Thrombospondin-derived peptide attenuates Sjögren's syndrome-associated ocular surface inflammation in mice.

Sjögren's syndrome is the second most common rheumatic disease in which autoimmune response targets exocrine glands (salivary and lacrimal glands) result in clinical symptoms of dry mouth and dry eye. Inflammation of the lacrimal gland induces tear abnormalities that contribute to the inflammation of the ocular surface, which includes ocular mucosa. Thrombospondin-1 (TSP-1) plays a critical regulatory role in the ocular mucosa and as such TSP-1-/- mice develop spontaneously chronic ocular surface inflammation associated with Sjögren's syndrome. The autoimmune pathology is also accompanied by a peripheral imbalance in regulatory (Treg ) and inflammatory Th17 effectors. In this study, we demonstrate an in-vitro effect of a CD47-binding TSP-derived peptide in the induction of transforming growth factor (TGF)-ß1-secreting forkhead box protein 2 (Foxp3+ ) Tregs from activated CD4+ CD25- T cells and the inhibition of pathogenic T helper type 17 (Th17)-promoting interleukin (IL)-23 derived from antigen-presenting cells. The in-vivo administration of this peptide promotes Foxp3+ Treg induction and inhibition of Th17 development. Consistent with these results, topical administration of CD47-binding TSP peptide, both before and after the onset of the disease, attenuates clinical symptoms of SS-associated dry eye in TSP-1-/- mice. Augmented expression of Foxp3 detected in the draining lymph nodes of TSP peptide -treated mice compared to those treated with control peptide suggests the ability of TSP peptide to restore peripheral immune imbalance. Thus, our results suggest that TSP-derived peptide attenuates Sjögren's syndrome-associated dry eye and autoimmune inflammation by preventing Th17 development while promoting the induction of Tregs . Collectively, our data identify TSP-derived peptide as a novel therapeutic option to treat autoimmune diseases.

Secondary allergic T cell responses are regulated by dendritic cell-derived thrombospondin-1 in the setting of allergic eye disease.

Allergic eye disease, as in most forms of atopy, ranges in severity among individuals from immediate hypersensitivity to a severe and debilitating chronic disease. Dendritic cells play a key role in stimulating pathogenic T cells in allergen re-exposure, or secondary responses. However, molecular cues by dendritic cells underpinning allergic T cell response levels and the impact that this control has on consequent severity of allergic disease are poorly understood. Here, we show that a deficiency in thrombospondin-1, a matricellular protein known to affect immune function, has subsequent effects on downstream T cell responses during allergy, as revealed in an established mouse model of allergic eye disease. More specifically, we demonstrate that a thrombospondin-1 deficiency specific to dendritic cells leads to heightened secondary T cell responses and consequent clinical disease. Interestingly, whereas thrombospondin-1-deficient dendritic cells augmented activity of allergen-primed T cells, this increase was not recapitulated with naïve T cells in vitro. The role of dendritic cell-derived thrombospondin-1 in regulating secondary allergic T cell responses was confirmed in vivo, as local transfer of thrombospondin-1-sufficient dendritic cells to the ocular mucosa of thrombospondin-1 null hosts prevented the development of augmented secondary T cell responses and heightened allergic eye disease clinical responses. Finally, we demonstrate that topical instillation of thrombospondin-1-derived peptide reduces T cell activity and clinical progression of allergic eye disease. Taken together, this study reveals an important modulatory role of dendritic cell-derived thrombospondin-1 on secondary allergic T cell responses and suggests the possible dysregulation of dendritic cell-derived thrombospondin-1 expression as a factor in allergic eye disease severity.

Modulation of conjunctival goblet cell function by inflammatory cytokines.

Ocular surface inflammation associated with Sjögren's syndrome is characterized by a loss of secretory function and alteration in numbers of mucin secreting goblet cells. Such changes are a prominent feature of ocular surface inflammatory diseases and are attributed to inflammation; however, the exact effect of the inflammatory cytokines on conjunctival goblet cell function remains largely unknown. In this study, we developed a primary culture of mouse goblet cells from conjunctival tissue and evaluated the effects on their function by inflammatory cytokines detected in the conjunctiva of mouse model of Sjögren's syndrome (Thrombospondin-1 deficient mice). We found that apoptosis of goblet cells was primarily induced by TNF-α and IFN-γ. These two cytokines also inhibited mucin secretion by goblet cells in response to cholinergic stimulation, whereas IL-6 enhanced such secretion. No changes in secretory response were detected in the presence of IL-13 or IL-17. Goblet cells proliferated to varying degrees in response to all the tested cytokines with the greatest response to IL-13 followed by IL-6. Our results therefore reveal that inflammatory cytokines expressed in the conjunctiva during an ocular surface disease directly disrupt conjunctival goblet cell functions, compromising the protective function of tears, thereby contributing to ocular surface damage.

Requirement for early donor cell chimerism during prolonged survival of murine skin allografts.

BACKGROUND: Posttransplantation infusion of donor bone marrow cells (BMC) can prolong allograft survival in antilymphocyte antibody- (ALA) treated recipients. This study examined the hypothesis that chimerism of donor BMC origin contributes to allograft unresponsiveness. METHODS: Survival of day 0 skin grafts from C3H (H2k) donors was prolonged on ALA-treated B6AF1 mice by day +7 infusion of BMC from C3H or C3H-H2o2 (H2K(d)I(d)D(k)) mice. To test for functional chimerism, depletional anti-H2Kd antibody was injected at intervals after C3H-H2o2 BMC infusion. To confirm the persistence of active cells in the recipients, cells harvested from bone marrow of ALA- and C3H BMC-treated primary recipients were transferred to secondary ALA-treated recipients. Other recipients were infused on day +21 with additional donor BMC or light-density BMC that had been cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to promote the differentiation of dendritic cells (DC). RESULTS: Injection of depletional antibody targeting infused BMC interrupted skin graft survival, whether the injection was made on day +7, 2 hr after BMC injection, or as late as day +28. Prolonged graft survival was transferred to secondary recipients with cells recovered from primary recipient marrow as long as 4 weeks after initial donor BMC treatment. Graft survival prolonged by day +7 BMC was further enhanced by infusion on day +21 of light density cultured BMC, although a second dose of unfractionated BMC was inactive. Repeated injection of cultured BMC at 2-week intervals after day +21 prolonged graft survival even further. CONCLUSIONS: These data directly demonstrate that at least short-term donor BMC-derived microchimerism is required for prolonged allograft survival. The data further suggest that the active chimeric cells persist in recipient marrow. Finally, the beneficial effect of late infusion of cultured cells containing partially differentiated DC suggest that DC may be the active chimeric cells.

Effect of monoclonal anti-CD4 and anti-CD8 on skin allograft survival in mice treated with donor bone marrow cells.

Allograft unresponsiveness can be induced by donor bone marrow cells (BMC) in antilymphocyte serum (ALS)-treated recipients. The effect of administering monoclonal anti-CD4 and -CD8 at several points has been examined in a mouse skin allograft model of this protocol. Brief peritransplant administration of anti-CD4 and -CD8 was used to replace ALS. Anti-CD4 treatment prolonged graft survival only slightly and conditioned recipients poorly for the effect of posttransplantation donor BMC infusion. Anti-CD8 was ineffective in both capacities. A mixture of anti-CD4 and anti-CD8 was at least as effective as ALS in prolonging graft survival and in promoting the beneficial effects of donor BMC. Like the monoclonal antibodies, ALS also depleted splenic and lymph node CD4+ and CD8+ cells. Injection of ALS, but not the monoclonal antibodies, altered the CD4/CD8 phenotype of thymocytes, although persistent binding of both types of antibody to thymocytes was demonstrated. Abrogation of the positive effect of BMC by reconstitution of normal spleen cells on day +3 after ALS treatment confirmed that cell depletion is a requirement of this system. Monoclonal antibodies were also given to ALS/BMC-treated recipients after their grafts had become established. Anti-CD8 injection at either 2 or 4 weeks after transplantation further prolonged graft survival. In contrast, anti-CD4 injection at 2 or 6 weeks after grafting precipitated rejection, which suggests that continued allograft survival following ALS and donor BMC treatment is due to the activity of a CD4+ cell population.

Effect of asialofetuin on prolongation of skin allograft survival by donor bone marrow cells.

Survival of skin allografts made to antilymphocyte serum (ALS)-treated recipients is prolonged by posttransplant intravenous injection of donor strain bone marrow cells (BMC). If asialofetuin (ASF) is coinjected with the BMC, the prolonged graft survival is augmented (e.g., median survival time increased from 43 days to 72 days by injection of ASF). We have confirmed that, like peanut agglutinin-binding stem cells, the active BMC are at risk for hepatic sequestration after injection, possibly via hepatic asialoglycoprotein receptors. A fraction enriched for these active cells binds to liver sections in vitro and localizes to liver in vivo. This binding and localization can be partly inhibited by ASF. Although injected cells could also be found in the spleen, the beneficial effect of ASF could be demonstrated in previously splenectomized mice. Also, splenectomy 2 hr after BMC injection (without ASF) had little effect on the BMC-induced prolonged graft survival, while transfer of cells from the removed spleens to secondary ALS-treated recipients did not transfer such prolongation. In contrast, transfer of BMC from primary, donor marrow-injected recipients did transfer graft-prolonging activity, especially if both primary and secondary recipients were ASF injected. Our results suggest that recipient marrow, but not spleen, is the site of short-term localization of graft survival-prolonging BMC. Augmentation of allograft survival with ASF in ALS-treated recipients appears to result from the diversion of BMC away from a microenvironment not conducive to the expression of their graft-prolonging activity.