CD47 Binding on Vascular Endothelial Cells Inhibits IL-17-Mediated Leukocyte Adhesion.

To address the conflicting role of thrombospondin (TSP)-1 reported in acute and chronic pathologies, this study investigated the role of TSP-1 in regulating leukocyte recruitment and regulation of VCAM-1 expression using mouse models of uveitis. The spontaneously increased VCAM-1 expression and leukocyte adhesion in retinas of TSP-1-deficient mice suggested a TSP-1-mediated regulation of VCAM-1 expression. In a chronic uveitis model, induced by immunizing wild-type mice with specific interphotoreceptor retinoid-binding protein (IRBP) peptide, topically applied TSP-1-derived CD47-binding peptide significantly reduced the clinical disease course and retinal leukocyte adhesion as compared to the control peptide-treated group. In contrast, in LPS-mediated acute uveitis, TSP-1 deficiency significantly reduced the retinal leukocyte adhesion. The results of our in vitro study, using vascular endothelial cell (EC) cultures, demonstrate that unlike TNF-α, VCAM-1 expression induced by IL-17 is associated with a reduced expression of endogenous TSP-1. Such reduced endogenous TSP-1 expression in IL-17-stimulated ECs helps limit the CD36-mediated increased VCAM-1 expression, while favoring CD47-mediated inhibition of VCAM-1 expression and leukocyte adhesion. Thus, our study identifies TSP-1:CD47 interaction as a molecular pathway that modulates IL-17-mediated VCAM-1 expression, contributing to its anti-inflammatory effect in chronic inflammatory conditions.

Immunology and Pathology in Ocular Drug Development.

Clear vision is dependent on features that protect the anatomical integrity of the eye (cornea and sclera) and those that contribute to internal ocular homeostasis by conferring hemangiogenic (avascular tissues and antiangiogenic factors), lymphangiogenic (lack of draining lymphatics), and immunologic (tight junctions that form blood-ocular barriers, immunosuppressive cells, and modulators) privileges. The later examples are necessary components that enable the eye to maintain an immunosuppressive environment that responds to foreign invaders in a deviated manner, minimizing destructive inflammation that would impair vision. These conditions allowed for the observations made by Medawar, in 1948, of delayed rejection of allogenic tissue grafts in the anterior chamber of mouse eye and permit the sequestration of foreign invaders (eg, Toxoplasma gondii) within the retina of healthy individuals. Yet successful development of intraocular drugs (biologics and delivery devices) has been stymied by adverse ocular pathology, much of which is driven by immune pathways. The eye can be intolerant of foreign protein irrespective of delivery route, and endogenous ocular cells have remarkable plasticity when recruited to preserve visual function. This article provides a review of current understanding of ocular immunology and the potential role of immune mechanisms in pathology observed with intraocular drug delivery.

Thrombospondin 1 polymorphism associated with decreased expression and increased risk of pterygium.

PURPOSE: To assess the potential association of a thrombospondin 1 gene (THBS1) single-nucleotide polymorphism (rs1478604) with thrombospondin 1 (TSP-1) mRNA expression, as well as the risk of pterygium, in a pilot study. METHODS: DNA and RNA were isolated from peripheral blood samples collected from normal volunteer subjects (n = 39). In addition, DNA was isolated from conjunctival tissue samples collected during pterygium excision surgeries (n = 42). Relative expression of TSP-1 mRNA was measured by quantitative RT-PCR, and rs1478604 genotype was determined using a TaqMan SNP genotyping assay. Genotype frequencies were compared with mRNA expression and between pterygium samples and normal controls. RESULTS: Expression of TSP-1 mRNA was significantly lower in the peripheral blood of normal subjects who were homozygous for the C allele of rs1478604 (CC) compared to TT and CT genotypes (p = 0.004). When we compared rs1478604 genotypes between normal and pterygium patients, we found that the CC genotype was also associated with an increased risk of pterygium compared to TT (odds ratio (OR) = 5.39, 95% CI [1.26-22.99], p = 0.028), CT (OR = 7.86, 95% CI [1.92-32.17], p = 0.003), and combined CT and TT genotypes (OR = 6.67; 95% CI = [1.75-25.37]; p = 0.003). CONCLUSIONS: We found that the C allele of rs1478604 was associated with both lower TSP-1 expression and higher risk of pterygium, possibly implicating TSP-1 in the pathogenesis of pterygium.

Mouse Models of Sjögren's Syndrome with Ocular Surface Disease.

Sjögren's syndrome (SS) is a systemic rheumatic disease that predominantly affects salivary and lacrimal glands resulting in oral and ocular dryness, respectively, referred to as sicca symptoms. The clinical presentation of ocular dryness includes keratoconjunctivitis sicca (KCS), resulting from the inflammatory damage to the ocular surface tissues of cornea and conjunctiva. The diagnostic evaluation of KCS is a critical component of the classification criteria used by clinicians worldwide to confirm SS diagnosis. Therapeutic management of SS requires both topical and systemic treatments. Several mouse models of SS have contributed to our current understanding of immunopathologic mechanisms underlying the disease. This information also helps develop novel therapeutic interventions. Although these models address glandular aspects of SS pathology, their impact on ocular surface tissues is addressed only in a few models such as thrombospondin (TSP)-1 deficient, C57BL/6.NOD.Aec1Aec2, NOD.H2b, NOD.Aire KO, and IL-2Rα (CD25) KO mice. While corneal and/or conjunctival damage is reported in most of these models, the characteristic SS specific autoantibodies are only reported in the TSP-1 deficient mouse model, which is also validated as a preclinical model. This review summarizes valuable insights provided by investigations on the ocular spectrum of the SS pathology in these models.

Dysregulated Marginal Zone B Cell Compartment in a Mouse Model of Sjögren's Syndrome with Ocular Inflammation.

The risk of developing lymphoma in patients with Sjögren's syndrome (SS) is 44 times higher than in the normal population with the most common lymphomas derived from marginal zone B (MZB) cells. Current understanding of the role of MZB cells in SS is primarily based on salivary gland pathology, while their contextual association with lacrimal glands and ocular manifestations largely remains unknown. We examined this possibility using a SS mouse model (thrombospondin-1 deficient (TSP1-/-)) with well-characterized ocular disease. We determined the frequency, localization, and cytokine profiles of MZB cells and their association with an antibody response in TSP1-/- mice treated with a TSP-derived peptide. A significantly increased frequency of MZB cells was detected in the spleens and lacrimal glands of TSP1-/- mice in comparison to wild-type tissues as detected by immunostaining. An altered cytokine profile of TSP1-/- MZB cells was supportive of T helper 17 (Th17)-related pathogenesis. A significantly reduced antibody response and the splenic MZB compartment against an eye-derived antigen were noted in TSP-derived peptide-treated mice. These changes correspond with the previously reported ability of the peptide to ameliorate SS-related ocular manifestations. Collectively, our results demonstrate dysregulation of MZB cells in TSP1-/- mice and highlight their role in the context of SS-related chronic ocular surface disease.

Topical Application of TGF-ß-Activating Peptide, KRFK, Prevents Inflammatory Manifestations in the TSP-1-Deficient Mouse Model of Chronic Ocular Inflammation.

Chronic inflammation of the ocular surface poses a risk of vision impairment. The understanding of the molecular mechanisms that are involved in the inflammatory response is critical to identify novel molecular targets. Recently, thrombospondin-1 (TSP-1) has emerged as a key player in ocular surface homeostasis that efficiently activates the TGF-ß2 isoform that is predominantly expressed in the ocular mucosa. Here, the potential of the peptide derived from TSP-1 (KRFK), that can activate TGF-ß, is proposed as a potentially applicable therapeutic for chronic ocular surface inflammatory disorders. Our in vitro results confirm that the chosen peptide activates TGF-ß, reducing the expression of co-stimulatory molecules on dendritic cells, driving them towards a tolerogenic phenotype. For the in vivo studies, the TSP-1-/- mouse is used as a pre-clinical model of chronic ocular inflammation. We observe that the topical application of KRFK alters the peripheral balance of effectors by reducing the proportion of pathogenic Th1 and Th17 cells while increasing Treg cell proportion in cervical lymph nodes. In line with these findings, the development of chronic ocular surface inflammation is significantly prevented in KRFK-treated TSP-1-/- mice, as assessed by clinical parameters and inflammatory cytokine expression in conjunctival and lacrimal gland tissues. Together, our results identify the KRFK peptide as a novel therapeutic option to prevent the development of chronic inflammatory manifestations of the ocular surface.

Thrombospondin-1 Is Necessary for the Development and Repair of Corneal Nerves.

Thrombospondin-1-deficient (TSP-1-/-) mice are used as an animal model of Sjögren's Syndrome because they exhibit many of the symptoms associated with the autoimmune type of dry eye found in primary Sjögren's Syndrome. This type of dry eye is linked to the inflammation of the lacrimal gland, conjunctiva, and cornea, and is thought to involve dysfunction of the complex neuronal reflex arc that mediates tear production in response to noxious stimuli on the ocular surface. This study characterizes the structural and functional changes to the corneal nerves that are the afferent arm of this arc in young and older TSP-1-/- and wild type (WT) mice. The structure and subtype of nerves were characterized by immunohistochemistry, in vivo confocal microscopy, and confocal microscopy. Cytokine expression analysis was determined by Q-PCR and the number of monocytes was measured by immunohistochemistry. We found that only the pro-inflammatory cytokine MIP-2 increased in young corneas of TSP-1-/- compared to WT mice, but tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) all increased in older TSP-1-/- mouse corneas. In contrast, CD11b+ pro-inflammatory monocytes did not increase even in older mouse corneas. Calcitonin gene-related peptide (CGRP)-, but not Substance P (SubP)-containing corneal nerves decreased in older, but not younger TSP-1-/- compared to WT mouse corneas. We conclude that CGRP-containing corneal sensory nerves exhibit distinct structural deficiencies as disease progresses in TSP-1-/- mice, suggesting that: (1) TSP-1 is needed for the development or repair of these nerves and (2) impaired afferent corneal nerve structure and hence function may contribute to ocular surface dysfunction that develops as TSP-1-/- mice age.

Sjögren's syndrome associated dry eye in a mouse model is ameliorated by topical application of integrin α4 antagonist GW559090.

Sjögren's syndrome is an autoimmune disease associated with inflammation of exocrine glands with clinical manifestations of dry eye and dry mouth. Dry eye in this disease involves inflammation of the ocular surface tissues - cornea and conjunctiva. While systemic blockade of adhesion molecules has been used to treat autoimmune diseases, the purpose of this study was to determine the therapeutic efficacy of topical application of an integrin α4 adhesion molecule antagonist in a mouse model of dry eye associated with Sjögren's syndrome. To assess this spontaneously developed ocular surface inflammation related to Sjögren's syndrome in TSP-1null mice (12 wks) was evaluated. Mice were treated with topical formulations containing 0.1% dexamethasone or 30 mg/ml GW559090 or vehicle control. Corneal fluorescein staining and conjunctival goblet cell density were assessed. Real-time PCR analysis was performed to assess expression of the inflammatory marker IL-1ß in the cornea and Tbet and RORγt in the draining lymph nodes. Ocular surface inflammation was detectable in TSP-1null mice (≥12 wk old), which resulted in increased corneal fluorescein staining indicative of corneal barrier disruption and reduced conjunctival goblet cell density. These changes were accompanied by increased corneal expression of IL-1ß as compared to WT controls and an altered balance of Th1 (Tbet) and Th17 (RORγt) markers in the draining lymph nodes. Topically applied dexamethasone and GW559090 significantly reduced corneal fluorescein staining compared to vehicle treatment (p = 0.023 and p < 0.001, respectively). This improved corneal barrier integrity upon adhesion molecule blockade was consistent with significantly reduced corneal expression of pro-inflammatory IL-1ß compared to vehicle treated groups (p < 0.05 for both treatments). Significant improvement in goblet cell density was also noted in mice treated with 0.1% dexamethasone and GW559090 (p < 0.05 for both). We conclude that similar to topical dexamethasone, topically administered GW559090 successfully improved corneal barrier integrity and inflammation in an established ocular surface disease associated with Sjögren's syndrome.

Alteration in cellular turnover and progenitor cell population in lacrimal glands from thrombospondin 1-/- mice, a model of dry eye.

The purpose of this study was to investigate the changes that occur in the lacrimal glands (LGs) in female thrombospondin 1 knockout (TSP1-/-) mice, a mouse model of the autoimmune disease Sjogren's syndrome. The LGs of 4, 12, and 24 week-old female TSP1-/- and C57BL/6J (wild type, WT) mice were used. qPCR was performed to measure cytokine expression. To study the architecture, LG sections were stained with hematoxylin and eosin. Cell proliferation was measured using bromo-deoxyuridine and immunohistochemistry. Amount of CD47 and stem cell markers was analyzed by western blot analysis and location by immunofluorescence microscopy. Expression of stem cell transcription factors was performed using Mouse Stem Cell Transcription Factors RT2 Profiler PCR Array. Cytokine levels significantly increased in LGs of 24 week-old TSP1-/- mice while morphological changes were detected at 12 weeks. Proliferation was decreased in 12 week-old TSP1-/- mice. Three transcription factors were overexpressed and eleven underexpressed in TSP1-/- compared to WT LGs. The amount of CD47, Musashi1, and Sox2 was decreased while the amount of ABCG2 was increased in 12 week-old TSP1-/- mice. We conclude that TSP1 is necessary for maintaining normal LG homeostasis. Absence of TSP1 alters cytokine levels and stem cell transcription factors, LG cellular architecture, decreases cell proliferation, and alters amount of stem cell markers.

Thrombospondin-1-dependent immune regulation by transforming growth factor-ß2-exposed antigen-presenting cells.

An important role of transforming growth factor-ß (TGF-ß) in the development of regulatory T cells is well established. Although integrin-mediated activation of latent TGF-ß1 is considered essential for the induction of regulatory T (Treg) cells by antigen-presenting cells (APCs), such an activation mechanism is not applicable to the TGF-ß2 isoform, which lacks an integrin-binding RGD sequence in its latency-associated peptide. Mucosal and ocular tissues harbour TGF-ß2-expressing APCs involved in Treg induction. The mechanisms that regulate TGF-ß activation in such APCs remain unclear. In this study, we demonstrate that murine APCs exposed to TGF-ß2 in the environment predominantly increase expression of TGF-ß2. Such predominantly TGF-ß2-expressing APCs use thrombospondin-1 (TSP-1) as an integrin-independent mechanism to activate their newly synthesized latent TGF-ß2 to induce Foxp3(+) Treg cells both in vitro and in vivo. Expression of Treg induction by TGF-ß2-expressing APCs is supported by a TSP-1 receptor, CD36, which facilitates activation of latent TGF-ß during antigen presentation. Our results suggest that APC-derived TSP-1 is essential for the development of an adaptive regulatory immune response induced by TGF-ß2-expressing APCs similar to those located at mucosal and ocular sites. These findings introduce the integrin-independent mechanism of TGF-ß activation as an integral part of peripheral immune tolerance associated with TGF-ß2-expressing tissues.

Polymorphism in THBS1 gene is associated with post-refractive surgery chronic ocular surface inflammation.

PURPOSE: To determine the association of single nucleotide polymorphisms (SNPs) of the thrombospondin 1 (THBS1) gene with development of chronic ocular surface inflammation (keratoconjunctivitis) after refractive surgery. DESIGN: Retrospective cohort study. PARTICIPANTS: Active duty U.S. Army soldiers (n = 143) who opted for refractive surgery. METHODS: Conjunctival impression cytology samples collected from participants before the surgery were used to harvest DNA for genotyping 5 THBS1 SNPs (rs1478604, rs2228262, rs2292305, rs2228262, and rs3743125) using the Sequenom iPLEX Gold platform (Sequenom, San Diego, CA). Samples collected after surgery were used to harvest RNA for gene expression analysis by real-time polymerase chain reaction (PCR). Participants were followed for 1 year after surgery to monitor the status of keratoconjunctivitis. MAIN OUTCOME MEASURES: Genetic basis of the development of chronic keratoconjunctivitis after refractive surgery. RESULTS: Carriers of minor alleles of 3 SNPs each were found to be more susceptible to developing chronic keratoconjunctivitis (rs1478604: odds ratio [OR], 2.5; 95% confidence interval [CI], 1.41-4.47; P = 2.5 × 10(-3); rs2228262 and rs2292305: OR, 1.9; 95% CI, 1.05-3.51; P = 4.8 × 10(-2)). Carriers of the rs1478604 minor allele expressed significantly reduced levels of thrombospondin 1 (TSP1) (P = 0.042) and increased levels of an inflammatory cytokine associated with keratoconjunctivitis, interleukin-1ß (P = 0.025), in their ocular surface epithelial cells compared with homozygous major allele controls. CONCLUSIONS: Genetic variation in the THBS1 gene that results in decreased expression of the encoded glycoprotein TSP1 in ocular surface epithelial cells significantly increases the susceptibility to develop chronic ocular surface inflammation after refractive surgery. Further investigation of THBS1 SNPs in a larger sample size is warranted.

Determining immune components necessary for progression of pigment dispersing disease to glaucoma in DBA/2J mice.

BACKGROUND: The molecular mechanisms causing pigment dispersion syndrome (PDS) and the pathway(s) by which it progresses to pigmentary glaucoma are not known. Mutations in two melanosomal protein genes (Tyrp1(b) and Gpnmb(R150X)) are responsible for pigment dispersing iris disease, which progresses to intraocular pressure (IOP) elevation and subsequent glaucoma in DBA/2J mice. Melanosomal defects along with ocular immune abnormalities play a role in the propagation of pigment dispersion and progression to IOP elevation. Here, we tested the role of specific immune components in the progression of the iris disease and high IOP. RESULTS: We tested the role of NK cells in disease etiology by genetically modifying the B6.D2-Gpnmb(R150X) Tyrp1(b) strain, which develops the same iris disease as DBA/2J mice. Our findings demonstrate that neither diminishing NK mediated cytotoxic activity (Prf1 mutation) nor NK cell depletion (Il2rg mutation) has any influence on the severity or timing of Gpnmb(R150X) Tyrp1(b) mediated iris disease. Since DBA/2J mice are deficient in CD94, an important immune modulator that often acts as an immune suppressor, we generated DBA/2J mice sufficient in CD94. Sufficiency of CD94 failed to alter either the iris disease or the subsequent IOP elevation. Additionally CD94 status had no detected effect on glaucomatous optic nerve damage. CONCLUSION: Our previous data implicate immune components in the manifestation of pigment dispersion and/or IOP elevation in DBA/2J mice. The current study eliminates important immune components, specifically NK cells and CD94 deficiency, as critical in the progression of iris disease and glaucoma. This narrows the field of possible immune components responsible for disease progression.

The Sjögren's Working Group: The 2023 OMERACT meeting and provisional domain generation.

Sjögren's disease (SjD) is a systemic autoimmune exocrinopathy with key features of dryness, pain, and fatigue. SjD can affect any organ system with a variety of presentations across individuals. This heterogeneity is one of the major barriers for developing effective disease modifying treatments. Defining core disease domains comprising both specific clinical features and incorporating the patient experience is a critical first step to define this complex disease. The OMERACT SjD Working Group held its first international collaborative hybrid meeting in 2023, applying the OMERACT 2.2 filter toward identification of core domains. We accomplished our first goal, a scoping literature review that was presented at the Special Interest Group held in May 2023. Building on the domains identified in the scoping review, we uniquely deployed multidisciplinary experts as part of our collaborative team to generate a provisional domain list that captures SjD heterogeneity.

Thrombospondin-1 derived from APCs regulates their capacity for allosensitization.

Thrombospondin (TSP)-1 is a matricellular glycoprotein with immunoregulatory properties, which include inhibition of APC function. We show in transplantation that TSP-1 inhibits T cell allosensitization and consequently suppresses immune rejection. This was revealed by comparing wild-type (WT) versus TSP-1 null allografts in corneal transplantation, as the cornea is a rich source of TSP-1. Compared with only 50% of rejected WT allografts, nearly all TSP-1 null allografts succumbed to rejection. This effect was reflected by donor-derived APCs, which exhibited a distinctively greater capacity for allosensitization in transplanted hosts. Corroborated in MLRs, greater proliferation levels and robust IFN-γ (but not IL-10)-positive T cells resulted from stimulation by TSP-1 null APCs relative to WT ones. Moreover, enhanced expression of MHC class II and B7 maturation markers were detected on TSP-1 null APCs during inflammation. Increased expression of CCR7 was further matched by enhanced lymph node migration of TSP-1 null APCs posttransplantation. We therefore conclude that APC-derived TSP-1 suppresses their capacity to allosensitize T cells, and this regulation stems from their resistance to taking on a mature form. Future strategies targeting APCs for TSP-1 upregulation may thus be effective in promoting allograft survival.

Reduced tear thrombospondin-1/matrix metalloproteinase-9 ratio can aid in detecting Sjögren's syndrome etiology in patients with dry eye.

Differentiating patients with Sjögren's syndrome (SS)-associated dry eye from non-SS dry eye is critical for monitoring and appropriate management of possible sight- or life-threatening extraglandular complications associated with SS. We tested whether reduced tear levels of immunoregulatory thrombospondin (TSP)-1, which also inhibits matrix metalloproteinase (MMP)-9, would reflect SS pathogenesis aiding the identification of patients with SS-dry eye. Total of 61 participants, including healthy controls (n = 20), patients with non-SS dry eye (n = 20) and SS-dry eye (n = 21) were enrolled prospectively. Tear TSP-1 and MMP-9 levels were measured using a custom magnetic bead-based multi-plex assay in a masked manner. Analyte concentrations were assessed further according to ocular surface and tear film parameters. Relative to median tear TSP-1 (308 ng/ml) and MMP-9 (1.9 ng/ml) levels in the control group, significantly higher proportion of patients with SS-dry eye than non-SS had lower tear TSP-1 levels (55% vs. 29%, odds ratio [OR] = 3, 95% confidence interval [CI] = 1.64 to 5.35, p < 0.05) and higher tear MMP-9 levels (65% vs. 24%, OR = 5.8, 95% CI = 4.46 to 19.81, p < 0.05), respectively. The tear TSP-1/MMP-9 ratio was significantly reduced in patients with SS-dry eye compared to non-SS (B = -2.36, 95% CI = -3.94 to -0.0.79, p < 0.05), regardless of tear MMP-9 levels. Patients with a lower ratio were 2.3 times more likely to have SS (OR = 0.28, 95% CI = 0.1 to 0.75, p < 0.05). This ratio showed significant inverse correlations with clinical parameters (conjunctival and corneal staining scores). Our results denote that tear TSP-1/MMP-9 ratio can be useful in identifying patients with dry eye with underlying SS and used as a screening test.

Conjunctival Goblet Cell Responses to TLR5 Engagement Promote Activation of Local Antigen-Presenting Cells.

Conjunctival epithelium forms a barrier between the ocular surface microbial flora and the ocular mucosa. In addition to secreting gel-forming mucins, goblet cells, located in the conjunctival epithelium, help maintain local immune homeostasis by secreting active TGFß2 and promoting tolerogenic phenotype of dendritic cells in the vicinity. Although dendritic cell subsets, characteristic of mucosal tissues, are found in the conjunctiva, previous studies provided limited information about their location within the tissue. In this study, we examine immunostained conjunctiva explants to determine the location of CD11c-positive dendritic cells in the context of MUC5AC-positive goblet cells. Considering that conjunctival goblet cells are responsive to signaling induced by pathogen recognition receptors, we also assess if their responses to microbial product, flagellin, can contribute to the disruption of ocular mucosal homeostasis that promotes activation of dendritic cells and results in chronic ocular surface inflammation. We find that dendritic cells in the conjunctiva with an increased microbial colonization are located adjacent to goblet cells. While their cell bodies in the stromal layer are immediately below the epithelial layer, several extensions of dendritic cells are projected across the epithelium towards the ocular surface. Such trans-epithelial dendrites are not detectable in healthy ocular mucosa. In response to topically applied flagellin, increased proportion of CD11c-positive cells in the conjunctiva strongly express MHC class II relative to the untreated conjunctiva. This change is accompanied by reduced immunoreactivity to TGFß-activating Thrombospondin-1 in the conjunctival epithelium. These findings are supported by in vitro observations in primary cultures of goblet cells that respond to the TLR5 stimulation with an increased expression of IL-6 and reduced level of active TGFß. The observed changes in the conjunctiva after flagellin application correspond with the development of clinical signs of chronic ocular mucosal inflammation including corneal epitheliopathy. Collectively, these findings demonstrate the ability of ocular mucosal dendritic cells to extend trans-epithelial dendrites in response to increased microbial colonization at the ocular surface. Moreover, this study provides key insight into how goblet cell responses to microbial stimuli may contribute to the disruption of ocular mucosal homeostasis and chronic ocular mucosal inflammation.

Surgical denervation of ocular sympathetic afferents decreases local transforming growth factor-beta and abolishes immune privilege.

Mounting evidence points to a role for the sympathetic nervous system in suppressing inflammation. This role might be of specific relevance for immune privilege in the eye, where, sporadically, patients with denervated sympathetic fibers develop chronic inflammation. The present study used mice to investigate whether the robust innervation of intraocular structures by the sympathetic system plays a role in maintaining ocular immune privilege. We first performed surgical removal of the superior cervical ganglion, which supplies sympathetic fibers to the eye, and studied the immune response generated against soluble antigens or allogeneic tumor cells injected into the ocular anterior chamber under these conditions. Our results show that in the absence of functional sympathetic fibers, the eye loses its ability to prevent either the immune rejection of intraocular allogeneic tumor cells or the suppression of delayed type hypersensitivity responses against soluble antigens injected in the anterior chamber. This loss of immune privilege is accompanied by a decrease in the concentration of transforming growth factor-beta in the aqueous humor. These results suggest that immune privilege is lost in the absence of a functional sympathetic innervation of the eye, allowing intraocular immune responses to become exaggerated. We conclude that ocular sympathetic nerves are critical for the generation and maintenance of immune privilege in the eye through the facilitation of local transforming growth factor-beta production.

Sjögren's syndrome-like ocular surface disease in thrombospondin-1 deficient mice.

Thrombospondin-1 (TSP-1) is a major activator of latent transforming growth factor-beta in vitro as well as in vivo. Mice deficient in TSP-1, despite appearing normal at birth, develop a chronic form of ocular surface disease that is marked by increased apoptosis and deterioration in the lacrimal gland, associated dysfunction, and development of inflammatory infiltrates that result in abnormal tears. The increase in CD4(+) T cells in the inflammatory infiltrates of the lacrimal gland, and the presence of anti-Sjögren's syndrome antigen A and anti-Sjögren's syndrome antigen B antibodies in the serum resemble autoimmune Sjögren's syndrome. These mice develop an ocular surface disorder dry eye that includes disruption of the corneal epithelial layer, corneal edema, and a significant decline in conjuctival goblet cells. Externally, several mice develop dry crusty eyes that eventually close. The inflammatory CD4(+) T cells detected in the lacrimal gland, as well as those in the periphery of older TSP-1 null mice, secrete interleukin-17A, a cytokine associated with chronic inflammatory diseases. Antigen-presenting cells, derived from TSP-1 null, but not from wild-type mice, activate T cells to promote the Th17 response. Together, these results indicate that TSP-1 deficiency results in a spontaneous form of chronic dry eye and aberrant histopathology associated with Sjögren's syndrome.

Increased IkappaB alpha expression is essential for the tolerogenic property of TGF-beta-exposed APCs.

IkappaB alpha is an inhibitor of the transcriptional factor NF-kappaB, and it is an essential component of the signaling pathways that lead to expression of inflammatory molecules. These include cytokines and costimulatory molecules associated with antigen presentation in an inflammatory immune response. In this study, we report that antigen-presenting cells exposed to TGF-beta induce peripheral tolerance by increasing IkappaB alpha expression. Exposure of antigen presenting cells (APCs) to TGF-beta is known to impair their ability to secrete IL-12, and such impairment correlated with reduced NF-kappaB activity as indicated by significantly reduced nuclear levels of p50, an essential subunit of NF-kappaB for IL-12 transcription. Blockade of increased nuclear IkappaB alpha in APCs by expression of small interfering RNA molecules (siRNAs) targeting IkappaB alpha transcripts prevented IL-12 impairment and the decline in nuclear p50 levels. Furthermore, such IkappaB alpha blockade also interfered with the tolerogenic property of TGF-beta-exposed APCs. However, increased expression of IkappaB alpha in APCs, independent of TGF-beta exposure, reduced nuclear p50 levels and permitted tolerance induction by APCs. Thus, our findings attribute a direct and significant role to IkappaB alpha in the tolerogenic potential of APCs. Increased IkappaB alpha expression in APCs may therefore offer a therapeutic approach to achieve antigen-specific immunomodulation.

Surprising up-regulation of P-selectin glycoprotein ligand-1 (PSGL-1) in endotoxin-induced uveitis.

P-selectin glycoprotein ligand-1 (PSGL-1) is constitutively expressed on leukocytes and was thought to be down-regulated with cell activation. However, this work shows the surprising finding of functional PSGL-1 up-regulation during acute inflammation. PSGL-1 function was studied in our autoperfusion assay, in which blood from a mouse carotid flows through a microchamber coated with a fixed density of P-selectin. Under the inflammatory conditions--uveitis induced by systemic lipopolysaccharide injection--we recorded significantly reduced leukocyte rolling velocity, which suggests PSGL-1 up-regulation; however, flow cytometry showed reduced PSGL-1. When bound leukocytes were released from the vasculature by PSGL-1 blockade, a large peripheral blood leukocyte (PBL) population showed elevated PSGL-1, which could account for the reduced PSGL-1 in the remaining unbound population. In the eye, systemic blockade of PSGL-1 with a monoclonal antibody or recombinant soluble PSGL-1 drastically reduced the severe manifestations of uveitis. Furthermore, PSGL-1 blockade was significantly more effective in reducing retinal leukostasis than was P-selectin blockade. Our results provide surprising evidence for functional PSGL-1 up-regulation in PBLs during acute inflammation. The temporal overlap between PSGL-1 and P-selectin up-regulation reveals an as yet unrecognized collaboration between this receptor-ligand pair, increasing efficiency of the first steps of the leukocyte recruitment cascade.