Immunolocalisation of leukaemia inhibitory factor in the cornea

ABSTRACT _Aim_ Leukaemia inhibitory factor (LIF) is a pleotrophic cytokine expressed in a variety of cell types, and have shown to regulate stem cell proliferation, vascular genesis,

inflammation, and immunity in various locations. Expression of LIF and its role in the cornea have not been studied previously. In this study, we examined the expression of LIF in the

cornea. _Materials and method_ Immunohistochemistry was performed using polyclonal LIF antibodies, and Avidin–Biotin ABC complex on cultured human corneal epithelium corneal fibroblasts and

wild-type murine corneal epithelium. _Results_ LIF was detected in the cytoplasm of murine corneal epithelium, cultured human corneal epithelium, and fibroblasts. The expression of LIF was

mainly cytoplasmic. _Conclusion_ LIF is expressed in the corneal epithelium and fibroblasts. It may have an important role in the maintenance of homeostasis of the corneal epithelium and

cornea stroma. Further studies are necessary to elucidate the role of LIF in the cornea. SIMILAR CONTENT BEING VIEWED BY OTHERS INTERFERON-Γ ELICITS THE OCULAR SURFACE PATHOLOGY MIMICKING

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Article Open access 22 October 2021 ROLE OF INTERFERON REGULATORY FACTOR 7 IN CORNEAL ENDOTHELIAL CELLS AFTER HSV-1 INFECTION Article Open access 13 August 2021 INTRODUCTION Leukaemia

inhibitory factor (LIF) is a pleotrophic cytokine expressed in a variety of cell types _in vitro_, which include activated T lymphocytes, monocytes, mast cells neuronal cells, dermal

keratinocytes, fibroblasts and endothelial cells, intestinal mucosal, respiratory mucosal, and retinal pigment epithelial cells.1, 2, 3, 4, 5 LIF belongs to a group of cytokines that

includes ciliary neurotropin factor, IL-6, Ct-1, OM, and IL-11.5 All the groups members elicit different biological effects through the GP-130 signal transducing subunit as a common

receptor.5 Li _et al_6, 7 first reported the expression of LIF mRNA in cultured human corneal epithelial cells and keratocytes. The regulatory function of LIF in a variety of locations

including stem cell proliferation, vascular genesis, inflammation, and immunity has been recently elucidated.8, 9, 10, 11 However, the role played by LIF in the cornea is largely unexplored.

In this study, we examined the corneal expression of LIF. MATERIAL AND METHODS CULTURING HUMAN CORNEAL EPITHELIAL CELLS Corneal epithelial cells were cultured according to accepted previous

methods.12, 13 Briefly, donor human corneal buttons maintained in organ culture media were obtained from the Manchester Eye bank. A measure of 2 mm explants from three different donors were

prepared after removing central 8 mm of cornea. Explants were incubated with 0.1% trypsin and EDTA for 2 h at 37°C in 5% CO2. Single cells were isolated and plated on growth-arrested 3T3-J2

feeder layer containing Green's media. The medium was changed every third day. The culture reached 70–80% confluence on day 14. 3T3 cells were removed by vigorous pipetting. The

remaining 3T3 cells were removed with 0.01% trypsin for 15 s. The epithelial cells were then allowed to reach confluence. After 3 weeks, the epithelial cells became stratified. Epithelial

sheets were separated from the culture dish with dispase (2 mg/ml in DMEM-Ca free and 10FCS) and fixed in 4% paraformaldehyde for 4 h. CULTURING HUMAN CORNEAL FIBROBLASTS Corneal fibroblasts

were cultured according to accepted previous methods.6, 14 Human corneas were obtained from Manchester Eye Bank. Central 8 mm corneal buttons from three different donors were fashioned

after removing the endothelium and the epithelium. The corneal stroma was cut into 2 × 2 mm pieces and incubated in fibroblast growth medium containing DMEM, 2 mM L-glutamine, 10% foetal

calf serum and 100 IU/ml penicillin and streptomycin (all from Sigma). Upon confluence, the cells were passaged into cell culture flasks. Human corneal fibroblasts (passage 4) were cultured

on cover slips (Sigma Grace Bio-Labs) according to manufacturer's instructions and incubated at 37°C in a CO2 incubator. After 48 h, the cells were washed in PBS twice and fixed in 4%

paraformaldehyde at room temperature for 15 min. After two washes in PBS, the cells were dehydrated through graded concentrations of ethanol. MOUSE CORNEA Six wild-type mice were killed by

cervical dislocation and their eyes were removed and fixed in 4% paraformaldehyde for 4 h. To facilitate sectioning, lenses were removed and the eyes were processed in paraffin wax. All

animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. HISTOLOGICAL AND IMMUNOLOGICAL TECHNIQUES Sections of eyes

and the epithelial sheets cut at 7 _μ_m were mounted on poly-L-lysine coated slides and deparaffinised sections were treated with 100% ethanol. For immunostaining, both the sections (eyes

and the epithelial sheath) on the slides and the fibroblasts on the cover slips were treated as follows.4 First, they were incubated with 3% hydrogen peroxide for 20 min to remove endogenous

peroxidase activity, then rehydrated with 70% ethanol, and washed in PBS. The specimens were treated with normal rabbit serum for 30 min to block the nonspecific binding of antibodies and

then incubated overnight at 4°C with primary LIF antibody (Santa Cruz Goat polyclonal IgG, stock diluted 1 : 500). The specimens were then treated for 30 min with secondary antibody

(biotinylated, anti-goat IgG, Vectastain ABC Kit) diluted 1 : 200 in blocking serum, washed in PBS, and then incubated for 30 min with ABC complex. After washing in PBS, the binding antibody

was visualised by 3,3′-diaminobenzidine (DAB, Sigma). All the specimens were then lightly counterstained with haematoxylin. Control slides and cover slips were incubated with normal rabbit

serum, but otherwise treated in the same way. RESULTS MOUSE CORNEA LIF expression was cytoplasmic and expressed in all layers of the corneal epithelium (Figure 1a and b). LIF expression was

detected in all samples exmained. The superficial layers showed intense LIF expression. The basal layers showed weaker staining for LIF. LIF expression was present in limbal, paracentral,

and central corneal epithelium. The endothelium and corneal stroma did not show LIF expression. Cultured corneal epithelial sheets expressed LIF in the cytoplasm and was detected in all

layers of the corneal epithelium(Figure 1c and d). Similar to murine cornea, the superficial layers showed intense LIF expression. In the cultured corneal fibroblasts, LIF expression was

detected in the cytoplasm (Figure 1e and f). DISCUSSION In this study, we were able to immunolocalise LIF in cultured human corneal epithelial cells and fibroblasts. The expression of LIF in

epithelial cells and fibroblasts, which were not subjected to injury or other stimulatory cytokines, was intriguing. LIF was also expressed in organ-cultured corneal epithelium (results not

shown). We then examined whether LIF is expressed in normal corneal epithelium. As we were not able to obtain fresh human corneal tissue, the mouse cornea was examined. This showed that LIF

is expressed _in vivo_ in the corneal epithelium. Recent studies have shown the regulatory importance of LIF in stem cell proliferation, vascular genesis, inflammation, and immunity.8, 9,

10, 11 However, the role played by LIF in the cornea is largely unexplored. The sources of the adult corneal epithelial cells are the stem cell population residing at the limbus. The factors

that regulate the corneal limbal stem cell have not been well elucidated. At lease two _in vitro_ LIF culture systems were shown to enhance stem cell proliferation. In a coculture system

containing murine fibroblast and embryonic stem cells, addition of LIF promotes the proliferation of ES cells by several fold.15 This property is unique to LIF and only occurred in the

presence of fibroblasts. Further addition of LIF, to human haematopoietic stem cells CD34(+) thy-1(+) and murine bone marrow-derived stromal cell coculture caused expansion of haematopoietic

stem cells _in vitro_.16 This stem cells expansion was specific to LIF and was observed only in the presence of fibroblast or stromal cells that are mesenchymal in origin. The action of LIF

is indirect and mediated by mesenchymal cells. We observed that coculturing human limbal epithelial cells and limbal fibroblasts gave more prolific colonies in the presence of LIF

(unpublished data). The interplay between corneal limbal stem cell and limbal fibroblast appears analogous to the interaction between haematopoietic stem cell and stromal cells, which is

modulated under LIF. A stem cell expansion-promoting factor may be the possible link between the fibroblasts and corneal stem cell under the modulation of LIF. The cornea is uniquely

organised to discourage the induction and expression of inflammatory responses, particularly immune effector mechanisms that inflict significant injury to adjacent cells. The creation of

such an immunological blind spot should render the cornea vulnerable to opportunistic infections and neoplasms. However, the conspicuous absence of neoplasm and the relatively low incidence

of opportunistic infections suggest the existence of an effective immunological surveillance at the corneal surface. This property may be attributed to the presence of LIF in the corneal

epithelial cells. Recently, the viral inhibitory potential of LIF has been demonstrated.17, 18 _In vitro_ LIF exerted a potent, GP130 dependent, inhibition of HIV-I replication.17 LIF also

inhibits HIV-I in placenta and thymus tissue grown in _ex vivo_ organ culture.17 Further, LIF is upregulated in human marrow stromal cells infected with human cytomegalovirus.18 In subacute

sclerosing panencephalitis, LIF mRNA expression is upregulated in neurones infected with measles virus.19 LIF may act as a pro- or anti-inflammatory factor depending on the profile of tissue

and other cytokines. This inflammatory role has been documented in CNS tissues and in skin.11, 19, 20, 21, 22, 23, 24 New vessel formation in the cornea is a significant factor contributing

to visual morbidity. Although factors such as VEGF, which modulates cornea, and new vascularisation has been characterised, the process of vascularisation is far from clear. Several studies

have indicated that LIF could play a role in the regulation of blood vessel formation,25, 26, 27, 28, 29 and LIF may have an implication in pathological vascularisation in ocular tissues.

Depending upon the tissue origin or stage of differentiation, LIF has been shown to exert different actions. LIF stimulates adrenal cortex endothelial cells and embryonic cells, while it

inhibit aortic endothelial cells.25, 26, 27 Embryonic endothelial cell line can be optimally induced to differentiate into capillary-like structures _in vitro_ by combined exposure to LIF

plus bFGF.26, 28 During the formation of capillary structures from embryonic endothelial cell, line two—Stat3 and P41/43MAPK cascade systems are activated.29 Stat3 acts as negative

regulators on capillary development, and inhibition of this pathway is accompanied by increases capillary outgrowth, while inhibition of P41/43MAPK pathway dramatically reduces the capillary

formation.29 Fine tuning of the extent of capillary growth depends on the regulation of these pathways and the cytokine profile. LIF can activate both cascades, thereby acting as a negative

as well as a positive regulator of vascular genesis.28, 29 Further studies examining the role of LIF in the corneal vascularisation may have therapeutic implications. LIF is constitutively

expressed in the corneal epithelium. It may play a role in modulating corneal stem cell dynamics, inflammation, and vascularisation. Further studies are necessary to determine the role of

LIF in cornea during health and pathological processes. REFERENCES * Hu J, Ono S, Katayama H, Imai T, Shimizu N, Nakagawa H . Leukemia inhibitory factor induces epidermal hyperplasia in

patients with amyotrophic lateral sclerosis. _J Invest Dermatol_ 2000; 115(3): 486–492. Article  CAS  Google Scholar  * Gillett NA, Lowe D, Lu L, Chan C, Ferrara N . Leukemia inhibitory

factor expression in human carotid plaques: possible mechanism for inhibition of large vessel endothelial regrowth. _Growth Factors_ 1993; 9(4): 301–305. Article  CAS  Google Scholar  *

Knight DA, Lydell CP, Zhou D, Weir TD, Robert Schellenberg R, Bai TR . Leukemia inhibitory factor (LIF) and LIF receptor in human lung. Distribution and regulation of LIF release. _Am J

Respir Cell Mol Biol_ 1999; 20(4): 834–841. Article  CAS  Google Scholar  * Paglia D, Kondo S, Ng KM, Sauder DN, McKenzie RC . Leukaemia inhibitory factor is expressed by normal human

keratinocytes _in vitro_ and _in vivo_. _Br J Dermatol_ 1996; 134(5): 817–823. Article  CAS  Google Scholar  * Gadient RA, Patterson PH . Leukemia inhibitory factor, Interleukin 6, and other

cytokines using the GP130 transducing receptor: roles in inflammation and injury. _Stem Cells_ 1999; 17(3): 127–137. Article  CAS  Google Scholar  * Li DQ, Tseng SC . Differential

regulation of cytokine and receptor transcript expression in human corneal and limbal fibroblasts by epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor

B, and interleukin-1 beta. _Invest Ophthalmol Vis Sci_ 1996; 37(10): 2068–2080. CAS  PubMed  Google Scholar  * Li DQ, Tseng SC . Three patterns of cytokine expression potentially involved

in epithelial-fibroblast interactions of human ocular surface. _J Cell Physiol_ 1995; 163(1): 61–79. Article  CAS  Google Scholar  * Shih CC, DiGiusto D, Forman SJ . _Ex vivo_ expansion of

transplantable human hematopoietic stem cells: where do we stand in the year 2000. _J Hematother Stem Cell Res_ 2000; 9(5): 621–628. Article  CAS  Google Scholar  * Shih CC, Hu MC, Hu J,

Weng Y, Yazaki PJ, Medeiros J et al. A secreted and LIF-mediated stromal cell-derived activity that promotes _ex vivo_ expansion of human hematopoietic stem cells. _Blood_ 2000; 95(6):

1957–1966. CAS  Google Scholar  * Smith AG, Nichols J, Robertson M, Rathjen PD . Differentiation inhibiting activity (DIA/LIF) and mouse development. _Dev Biol_ 1992; 151(2): 339–351.

Article  CAS  Google Scholar  * McKenzie RC, Paglia D, Kondo S, Sauder DN . A novel endogenous mediator of cutaneous inflammation: leukemia inhibitory factor. _Acta Derm Venereol_ 1996;

76(2): 111–114. CAS  PubMed  Google Scholar  * Pellegrini G, Bondanza S, Guerra L, De Luca M . Cultivation of human keratinocyte stem cells: current and future clinical applications. _Med

Biol Eng Comput_ 1998; 36(6): 778–790. Article  CAS  Google Scholar  * Rheinwald JG . Serial cultivation of normal human epidermal keratinocytes. _Methods Cell Biol_ 1980; 21A: 229–254.

Article  CAS  Google Scholar  * Li DQ, Tseng SC . Differential regulation of keratinocyte growth factor and hepatocyte growth factor/scatter factor by different cytokines in human corneal

and limbal fibroblasts. _J Cell Physiol_ 1997; 172(3): 361–372. Article  CAS  Google Scholar  * Nichols J, Evans EP, Smith AG . Establishment of germ-line-competent embryonic stem (ES) cells

using differentiation inhibiting activity. _Development_ 1990; 110(4): 1341–1348. CAS  PubMed  Google Scholar  * Shih CC, Hu MC, Hu J, Medeiros J, Forman SJ . Long-term _ex vivo_

maintenance and expansion of transplantable human hematopoietic stem cells [see comments]. _Blood_ 1999; 94(5): 1623–1636. CAS  Google Scholar  * Patterson BK, Behbahani H, Kabat WJ,

Sullivan Y, O'Gorman MR, Landay A et al. Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women. _J Clin Invest_ 2001; 107(3):

287–294. Article  CAS  Google Scholar  * Lagneaux L, Delforge A, Snoeck R, Bosmans E, Moreau JF, Taupin JL et al. Human cytomegalovirus increases constitutive production of interleukin- 6

and leukemia inhibitory factor by bone marrow stromal cells. _Blood_ 1996; 87(1): 59–66. CAS  PubMed  Google Scholar  * McQuaid S, Campbell R, Isserte S, Cosby SL . Leukaemia inhibitory

factor mRNA is expressed in the brains of patients with subacute sclerosing panencephalitis. _J Neuroimmunol_ 1997; 77(1): 57–62. Article  CAS  Google Scholar  * Banner LR, Patterson PH .

Major changes in the expression of the mRNAs for cholinergic differentiation factor/leukemia inhibitory factor and its receptor after injury to adult peripheral nerves and ganglia. _Proc

Natl Acad Sci USA_ 1994; 91(15): 7109–7113. Article  CAS  Google Scholar  * Banner LR, Moayeri NN, Patterson PH . Leukemia inhibitory factor is expressed in astrocytes following cortical

brain injury. _Exp Neurol_ 1997; 147(1): 1–9. Article  CAS  Google Scholar  * Curtis R, Scherer SS, Somogyi R, Adryan KM, Ip NY, Zhu Y et al. Retrograde axonal transport of LIF is increased

by peripheral nerve injury: correlation with increased LIF expression in distal nerve. _Neuron_ 1994; 12(1): 191–204. Article  CAS  Google Scholar  * Kurek JB, Austin L, Cheema SS, Bartlett

PF, Murphy M . Up-regulation of leukaemia inhibitory factor and interleukin-6 in transected sciatic nerve and muscle following denervation. _Neuromuscul Disord_ 1996; 6(2): 105–114. Article

  CAS  Google Scholar  * Kurek JB, Bennett TM, Bower JJ, Muldoon CM, Austin L . Leukaemia inhibitory factor (LIF) production in a mouse model of spinal trauma. _Neurosci Lett_ 1998; 249(1):

1–4. Article  CAS  Google Scholar  * Pepper MS, Ferrara N, Orci L, Montesano R . Leukemia inhibitory factor (LIF) inhibits angiogenesis _in vitro_. _J Cell Sci_ 1995; 108(Part 1): 73–83. CAS

  PubMed  Google Scholar  * Gendron RL, Tsai FY, Paradis H, Arceci RJ . Induction of embryonic vasculogenesis by bFGF and LIF _in vitro_ and _in vivo_. _Dev Biol_ 1996; 177(1): 332–346.

Article  CAS  Google Scholar  * Ferrara N, Winer J, Henzel WJ . Pituitary follicular cells secrete an inhibitor of aortic endothelial cell growth: identification as leukemia inhibitory

factor. _Proc Natl Acad Sci USA_ 1992; 89(2): 698–702. Article  CAS  Google Scholar  * Paradis H, Arceci RJ, Adams LC, Gendron RL . Differentiation responses of embryonic endothelium to

leukemia inhibitory factor. _Exp Cell Res_ 1998; 240(1): 7–15. Article  CAS  Google Scholar  * Paradis H, Gendron RL . LIF transduces contradictory signals on capillary outgrowth through

induction of stat3 and (P41/43)MAP kinase. _J Cell Sci_ 2000; 113(Part 23): 4331–4339. CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by

Ophthalmology research grant- Royal College of Surgeons Edinburgh. Sponsored by Royal Blind Asylum & Scottish National Institution for war Blind. (K Ramaesh, J West. B Dhillon: Leukaemia

inhibitory factor and corneal epithelial stem cell proliferation) AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Tennent Institute of Ophthalmology, Gartnaval General Hospital, 1053, Great

Western Road, Glasgow, G12 0YN, UK K Ramaesh * Princess Alexandra Eye Pavilion, Chalmers street, EH3 9HA, Edinburgh, UK T Ramaesh & B Dhillon * Department of Developmental Sciences,

University of Edinburgh, Hugh Robson Building, EH8 9DX, George square, UK T Ramaesh & J D West Authors * K Ramaesh View author publications You can also search for this author inPubMed 

Google Scholar * T Ramaesh View author publications You can also search for this author inPubMed Google Scholar * J D West View author publications You can also search for this author

inPubMed Google Scholar * B Dhillon View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to K Ramaesh. RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ramaesh, K., Ramaesh, T., West, J. _et al._ Immunolocalisation of leukaemia inhibitory factor in the cornea. _Eye_

18, 1006–1009 (2004). https://doi.org/10.1038/sj.eye.6701394 Download citation * Received: 27 August 2003 * Accepted: 06 November 2003 * Published: 02 April 2004 * Issue Date: 01 October

2004 * DOI: https://doi.org/10.1038/sj.eye.6701394 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link

is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * leukaemia inhibitory factor * corneal epithelium

* and fibroblasts