidative stress, inflammation, and overall retinal and choroidal health. We propose an important role for thrombospondin-1 (TSP1), a major ocular angioinflammatory factor, in regulation of choroidal mast cell homeostasis and activation in AMD pathogenesis. Drawing from limited studies, this review underscores the need for further comprehensive studies aimed at understanding the precise roles changes in TSP1 levels and choroidal mast cell activity play in pathophysiology of AMD. We will also propose potential therapeutic strategies targeting these regulatory pathways, and highlighting the promise they hold for curbing AMD progression through modulation of mast cell activity. In conclusion, the evolving understanding of the role of choroidal mast cells in AMD pathogenesis will not only offer deeper insights into the underlying mechanisms but will also offer opportunities for development of novel preventive strategies.

Assessment of Choroidal Vasculature and Innate Immune Cells in the Eyes of Albino and Pigmented Mice.

The visualization of choroidal vasculature and innate immune cells in the eyes of pigmented mice has been challenging due to the presence of a retinal pigment epithelium (RPE) layer separating the choroid and retina. Here, we established methods for visualizing the choroidal macrophages, mast cells, and vasculature in eyes of albino and pigmented mice using cell type-specific staining. We were able to visualize the choroidal arterial and venous systems. An arterial circle around the optic nerve was found in mice similar to the Zinn–Haller arterial circle that exists in humans and primates. Three different structural patterns of choriocapillaris were observed throughout the whole choroid: honeycomb-like, maze-like, and finger-like patterns. Choroidal mast cells were relatively few but dense around the optic nerve. Mast cell distribution in the middle and periphery was different among strains. Macrophages were found in all layers of the choroid. Thus, utilizing the simple and reliable methods described herein will allow the evaluation of transgenic and preclinical mouse models of ocular diseases that affect the choroid, including age-related macular degeneration (AMD), diabetic choroidopathy, and retinopathy of prematurity. These studies will advance our understanding of the pathophysiology, and molecular and cellular mechanisms that can be targeted therapeutically, in these diseases.

Topical Ketotifen Fumarate Inhibits Choroidal Mast Cell Degranulation and Loss of Retinal Pigment Epithelial Cells in Rat Model for Geographic Atrophy.

PurposeThis study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model.MethodsPharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function.ResultsPK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF.ConclusionsBoth 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA.Translational RelevanceThe results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.

Retinal Distribution and Extracellular Activity of Granzyme B: A Serine Protease That Degrades Retinal Pigment Epithelial Tight Junctions and Extracellular Matrix Proteins.

Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. In vitro studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

Single-cell profiling reveals an endothelium-mediated immunomodulatory pathway in the eye choroid.

The activity and survival of retinal photoreceptors depend on support functions performed by the retinal pigment epithelium (RPE) and on oxygen and nutrients delivered by blood vessels in the underlying choroid. By combining single-cell and bulk RNA sequencing, we categorized mouse RPE/choroid cell types and characterized the tissue-specific transcriptomic features of choroidal endothelial cells. We found that choroidal endothelium adjacent to the RPE expresses high levels of Indian Hedgehog and identified its downstream target as stromal GLI1+ mesenchymal stem cell-like cells. In vivo genetic impairment of Hedgehog signaling induced significant loss of choroidal mast cells, as well as an altered inflammatory response and exacerbated visual function defects after retinal damage. Our studies reveal the cellular and molecular landscape of adult RPE/choroid and uncover a Hedgehog-regulated choroidal immunomodulatory signaling circuit. These results open new avenues for the study and treatment of retinal vascular diseases and choroid-related inflammatory blinding disorders.

Increased choroidal mast cells and their degranulation in age-related macular degeneration

Background/aimsInflammation has been implicated in age-related macular degeneration (AMD). This study investigates the association of mast cells (MCs), a resident choroidal inflammatory cell, with pathological changes in AMD.MethodsHuman donor eyes...

Choroidal Mast Cells in Retinal Pathology: A Potential Target for Intervention

Mast cells are important in the initiation of ocular inflammation, but the consequences of mast cell degranulation on ocular pathology remain uncharacterized. We induced mast cell degranulation by local subconjunctival injection of compound 48/80. Initial degranulation of mast cells was observed in the choroid 15 minutes after the injection and increased up to 3 hours after injection. Clinical signs of anterior segment inflammation paralleled mast cell degranulation. With the use of optical coherence tomography, dilation of choroidal vessels and serous retinal detachments (SRDs) were observed and confirmed by histology. Subconjunctival injection of disodium cromoglycate significantly reduced the rate of SRDs, demonstrating the involvement of mast cell degranulation in posterior segment disorders. The infiltration of polymorphonuclear and macrophage cells was associated with increased ocular media concentrations of tumor necrosis factor-α, CXCL1, IL-6, IL-5, chemokine ligand 2, and IL-1β. Analysis of the amounts of vascular endothelial growth factor and IL-18 showed an opposite evolution of vascular endothelial growth factor compared with IL-18 concentrations, suggesting that they regulate each other's production. These findings suggest that the local degranulation of ocular mast cells provoked acute ocular inflammation, dilation, increased vascular permeability of choroidal vessels, and SRDs. The involvement of mast cells in retinal diseases should be further investigated. The pharmacologic inhibition of mast cell degranulation may be a potential target for intervention.

Role of interleukin 33/ST2 axis in the immune-mediated pathogenesis of age-related macular degeneration

BackgroundAge-related macular degeneration is a leading cause of irreversible blindness. Altered immune responses drive degeneration, in response to oxidative stress and hypoxia-induced regulation of metabolism. We tested the hypothesis that toll-like receptor activation of retinal pigment epithelium and cellular metabolic switch upregulate interleukin 33, which acts through its receptor ST2 to activate both choroidal stromal fibroblasts and mast cells. By such mechanisms, the fibrosis and insidious degeneration, which we observe clinically, is accentuated. MethodsRetinal pigment epithelial cells (ARPE-19 and B6-RPE07) were stimulated with toll-like receptor ligands, and energetic pathways were assessed through lactate production and the expression of glycolytic enzymes. Expression profile and secretion of interleukin 33 were determined by RT-PCR and western blots. Function and expression profile of bone-marrow-derived mast cells and human choroidal fibroblasts were also assessed. FindingsThe production of lactate, determining aerobic glycolysis, increased after stimulation of retinal pigment epithelial cells with LPS or poly(I:C), indicating an increase in the glycolytic activity after toll-like receptor stimulation. Increased levels of GLUT1 transcripts, and upregulation of GAPDH expression corroborated this finding. Furthermore, increased expression of interleukin 33 was dependent on a glycolytic metabolic switch and was enhanced under hypoxic conditions. ST2 was highly expressed in retinal pigment epithelium, choroidal mast cells, and choroidal fibroblasts in mouse and man. ST2+ bone-marrow-derived mast cells generated a spectrum of inflammatory cytokines and PGS2 when cultured with interleukin-33-rich retinal pigment epithelium supernatant. Interleukin-33 treatment impaired fibroblast migration and gel contraction alongside suppression of MMP-2 and MMP-9 expression. InterpretationOur data highlight an unrecognised link between retinal pigment epithelium bioenergetic status and tissue remodelling of choroidal stroma. Our findings suggest that the interleukin 33/ST2 axis and changing bioenergetic sources are potential therapeutic targets to inhibit progression of age-related macular degeneration. FundingNational Institute for Health Research, National Eye Research Centre.

Secretory response of mast cells contained in monodispersed human choroidal preparations.

Mast cells have been identified in the choroid of numerous species including man. However, functional studies involving these human mast cells have not been reported. In the current studies, the secretory response of human choroidal mast cells to various stimuli was examined using monodispersed choroidal cell preparations. Monodispersed cell suspensions of human choroid were prepared from eye bank globes and the number, histamine content, and secretory response of mast cells in these preparations were determined. Choroids from 27 donors were used for these experiments. Cell suspensions contained an average of 15% mast cells. Mast cells stained positively with toluidine blue and exhibited the classical granular appearance upon electron microscopy. The amount of histamine contained in each mast cell was calculated to be 2.74+/-0.17 pg. Significant histamine release was observed following treatment with anti-human IgE, calcium ionophore A23187, concanavalin A, compound 48/80 and morphine. A method has been developed for obtaining monodispersed human choroidal mast cell preparations. The cells were functional as evidenced by their ability to release histamine upon immunological and nonimmunological stimulation. The degranulation noted following compound 48/80 and morphine challenge suggests that these human choroidal mast cells are analogous to connective tissue or chymase/tryptase-positive mast cells.

Distribution and characterisation of rat choroidal mast cells.

Despite the implication that choroidal mast cells are involved in the onset of experimental autoimmune uveoretinitis (EAU), a widely used animal model of uveoretinitis, little is known of these cells. In the present study the distribution, total number, regional density, and phenotype of choroidal mast cells were examined in Lewis, Wistar Furth, PVG/c, and brown Norway rats. Choroidal mast cells were predominantly associated with arteries and arterioles of more than 30 microns diameter which lie in the outer (sclerad) choroid. The density of mast cells was greatest in the posterior choroid with density diminishing anteriorly. The choroid of male Lewis rats contained significantly greater number of mast cells than that of females (p < 0.01). Histochemical (Alcian blue/safranin) and immunohistochemical (anti-rat mast cell protease I and II monoclonal antibodies) studies revealed choroidal mast cells were of the connective tissue type. However, granule proteinase content appeared less than that of well characterised connective tissue mast cell populations such as those in mesentery and skin. Lewis rats exhibited the highest density of choroidal mast cells (23.6 (SD 1.2)/mm2), Wistar Furth approximately half that of Lewis (13.5 (0.7)/mm2) while PVG/c and brown Norway rats had very low densities (3.06(0.3); 1.95(0.2/mm2 respectively). These studies provide valuable choroidal mast cell data for rats which may have implications for our understanding of experimental models of intraocular inflammation and clinical uveitis.

Choroidal mast cell dynamics during experimental autoimmune uveoretinitis in rat strains of differing susceptibility

Choroidal mast cells have been implicated in the pathogenesis of experimental autoimmune uveoretinitis (EAU). The aim of the present study was to examine the dynamics of choroidal mast cells during the course of EAU in rat strains of varying susceptibility. Histochemical staining showed choroidal mast cell degranulation in Lewis rats, a highly susceptible strain, commenced nine days post-immunisation, and peaked at day 11, at which time the percentage of degranulated choroidal mast cells (32. 5± 4%) was significantly higher than controls (15. 3 ± 3%; p <0.05). At day 14, mean choroidal mast cell density was significantly reduced (from 23. 6 ± 1/mm(2) tot 16. 2 ± 2/mm(2); p<0.05) and early signs of choroidal mast cell regeneration were evident. Immunisation of PVG/C (moderate susceptibility) and Brown Norway (very low susceptibility) rats produced a similar pattern of morphological changes. Onset of clinical signs in Lewis rats, which possess approximately 1100 to 1800 choroidal mast cells per eye, occurred one day following commencement of choroidal mast cell degranulation but prior to the peak of degranulation. However, in PVG/C and Brown Norway rats, which possess only approximately 70 and 110 choroidal mast cells per eye respectively, onset of disease was not temporally linked to commencement of degranulation. Production of antigen-specific IgE during the course of EAU was extremely low in all three strains. These results indicate that choroidal mast cells may be important in the pathogenesis of EAU in Lewis rats but not in PVG/C or Brown Norway rats and that non-IgE mediated degranulation may play a role in disease induction.

An association between susceptibility to experimental autoimmune uveitis and choroidal mast cell numbers.

An association was found in rats of different strains between the susceptibility to EAU and the number of mast cells in the choroid of the eye. High responder rats (Lewis, CAR) had strikingly more choroidal mast cells than the low responder BN animals, whereas intermediate numbers of mast cells were found in the F1 hybrids of Lewis and BN (LBNF), which exhibited an average level of susceptibility to EAU. LeR rats, derived from the Lewis strain, developed EAU only when treated with B. pertussis, and their number of choroidal mast cells was only about 1/5 of that found in the Lewis rats. Unlike the differences in the number of choroidal mast cells, small variations were found in the skin mast cell numbers of the tested rats. It is proposed that the number of local mast cells may be one mechanism by which the susceptibility to an organ-specific autoimmune disease is genetically regulated.

Evidence for immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis.

Evidence is presented for the involvement of immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis, an ocular inflammatory disease induced in Lewis rats by injection of the purified S antigen from bovine retina. Several parameters at various dates after immunization have been studied. In vivo degranulation of choroidal mast cells, in vitro degranulation of peritoneal mast cells in the presence of S antigen and cutaneous anaphylaxis were observed as early as 6-8 days after immunization. Serum antibodies were detected by passive hemagglutination from day 7 on. These phenomena preceded the onset of the ocular inflammation, consisting of a transient polymorphonuclear leukocyte infiltration, a long-lasting lymphoid cell infiltration and extensive damage to the retina and surrounding tissues. Such time course of events may suggest a role for reaginic antibody as a trigger for vascular changes in the target ocular tissue, favoring the development of inflammatory lesions.

Effect of Photon ( 60 Co) Irradiation on Rabbit Choroidal Mast Cells

The number of choroidal mast cells in the uvea of the albino rabbit was decreased at 3 days after a single dose of60 Co gamma radiation ranging from 2,400 to 19,400 R. A return to normal levels generally occurred within 8 days postirradiation. This decrease in the number of mast cells could be a direct effect on the cell or an effect on their cytoplasmic granules. 850 R given abdominally, with the head shielded, did not change the mast cell population and indicates an indirect effect was not the cause of the observed fall in number when their heads were irradiated. A dose-response relationship was not found. A dose as high as 19,000 R did not change this population remarkably; therefore, this cell is very radioresistant.

The Effect of Cortisone Acetate on Albino Rabbit Choroidal Mast Cells

The choroidal mast cell density gradient of the albino was used to determine the effect of cortisone acetate on this cell. Large doses did reduce mast cell numbers to a statistically significant level