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Conjunctiva-associated tissue (CALT) is assumed to play a crucial role in the immune system of the ocular surface. Its function in several ocular surface diseases (OSD) is still not fully understood. This study investigates the function of CALT in mouse models of dry-eye disease and ocular allergy. Since antigen-presentation is the central similarity in the pathologies, this study focuses on antigen-presentation in CALT Morphology and the expression of CALT, which was investigated in mice after induction of dry-eye, ocular allergy, topical antigen-stimulation, and after local depletion of phagocytic cells. Antigen uptake was investigated after the application of fluorescent ovalbumin (OVA). OSD influences the appearance and morphology of CALT in a disease-dependent manner. Ocular allergy leads to an increase and dry-eye disease to a decrease in number and size of CALT. The development of CALT is dependent on the presence of APCs. Professional APCs are present in CALT, and soluble antigen is transported into the follicle. CALT appearance is disease-specific and indicative of differing functions. Although the specific involvement of CALT in OSD needs further study, the existence of functional APCS and antigen-uptake supports the hypothesis that CALT is an immunological key player at the ocular surface.

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model., Methods: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy., Results: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min., Conclusions: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

Human beta-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of beta-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human beta-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse beta-defensins-2, -3 and -4 (mBD2-4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of beta-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1beta is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that beta-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of beta-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of beta-defensins at the ocular surface and lacrimal apparatus and show how beta-defensins are regulated specifically.

The conjunctiva contains organized lymphoid tissue that consists of intraepithelial lymphocytes, subepithelial lymphoid follicles and adjacent lymphatics and blood vessels. This conjunctiva-associated lymphoid tissue (CALT) is assumed to play a key role in the protection of the ocular surface by initiating and regulating immune responses. The article reviews the current knowledge of CALT in humans and compares its structure and functions with mucosa-associated lymphoid tissues at other mucosal sites. An update is given on CALT animal models and on functional studies on the transepithelial transport of microorganisms and antigenic substances by specialized epithelial cells. The use of two-photon microscopy is reviewed regarding its importance to the understanding of dynamic processes going on within CALT.

Purpose: Conjunctiva-associated lymphoid tissue (CALT) is assumed to be a key location for the generation of adaptive immune mechanisms of the ocular surface, but functional studies of CALT are still lacking. The purpose of this study was to establish an animal model that enables functional analysis of immune mechanisms going on within CALT. In addition, the use of two-photon microscopy, a new optical method, was evaluated for examining complex immunologic interactions of CALT by volume (three-dimensional [3-D]) and time-dependence (four-dimensional [4-D]) in vivo., Methods: The conjunctiva of female BALB/c mice was repeatedly challenged with topical Chlamydia trachomatis serovar C or a solution of ovalbumin and cholera toxin B. Two-photon microscopy was conducted on explanted, unfixed, and unstained eyes with adjacent nictitating membranes., Results: After three to five stimulations, CALT was detected exclusively in the nictitating membrane of 73% (C. trachomatis) or 70% (ovalbumin/ cholera toxin) of the animals. CALT mainly consisted of CD45R/B220+ B cells and CD4+ and CD8+ T cells. Electron microscopy showed intraepithelial lymphocytes and follicles consisting of lymphocytes, dendritic cells, and macrophages. Two-photon microscopy based on tissue autofluorescence allowed all components of CALT to be detected three dimensionally. High-resolution images were generated in tissue depths of 65 microm below the mucosal surface., Conclusions: This study introduces a novel mouse model for functional investigations of CALT. Topical stimulation with C. trachomatis or ovalbumin/cholera toxin B reliably leads to CALT generation at the nictitating membrane. The use of two-photon microscopy enables groundbreaking 3-D and, in the future, intravital 4-D investigations of immunologic processes initiated in CALT.