Driss Zoukhri

Mechanisms of Murine Lacrimal Gland Repair after Experimentally Induced Inflammation

PURPOSEnThe authors recently reported that a severe inflammatory response resulting in substantial loss of acinar cells was induced by a single injection of interleukin-1alpha into the lacrimal gland and that this effect was reversible. The purpose of the present study was to determine the mechanisms involved in lacrimal gland injury and repair.nnnMETHODSnInflammation was induced by direct injection of recombinant human interleukin-1alpha (IL-1alpha, 1 microg in 2 microL) into the exorbital lacrimal glands of anesthetized female BALB/c mice. Animals were killed 1, 2, 3, 4, 5, 6, or 7 days after injection. Exorbital lacrimal glands were then removed and processed for measurement of protein secretion, histology, immunohistochemistry, and Western blotting.nnnRESULTSnThe results show that lacrimal gland acinar cells are lost through programmed cell death (apoptosis) and autophagy. They also show that the number of nestin (a stem cell marker)-positive cells increased 2 to 3 days after injury and that some of these cells were also positive for Ki67 (a cell proliferation marker) and alpha-smooth muscle actin (a marker of myoepithelial cells). Finally, they show that the amount of phosphorylated Smad1/5/8 (effector molecules of bone morphogenetic protein 7 [BMP7]) increased 2 to 3 days after injury and could also be detected in nestin-positive cells.nnnCONCLUSIONSnThe lacrimal gland contains stem/progenitor cells capable of tissue repair after injury. Programmed cell death after injury triggers proliferation and differentiation of these cells, presumably through activation of the BMP7 pathway.

TFOS DEWS II pathophysiology report

The TFOS DEWS II Pathophysiology Subcommittee reviewed the mechanisms involved in the initiation and perpetuation of dry eye disease. Its central mechanism is evaporative water loss leading to hyperosmolar tissue damage. Research in human disease and in animal models has shown that this, either directly or by inducing inflammation, causes a loss of both epithelial and goblet cells. The consequent decrease in surface wettability leads to early tear film breakup and amplifies hyperosmolarity via a Vicious Circle. Pain in dry eye is caused by tear hyperosmolarity, loss of lubrication, inflammatory mediators and neurosensory factors, while visual symptoms arise from tear and ocular surface irregularity. Increased friction targets damage to the lids and ocular surface, resulting in characteristic punctate epithelial keratitis, superior limbic keratoconjunctivitis, filamentary keratitis, lid parallel conjunctival folds, and lid wiper epitheliopathy. Hybrid dry eye disease, with features of both aqueous deficiency and increased evaporation, is common and efforts should be made to determine the relative contribution of each form to the total picture. To this end, practical methods are needed to measure tear evaporation in the clinic, and similarly, methods are needed to measure osmolarity at the tissue level across the ocular surface, to better determine the severity of dry eye. Areas for future research include the role of genetic mechanisms in non-Sjögren syndrome dry eye, the targeting of the terminal duct in meibomian gland disease and the influence of gaze dynamics and the closed eye state on tear stability and ocular surface inflammation.

Isolation and Propagation of Mesenchymal Stem Cells From the Lacrimal Gland

PURPOSEnPreviously, it was reported that the murine lacrimal gland is capable of repair after experimentally induced injury and that the number of stem/progenitor cells was increased during the repair phase (2-3 days after injury). The aim of the present study was to determine whether these cells can be isolated from the lacrimal gland and propagated in vitro.nnnMETHODSnLacrimal gland injury was induced by injection of interleukin (IL)-1, and injection of saline vehicle served as control. Two and half days after injection, the lacrimal glands were removed and used to prepare explants or acinar cells for tissue culture. Cells derived from the explants and the acinar cells were grown in DMEM supplemented with 10% fetal bovine serum. Cells were stained for the stem cells markers, nestin, vimentin, ABCG2, and Sca-1. Cell proliferation was measured using an antibody against Ki67 or a cell-counting kit. The adipogenic capability of these cells was also tested in vitro.nnnRESULTSnResults show that nestin-positive cells can be isolated from IL-1-injected, but not saline-injected, lacrimal glands. A population of nestin-positive cells was also positive for vimentin, an intermediate filament protein expressed by mesenchymal cells. In addition, cultured cells expressed two other markers of stem cells, ABCG2 and Sca-1. These cells proliferated in vitro and can be induced to form adipocytes, attesting to their mesenchymal stem cell property.nnnCONCLUSIONSnMurine lacrimal glands contain mesenchymal stem cells that seem to play a pivotal role in tissue repair.

Mechanisms Involved in Injury and Repair of the Murine lacrimal Gland: Role of Programmed Cell Death and Mesenchymal Stem Cells

The non-keratinized epithelia of the ocular surface are constantly challenged by environmental insults, such as smoke, dust, and airborne pathogens. Tears are the sole physical protective barrier for the ocular surface. Production of tears in inadequate quantity or of inadequate quality results in constant irritation of the ocular surface, leading to dry eye disease, also referred to as keratoconjunctivitis sicca (KCS). Inflammation of the lacrimal gland, such as occurs in Sjogren syndrome, sarcoidosis, chronic graft-versus-host disease, and other pathological conditions, results in inadequate secretion of the aqueous layer of the tear film and is a leading cause of dry eye disease. The hallmarks of lacrimal gland inflammation are the presence of immune cell infiltrates, loss of acinar epithelial cells (the secreting cells), and increased production of proinflammatory cytokines. To date, the mechanisms leading to acinar cell loss and the associated decline in lacrimal gland secretion are still poorly understood. It is also not understood why the remaining lacrimal gland cells are unable to proliferate in order to regenerate a functioning lacrimal gland. This article reviews recent advances in exocrine tissue injury and repair, with emphasis on the roles of programmed cell death and stem/progenitor cells.

c-Jun NH2-terminal kinase mediates interleukin-1beta-induced inhibition of lacrimal gland secretion.

Sjögrens syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear‐deficient type of dry eye. We previously showed that interleukin‐1β (IL‐1β) protein is up regulated in the lacrimal gland of a murine model of Sjögrens syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c‐Jun NH2‐terminal kinase (JNK) in IL‐1β‐mediated inhibition of lacrimal gland secretion and tear production. Inu2003vitro, IL‐1β induced a time‐dependent activation of JNK with a maximum 7.5‐fold at 30u2003min. SP600125, a JNK inhibitor, inhibited, in a concentration‐dependent manner, IL‐1β‐induced activation of JNK with a maximum of 87% at 10−4u2003m. Inu2003vivo, IL‐1β stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL‐1β inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL‐1β on KCl‐ and agonist‐induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7u2003days with SP600125 increased tear production in a murine model of Sjögrens syndrome dry eye. We conclude that JNK plays a pivotal role in IL‐1β‐mediated inhibition of lacrimal gland secretion and subsequent dry eye.

Role of Epithelial-Mesenchymal Transition in Repair of the Lacrimal Gland after Experimentally Induced Injury

PURPOSEnOngoing studies demonstrate that the murine lacrimal gland is capable of repair after experimentally induced injury. It was recently reported that repair of the lacrimal gland involved the mobilization of mesenchymal stem cells (MSCs). These cells expressed the type VI intermediate filament protein nestin whose expression was upregulated during the repair phase. The aim of the present study was to investigate the roles of vimentin, a type III intermediate filament protein and a marker of epithelial-mesenchymal transition (EMT) in repair of the lacrimal gland.nnnMETHODSnInjury was induced by direct injection of interleukin (IL)-1 into the exorbital lacrimal gland. MSCs were prepared from injured glands using tissue explants. Expression of vimentin and the transcription factor Snai1, a master regulator of EMT, was determined by RT-PCR, Western blotting analysis, and immunofluorescence.nnnRESULTSnThese data show that vimentin expression, at both the mRNA and the protein levels, was upregulated during the repair phase (2-3 days postinjury) and returned to the control level when repair ended. Temporal expression of Snai1 mirrored that of vimentin and was localized in cell nuclei. Cultured MSCs isolated from injured lacrimal glands expressed Snai1 and vimentin alongside nestin and alpha smooth muscle actin (another biomarker of EMT). There was a strong positive correlation between Snai1 expression and vimentin expression.nnnCONCLUSIONSnIt was found that EMT is induced during repair of the lacrimal gland to generate MSCs to initiate repair, and that mesenchymal-epithelial transition is then activated to form acinar and ductal epithelial cells.

Immunolocalization of Lacrimal Gland PKC Isoforms. Effect of Phorbol Esters and Cholinergic Agonists on Their Cellular Distribution

Abstract. In previous studies, we showed that lacrimal gland acini express three isoforms of protein kinase C (PKC): PKCα,-δ, and -ε. In the present study, we report the identification of two other PKC isoforms, namely PKCμ and -ι/λ. Using immunofluorescence techniques, we showed that these isoforms are differentially located. PKCα and -μ showed the most prominent membrane localization, whereas PKCδ, -ε and -ι/λ were mainly cytosolic. Using cell fractionation and western blotting techniques, we showed that the phorbol ester, phorbol 12, 13-dibutyrate (PdBu, 10−6m), translocated all PKC isoforms, except PKCι/λ, from the soluble fraction into the particulate fraction. The effect was maximum at 5 min and persisted at 10 min. PKCε was the most responsive to PdBu reaching almost maximal translocation at a PdBu concentration as low as 10−9m. The cholinergic agonist, carbachol (10−5 and 10−3m), induced translocation which was transient for PKCδ, and -μ, but persisted for 10 min for PKCε. Carbachol did not translocate PKCα and, like PdBu, did not translocate PKCι/λ. We concluded that lacrimal gland PKC isoforms are differentially localized and that they translocate differentially in response to phorbol esters and cholinergic agonists.n

Role of protein kinase C in cholinergic stimulation of lacrimal gland protein secretion

The purpose of this study was to determine the role of protein kinase C (PKC) isozymes in carbachol‐induced protein secretion in the lacrimal gland. Three isoforms of PKC are present in rat lacrimal gland: PKC‐α, ‐δ and ‐g3. Carbachol translocated PKC‐ε during 5 s incubation. Pretreatment with PdBu for 0 to 4 h down‐regulated PKC‐α by 31% at 20 min, PKC‐ε by 36% at 2 h, and PKC‐δ by 37% at 4 h. A 2 h phorbol ester treatment inhibited carbachol‐induced secretion completely at 1 min and partially at 5, and 20 min, but did not alter the carbachol‐induced increase in the intracellular [Ca2+]. We conclude that PKC‐α and ‐g3, but not PKC‐δ, are implicated in cholinergic agonist‐induced protein secretion in rat lacrimal gland.

Direct evidence of cytoplasmic delivery of PKC-α, -ϵ and -ζ pseudosubstrate lipopeptides: study of their implication in the induction of apoptosis

Protein kinases C (PKC) are serine/threonine kinase enzymes involved in the mechanism of cell survival. Their pseudosubstrate sequences are autoinhibitory domains, which maintain the enzyme in an inactive state in the absence of allosteric activators, thus representing an attractive tool for the modulation of different PKC isoforms. Here, we report the use of palmitoylated modified PKC‐α, ‐ϵ, and ‐ζ pseudosubstrate peptides, and determine their intracellular distribution together with their respective PKC isoenzymes. Finally, we propose that the differential distribution of the peptides is correlated with a selective induction of apoptosis and therefore argues for different involvement of PKC isoforms in the anti‐apoptotic program.

Transcription Factors Runx1 to 3 Are Expressed in the Lacrimal Gland Epithelium and Are Involved in Regulation of Gland Morphogenesis and Regeneration

PURPOSEnLacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration.nnnMETHODSnExpression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT(2)Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration.nnnRESULTSnWe found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation.nnnCONCLUSIONSnRunx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration.