Fabian Garreis

Intestinal Trefoil Factor/TFF3 Promotes Re-epithelialization of Corneal Wounds

Disorders of wound healing characterized by impaired or delayed re-epithelialization are a serious medical problem. These conditions affect many tissues, are painful, and are difficult to treat. In this study using cornea as a model, we demonstrate the importance of trefoil factor 3 (TFF3, also known as intestinal trefoil factor) in re-epithelialization of wounds. In two different models of corneal wound healing, alkali- and laser-induced corneal wounding, we analyzed the wound healing process in in vivo as well as in combined in vivo/in vitro model in wild type (Tff3+/+) and Tff3-deficient (Tff3-/-) mice. Furthermore, we topically applied different concentrations of recombinant human TFF3 (rTFF3) peptide on the wounded cornea to determine the efficacy of rTFF3 on corneal wound healing. We found that Tff3 peptide is not expressed in intact corneal epithelium, but its expression is extensively up-regulated after epithelial injury. Re-epithelialization of corneal wounds in Tff3-/- mice is significantly prolonged in comparison to Tff3+/+ mice. In addition, exogenous application of rTFF3 to the alkali-induced corneal wounds accelerates significantly in in vivo and in combined in vivo/in vitro model wound healing in Tff3+/+ and Tff3-/- mice. These findings reveal a pivotal role for Tff3 in corneal wound healing mechanism and have broad implications for developing novel therapeutic strategies for treating nonhealing wounds.

Schirmer strip vs. capillary tube method: Non-invasive methods of obtaining proteins from tear fluid

Human tear fluid is a complex mixture containing over 500 solute proteins, lipids, electrolytes, mucins, metabolites, hormones and desquamated epithelial cells as well as foreign substances from the ambient air. Little is known to date about the function of most tear components. The efficient and gentle collection of tear fluid facilitates closer investigation of these matters. The objective of the present paper was to compare two commonly used methods of obtaining tear fluid, the capillary tube and Schirmer strip methods, in terms of usefulness in molecular biological investigation of tear film. The comparative protein identification methods Bradford and Western Blot were used in the analyses to this end. The surfactant proteins (SP) A-D recently described as present on the eye surface were selected as the model proteins. Both methods feature sufficient uptake efficiency for proteins in or extraction from the sampling means used (capillary tube/Schirmer strip). The total protein concentration can be determined and the proteins in the tears can be detected - besides the hydrophilic SP-A and D also the non-water-soluble proteins of smaller size such as SP-B and C. Thus both methods afford a suitable basis for comparative analysis of the physiological processes in the tear fluid of healthy and diseased subjects. On the whole, the Schirmer strip has several advantages over the capillary tube.

Thermosensitive transient receptor potential channels in human corneal epithelial cells

Thermosensitive transient receptor potential (TRP) proteins such as TRPV1–TRPV4 are all heat‐activated non‐selective cation channels that are modestly permeable to Ca2+. TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca 2+]i transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT‐PCR and TRPV2 transcripts were also detected. In fura2‐loaded HCEC, a TRPV1–3 selective agonist, 100 µM 2‐aminoethoxydiphenyl borate (2‐APB), induced intracellular Ca2+ transients and an increase in non‐selective cation outward currents that were suppressed by ruthenium‐red (RuR) (10–20 µM), a non‐selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40°C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La3+) (100 µM) suppressed these temperature‐induced Ca2+ increases. Planar patch‐clamp technique was used to characterize the currents underlying Ca2+ transients. Increasing the temperature to over 40°C induced reversible rises in non‐selective cation currents. Moreover, a hypotonic challenge (25%) increased non‐selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermo‐sensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress. J. Cell. Physiol. 226: 1828–1842, 2011.

Roles of human β-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells

Human β-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 β-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human β-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 β-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 β-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1β 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 β-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 β-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of β-defensins at the ocular surface and lacrimal apparatus and show how β-defensins are regulated specifically.

Expression and Regulation of Antimicrobial Peptide Psoriasin (S100A7) at the Ocular Surface and in the Lacrimal Apparatus

PURPOSE Psoriasin, originally isolated from psoriasis as an overexpressed molecule of unknown function, has recently been identified as a principal Escherichia coli-killing antimicrobial peptide of healthy skin. The purpose of this study was to investigate the expression and antimicrobial role of psoriasin at the ocular surface and in the lacrimal apparatus. METHODS Different tissues of the lacrimal apparatus and ocular surface were systematically analyzed by means of RT-PCR, Western blot, and immunohistochemistry for their ability to express and produce psoriasin. The inducibility and regulation of psoriasin were studied in human corneal as well as conjunctival epithelial cell lines after challenge with ocular pathogens and proinflammatory cytokines. Real-time RT-PCR was performed to evaluate the expression and induction of psoriasin. In addition, tear fluid obtained from different healthy volunteers was examined by ELISA for its psoriasin concentration. RESULTS RT-PCR and Western blot analyses revealed a constitutive expression of psoriasin in cornea, conjunctiva, nasolacrimal ducts, and lacrimal gland. Immunohistochemistry showed strong staining of meibomian glands for psoriasin. No induction of psoriasin was observed after stimulation with supernatants of E. coli, whereas supernatants of Staphylococcus aureus and Haemophilus influenzae significantly increased the psoriasin mRNA expression. Stimulation with IL-1β and VEGF also strongly increased psoriasin transcription. The highest amounts of psoriasin protein were detected in the tear fluid (~170 ng/mL) of healthy volunteers. CONCLUSIONS The results suggest that psoriasin is produced by the structures of the ocular surface and is part of the innate immune system at the ocular surface and tear film.

Human parotid and submandibular glands express and secrete surfactant proteins A, B, C and D

The oral cavity and the salivary glands are open to the oral environment and are thus exposed to multiple microbiological, chemical and mechanical influences. The existence of an efficient defense system is essential to ensure healthy and physiological function of the oral cavity. Surfactant proteins play an important role in innate immunity and surface stability of fluids. This study aimed to evaluate the expression and presence of surfactant proteins (SP) A, B, C, and D in human salivary glands and saliva. The expression of mRNA for SP-A, -B, -C and -D was analyzed by RT-PCR in healthy parotid and submandibular glands. Deposition of all surfactant proteins was determined with monoclonal antibodies by means of Western blot analysis and immunohistochemistry in healthy tissues and saliva of volunteers. Our results show that all four surfactant proteins SP-A, SP-B, SP-C and SP-D are peptides of saliva and salivary glands. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D appear to be involved in immune defense inside the oral cavity. Furthermore, by lowering surface tension between saliva and the epithelial lining of excretory ducts, SP-B and SP-C may assist in drainage and outflow into the oral cavity. Further functions such as pellicle formation on teeth have yet to be determined.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes

OBJECTIVE Trefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage. METHODS We used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay. RESULTS Trefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis. CONCLUSION In this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

MUC16 in the lacrimal apparatus

The aim of the present study was to determine the possible expression of the mucin MUC16 in the lacrimal apparatus. Expression and distribution of MUC16 in lacrimal gland, accessory lacrimal glands, and nasolacrimal ducts was monitored by RT-PCR and immunohistochemistry. MUC16 was expressed and detected in all tissues investigated. Comparable to conjunctiva and cornea it was membrane-anchored in accessory lacrimal glands whereas in lacrimal gland acinar cells and columnar cells of the nasolacrimal ducts it was stored in intracytoplasmic vesicles without membrane-association. Subepithelial serous glands of the nasolacrimal ducts revealed staining of the secretion product. Intracelluar production of MUC16 is present in lacrimal gland and epithelial cells of the nasolacrimal ducts but it is not clear whether this MUC16 is secreted. MUC16 seems to be shedded or secreted from the epithelial surface of subepithelial serous glands of the nasolacrimal ducts. Our results show that MUC16 is present in the whole lacrimal apparatus. Its distribution pattern suggests different physiological functions with regard to tear film physiology and tear outflow. Moreover, the results demonstrate the existence of so far not recognized qualitative differences in the secretion product of main lacrimal gland and accessory lacrimal glands (glands of Krause).

Calcium regulation by temperature-sensitive transient receptor potential channels in human uveal melanoma cells

Uveal melanoma (UM) is both the most common and fatal intraocular cancer among adults worldwide. As with all types of neoplasia, changes in Ca(2+) channel regulation can contribute to the onset and progression of this pathological condition. Transient receptor potential channels (TRPs) and cannabinoid receptor type 1 (CB1) are two different types of Ca(2+) permeation pathways that can be dysregulated during neoplasia. We determined in malignant human UM and healthy uvea and four different UM cell lines whether there is gene and functional expression of TRP subtypes and CB1 since they could serve as drug targets to either prevent or inhibit initiation and progression of UM. RT-PCR, Ca(2+) transients, immunohistochemistry and planar patch-clamp analysis probed for their gene expression and functional activity, respectively. In UM cells, TRPV1 and TRPM8 gene expression was identified. Capsaicin (CAP), menthol or icilin induced Ca(2+) transients as well as changes in ion current behavior characteristic of TRPV1 and TRPM8 expression. Such effects were blocked with either La(3+), capsazepine (CPZ) or BCTC. TRPA1 and CB1 are highly expressed in human uvea, but TRPA1 is not expressed in all UM cell lines. In UM cells, the CB1 agonist, WIN 55,212-2, induced Ca(2+) transients, which were suppressed by La(3+) and CPZ whereas CAP-induced Ca(2+) transients could also be suppressed by CB1 activation. Identification of functional TRPV1, TRPM8, TRPA1 and CB1 expression in these tissues may provide novel drug targets for treatment of this aggressive neoplastic disease.

Antimicrobial Peptides as a Major Part of the Innate Immune Defense at the Ocular Surface

The ocular surface is in constant contact with the environment (e.g. when using ones fingers to insert a contact lens) and thus also with diverse bacteria, bacterial components and their pathogen associated molecules. Dysfunctions of the tear film structure or decreased moistening of the ocular surface, as in dry eye (keratoconjunctivitis sicca) for example, often lead to inflammatory and infectious complications resulting in severe functional disorders, particularly concerning the cornea. Besides different protective antimicrobial substances in the tear fluid (mucins, lysozyme, lactoferrin), the epithelia of cornea and conjunctiva can also protect themselves from microbial invasion by producing an arsenal of antimicrobial peptides (AMPs). A number of different studies have revealed that small cationic AMPs, which display antimicrobial activity against a broad spectrum of microorganisms, are a major component of the innate immune system at the human ocular surface. Furthermore, several AMPs modulate cellular activation processes like migration, proliferation, chemotaxis and cytokine production, and in this way also affect the adaptive immune system. In this article, we have summarized current knowledge of the mechanisms of activity and functional roles of AMPs, with a focus on potential multifunctional roles of human beta-defensins and S100 peptide psoriasin (S100A7) at the ocular surface.