Marta Sloniecka

Substance P Enhances Keratocyte Migration and Neutrophil Recruitment through Interleukin-8

Keratocytes, the resident cells of the corneal stroma, are responsible for maintaining turnover of this tissue by synthesizing extracellular matrix components. When the cornea is injured, the keratocytes migrate to the wounded site and participate in the stromal wound healing. The neuropeptide substance P (SP), which is also known to be produced by non-neuronal cells, has previously been implicated in epithelial wound healing after corneal injury. Corneal scarring, which occurs in the stroma when the process of wound healing has malfunctioned, is one of the major causes of preventable blindness. This study aimed to elucidate the potential role of SP in keratocyte migration and therefore in stromal wound healing. We report that the expression and secretion of SP in human keratocytes are increased in response to injury in vitro. Moreover, SP enhances the migration of keratocytes by inducing the actin cytoskeleton reorganization and focal adhesion formation through the activation of the phosphatidylinositide 3-kinase and Ras-related C3 botulinum toxin substrate 1/Ras homolog gene family, member A pathway. Furthermore, SP stimulation leads to upregulated expression of the proinflammatory and chemotactic cytokine interleukin-8 (IL-8), which also contributes significantly to SP-enhanced keratocyte migration and is able to attract neutrophils. In addition, the preferred SP receptor, the neurokinin-1 receptor, is necessary to induce keratocyte migration and IL-8 secretion. In conclusion, we describe new mechanisms by which SP enhances migration of keratocytes and recruits neutrophils, two necessary steps in the corneal wound-healing process, which are also likely to occur in other tissue injuries.

Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ

Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides/neurotransmitters are involved in cell proliferation, migration, and angiogenesis, it is possible that they play a role in corneal wound healing.

Acetylcholine enhances keratocyte proliferation through muscarinic receptor activation.

Acetylcholine (ACh), a classical neurotransmitter, has been shown to be present in various non-neuronal cells, including cells of the eye, such as corneal epithelium and endothelium, and to have widespread physiological effects such as cytoskeleton reorganization, cellular proliferation, differentiation, and apoptosis. The aim of this study was to investigate the effect of ACh on corneal keratocyte proliferation, and the underlying mechanisms, in order to explore its possible effect in corneal wound healing. Primary culture of human keratocytes was established from donated corneas. Cell viability and fraction of proliferating cells were detected by MTS assay and BrdU incorporation ELISA, respectively. Expression of proliferative markers, PCNA and Ki-67, was detected by western blot and immunocytochemistry. Activation of the MAPK/Erk signaling pathway and its involvement in ACh-enhanced proliferation was determined by western blot analysis, MTS, and BrdU ELISA. We found that ACh enhanced keratocyte proliferation even at low concentrations. Stimulation of proliferation was mediated through activation of muscarinic ACh receptors (mAChRs). Western blot analysis revealed that ACh stimulation of keratocytes upregulated the expression of PCNA and Ki-67, and Ki-67 immunocytochemistry showed that ACh-treated cells were in an active phase of the cell cycle. ACh activated MAPK signaling, and this step was crucial for the ACh-enhanced proliferation, as inhibition of the MAPK pathway resulted in ACh having no proliferative effect. In conclusion, ACh enhances keratocyte proliferation and might thus play a role in proper corneal wound healing.

Antiapoptotic Effect of Acetylcholine in Fas-Induced Apoptosis in Human Keratocytes.

Purpose To investigate the possible antiapoptotic effect of acetylcholine (ACh) in Fas-mediated apoptosis of primary human keratocytes in vitro, and to explore the underlying mechanism. Methods Primary human keratocytes were isolated from healthy corneas. Fas ligand (FasL) was used to induce apoptosis in keratocytes. Cell death was assessed by ELISA. Activity of caspase-3, -7, -8, and -9 was measured with luminescent caspase activity assays. Expression of nuclear factor-κB (NF-κB) gene was assessed with RT-quantitative (q)PCR. Cytochrome c release apoptosis assay kit was used to extract mitochondria and cytosol. Cytochrome c release, cleavage of Bid, and expression of B-cell lymphoma 2 (Bcl-2) were determined by Western blot. Results Cell death ELISA revealed that ACh is able to reduce Fas-induced apoptosis in keratocytes. Analysis of the activity of effector caspases-3 and -7 showed that ACh, when added to Fas-treated cells, decreases the activation of both these enzymes. The activity of initiator caspases -8 and -9 also decreased when ACh was added to Fas-treated cells. This antiapoptotic effect of ACh was dependent on ACh concentration and activation of muscarinic ACh receptors. Analysis of the antiapoptotic mechanisms triggered by ACh showed that ACh downregulates expression of FasL-induced NF-κB RNA expression, upregulates expression of antiapoptotic protein Bcl-2, downregulates expression of proapoptotic protein Bad, reduces cytochrome c release, and prevents proapoptotic Bid protein cleavage. Conclusions Acetylcholine has an antiapoptotic effect in a Fas-apoptosis model of human primary keratocytes in vitro. It is therefore possible that ACh may play a role in corneal wound healing, by modulating its initiation phase.

Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes.

ABSTRACT Purpose: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea – such as stimulating epithelial cell proliferation – processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis. Methods: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR). Results: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor. Conclusions: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.