Downloaded from http://bjo.bmj.com/ on August 30, 2016 - Published by group.bmj.com Laboratory science and are expressed by human corneal stromal dendritic cells Adam Wilkinson, Naomi Kawaguchi, Carolyn Geczy, Nick Di Girolamo

▸ Additional material is ABSTRACT but are also found within the corneal epithelium6 published online only. To view Background/aims The limbus is a remarkable and play a pivotal role in immune surveillance. please visit the journal online (http://dx.doi.org/10.1136/ anatomical site endowed with specialised functions to DCs located within the limbus are strategically bjophthalmol-2016-308827). ensure corneal health and transparency, which is placed within a region prone to infection, inflam- essential for exquisite vision. Cell types that contribute to mation and tumourigenesis. School of Medical Sciences, University of New South Wales, homeostasis and the disease-free state of the cornea S100 proteins are involved in regulating numerous Sydney, New South Wales, include epithelial and stromal stem cells, and antigen- cellular functions including differentiation, prolifer- Australia presenting dendritic cells (DCs). DCs are found ation and inflammation.7 S100A8 (A8) and S100A9 throughout the corneal epithelium and stroma, but the (A9) are constitutively expressed in myeloid-derived Correspondence to markers that discriminate between cells in cells including and monocytes, and are Dr Nick Di Girolamo, School of 8 Medical Sciences, University of different locations have not been properly identified. induced in , capillary endothelial and New South Wales, Sydney, S100 proteins are expressed in normal and diseased epithelial cells by Toll-like receptor agonists.7 These NSW 2052 Australia; ocular surfaces and are implicated in DC differentiation. proteins promote pro-inflammatory7 and immuno- [email protected] Methods This study used transplant quality human protective responses,89and are linked to antioxidant fl 10 7 Received 5 April 2016 cadaveric donor corneas (n=6) and immuno uorescence defence. The A8/A9 complex, also known as cal- 11 Revised 6 June 2016 to determine the spatial distributions of S100A8 (A8) protectin, can suppress acute inflammation and has Accepted 19 June 2016 and S100A9 (A9), and to characterise the cell types antimicrobial activities.12 A8 and A9 overexpression Published Online First expressing these proteins. has been linked to dry eye, pterygium, ocular surface 7 July 2016 Results A8-expressing and A9-expressing cells were squamous neoplasia13 and corneal neovascularisa- predominantly confined to the limbal stroma and tion,14 and the absence of these proteins is asso- represented 0.25%±0.1% and 0.39%±0.1%, ciated with intraocular inflammation,15 although A9 respectively, of the total stromal cell population. They was suggested as an early marker of inflammation were phenotyped as CD45+/HLA-DR+/CD11c+, markers within the limbal stem cell niche.15 Interestingly, A8 characteristic of DCs. Interestingly, A8 and A9 and A9 proteins have been detected in various popu- – immunoreactivity was only associated with stromal DCs, lations of DCs.16 18 In the current study, we investi- but not those entrenched in the epithelium. gated the distribution of A8 and A9 in cadaveric Conclusions A8 and A9 expression may distinguish donor human corneas and discovered their expres- between subpopulations of DC that reside in different sion in a population of DCs specifically residing in regions of the human cornea and may influence their the corneal stroma. maturation status.

METHODS INTRODUCTION Human tissue and isolation of white blood cells The cornea is a transparent collagenous organ cir- Transplant quality donor corneas (n=6) were cumscribed by a narrow transition zone known as sourced from Lions NSW Eye Bank (Sydney) (four the limbus that partitions it from the conjunctiva. males, two females; average age, 59 years; age This collar of tissue is endowed with specialised range 39–86 years). Peripheral blood (10 mL) from functions including (i) maintenance of epithelial healthy donors (n=3) was collected in acid citrate- mass through the activity of limbal epithelial stem dextrose and depleted of erythrocytes and neutro- cells, (ii) supplying nutrients and chemical media- phils by sedimentation on 4.5% Dextran T-500 tors to the avascular cornea through its extensive solution (Sigma, Sydney, Australia). Density sedi- vascular network1 and (iii) immune surveillance by mentation over Lymphoprep (Axis Shield, Oslo, inflammatory cells.2 The corneal stroma comprises Norway) was performed by centrifugation for ∼90% of corneal thickness and contains a hetero- 1 hour at 1500 rpm. The intermediate layer was geneous population of cells, the vast majority of collected, washed twice with phosphate buffered which are resident neural crest-derived keratocytes, saline (PBS), centrifuged for 10 min at 1500 rpm and a rarer population of primitive fibrocytes that and the pellet containing polymorphonuclear cells reside proximal to the limbus and express markers reconstituted in PBS. Peripheral blood mononuclear of bone marrow-derived mesenchymal stem cells.3 cells (PBMCs) and neutrophils were counted in a These cells are responsible for regenerating the haemocytometer and resuspended to a final con- corneal stroma under steady state,3 and following centration of 5×105 cells/mL. A 200 μL cell sus- pathological insults, they regulate inflammation pension was placed into disposable cuvettes and 3 To cite: Wilkinson A, through their immunosuppressive properties. centrifuged onto glass slides at 900 rpm for 5 min Kawaguchi N, Geczy C, Dendritic cells (DCs) are also resident to the (Thermo Shandon, Pittsburgh, Pennsylvania, USA). et al. Br J Ophthalmol cornea;45these ‘professional’ antigen-presenting Slides were fixedfor10minin4% – 2016;100:1304 1308. cells tend to congregate within the limbal stroma paraformaldehyde.

1304 Wilkinson A, et al. Br J Ophthalmol 2016;100:1304–1308. doi:10.1136/bjophthalmol-2016-308827 ikno A, Wilkinson tal et . rJOphthalmol J Br Table 1 Antibodies used for immunological analysis Antibody Isotype Source Clone no. Host Stock (mg/mL) Dilution factor

19 S100A8 IgG In-house Polyclonal (NS) Rabbit 1.0 1:500 Downloaded from 2016; S100A9 IgG In-house19 Polyclonal (NS) Rabbit 1.5 1:1500

100 CD34 IgG1 Chemicon; Billerica, Massachusetts, USA QBEND/10 Mouse 1.0 1:10

:1304 CD45 IgG1 BD Biosciences; San Jose, California, USA 2D1 Mouse NS 1:50 HLA-DR IgG2κ BD Biosciences L243 Mouse 0.025 1:300 –

38 o:013/jptaml21-0871305 doi:10.1136/bjophthalmol-2016-308827 1308. CD11c IgG1κ BD Biosciences B-ly6 Mouse NS 1:50

Mouse IgG IgG1 IgG1; R&D Systems, Minneapolis, Minnesota, USA 11711 Mouse 1.0 1:10–1:1000 http://bjo.bmj.com/ Rabbit IgG IgG Santa Cruz Biotech; Dallas, Texas, USA SC-2027 Rabbit 0.4 1:10–1:1000 NS, not specified. onAugust30,2016-Publishedby el.Aliae eetkna a at taken stromal incubated were immunoreactive section indicate images a arrows All in White cells. reactivity IgG. no rabbit shows control (B) with The Inset (blue). sections. DAPI magni serial with is from counterstained (B) and in red), box C, hatched and (B A9 specimens and independent two B from and corneas (A of Sections cornea. human 1 Figure asidct 10 indicate bars – ,rsetvl)wr muotie o 8( n ,red) D, and (A A8 for immunostained were respectively) D, oaiaino 108(8 n 109(9 ntehealthy the in (A9) S100A9 and (A8) S100A8 of Localisation m m. group.bmj.com fi di C.Iae nB) in Images (C). in ed fi a magni nal aoaoyscience Laboratory fi aino 10,scale ×1000, of cation – D r derived are (D) Downloaded from http://bjo.bmj.com/ on August 30, 2016 - Published by group.bmj.com Laboratory science

Immunocytochemistry microscope (Olympus BX51), images were acquired as tiff or Cells deposited on glass slides were permeabilised with 0.1% jpg files with DP Controller (Olympus, V.1.1.1.65) and DP Saponin (Sigma) in tris-buffered saline (TBS), blocked with 20% Manager (Olympus, V.1.1.1.71), and processed with Adobe goat serum in 2% bovine serum albumin (BSA) in TBS, incu- Photoshop (V.10.0). Some slides were digitally scanned using bated overnight with rabbit antihuman A8 or A9 IgG produced ScanScope FL (Aperio Technologies, Vista, California, USA) at in the host laboratory (table 1),19 and the reactivity detected ×20 resolution. A8-positive and A9-positive cells were counted with a biotinylated goat antirabbit IgG secondary antibody on representative corneas; each sample contained two or more (Dako, Sydney, Australia). Cells were stained using a Vectastain sections. The average number of S100-positive cells over the ABC-AP kit (Vector Laboratories, Burlingame, California, USA), total number of DAPI-stained nuclei within the stroma was counterstained with Mayer’s haematoxylin (Sigma) and recorded from limbus to limbus in transverse sections. mounted in DPX (Fronine Laboratory Supplies, Sydney, Australia). Reagent controls included replacing the primary anti- RESULTS body with a relevant isotype IgG antibody (table 1). S100A8 and S100A9 expression in normal human cornea Faint positive staining for A8 and A9 was associated with the Immunofluorescence and cell counting superficial corneolimbal epithelium (not shown). In addition, Fresh-frozen human corneal sections were cut (5 μm), fixed in both proteins were abundantly expressed in a rare population of ice-cold 95% ethanol and allowed to equilibrate room tempera- stromal cells located within the limbal domain (figure 1A, B). As ture. Tissue was placed in TBS and blocked with 20% goat indicated in serial sections, cells expressing A8 were also immu- serum in 2% BSA/TBS for 40 min at room temperature. noreactive for A9 (figure 1C, D). The proportion (mean±SD) Predetermined concentrations of primary antibodies (table 1) of A8+ and A9+ over total stromal cell population (ie, DAPI+ were added to respective sections prior to incubating overnight cells) in human corneas (n=3) represented 0.25%±0.1% and at 4°C. Goat antimouse Alexa-Fluoro488 and goat antirabbit 0.39%±0.1%, respectively. Notably, only the occasional S100+ Alexa-Fluoro568 (Molecular Probes, Eugene, Oregon, USA) sec- cell was detected in the central corneal stroma (data not shown). ondary antibodies were added (simultaneously for double stain- To ensure specificity of the anti-S100 antibodies, they were ing) for 30 min. Sections were washed, then cover-slipped after tested on cytospin preparations of whole-blood PBMC and iso- adding Prolong Gold antifade reagent containing DAPI lated neutrophils, which express both proteins.7 As expected, (Molecular Probes). Tissue was viewed under a fluorescence monocyte-like cells in the PBMC fraction (see online

Figure 2 S100A8 (A8)/A9+ (A9) cells coexpress CD45. Tissue sections from three individual corneas (A–C, D and E–G, respectively) were immunostained for A9 (A, red), A8 (E, red), CD45 (B and D), HLA-DR (F, green) and counterstained with DAPI (C, D, and G, blue). Red asterisks in A)–(C) indicate the location of the epithelium. White asterisks (E–G) indicate the location of HLA-DR+ cells within the epithelium. The white lines in all panels demarcate the epithelial basement membrane. Immunofluorescence was visualised using appropriate filters and images were merged (C, D and G). All photomicrographs were taken at a final magnification of ×1000 under oil immersion and scale bars indicate 10 mm.

1306 Wilkinson A, et al. Br J Ophthalmol 2016;100:1304–1308. doi:10.1136/bjophthalmol-2016-308827 Downloaded from http://bjo.bmj.com/ on August 30, 2016 - Published by group.bmj.com Laboratory science supplementary figure S1A–C) and neutrophils (see online DISCUSSION supplementary figure S1D–F) expressed both A8 and A9. The cornea is often referred to as an ‘immune-privileged’ site.20 However, it is constantly exposed to environmental stimuli and external pathogens, and immunodefence must occur continuously Characterising S100A8+/A9+ corneal stromal cells in order to maintain tissue homeostasis. To achieve this effect- Because of the rare occurrence of these cells and their odd mor- ively, resident immune cells such as DCs are strategically posi- – phological appearance, we theorised that they may be specia- tioned to sample foreign antigen4 6 and play an integral role in lised immune cells. First, we established that host defence against microbes. They contribute to self-tolerance A8-immunoreactive and A9-immunoreactive cells were not by interacting with T cells through a network of peripherally haematopoietic in origin, but despite detecting individual located blood and lymphatic vessels,1 thereby controlling inflam- CD34+ cells, and blood vessel-associated CD34 reactivity mation. Notably, antigen-presenting cells are stratified into bone within the limbal stroma, these cell types did not express A8 marrow-derived monocytic and dendritic lineages that are present and A9 (see online supplementary figure S2). S100-positive within the murine621and human22 cornea. corneal stromal cells were CD45+ (figure 2A–C), HLA-DR+ This study was conducted to map the spatial distribution of (figure 2E–G) and CD11c+ (figure 3A–F), suggesting that a two myeloid-derived members of the family population of DCs was being marked. Although CD45+/ within the normal human cornea. Herein, we detected a rare HLA-DR+/CD11c+ cells were detected within the corneolimbal population of peripherally located corneal stromal cells that epithelium (figures 2D, F and 3B, E), these cells did not express coexpressed A8 and A9 (figure 1) in conjunction with pheno- A8 and A9. typic markers typical of DCs. CD45+/HLA-DR+/CD11c+ DCs

Figure 3 S100-positive stromal cells are dendritic cells (DCs). Tissue sections from two individual corneas (A–C and D–F, respectively) were immunostained for S100A8 (A8) (A and D, red) and CD11c (B and E, green), and counterstained with DAPI (C and F, blue). White asterisks and arrows indicate the location of CD11c+ DCs within the epithelium and stroma, respectively. The white line in all panels demarcates the epithelial basement. All images were taken at a final magnification of ×1000 under oil immersion and scale bars indicate 10 mm.

Wilkinson A, et al. Br J Ophthalmol 2016;100:1304–1308. doi:10.1136/bjophthalmol-2016-308827 1307 Downloaded from http://bjo.bmj.com/ on August 30, 2016 - Published by group.bmj.com Laboratory science were detected within the corneal epithelium, but these superfi- Ethics approval University of New South Wales Human Research Ethics cially located DCs did not express A8 and A9 (figures 2 and 3), Committee. suggesting that a distinct subpopulation was discriminated by Provenance and peer review Not commissioned; externally peer reviewed. their S100 content. Recent studies describe DCs as heteroge- neous, with functions dependent on their anatomical location and on the pathogens or conditions to which they are exposed; REFERENCES some DC subsets, such as those in the skin, contribute to tolero- 1Ecoiffier T, Yuen D, Chen L. Differential distribution of blood and genic mechanisms that maintain immune homeostasis.23 lymphatic vessels in the murine cornea. Invest Ophthalmol Vis Sci 2010;51: + + 2436–40. Interestingly, CD45 /CD11c DCs within the corneal epithe- 2 Niederkorn JY. Immune privilege and immune regulation in the eye. Adv Immunol lium exhibited a morphologically distinct dendritiform shape, 1990;48:191–226. whereas those located within the anterior limbal stroma 3 Hertsenberg AJ, Funderburgh JL. Stem cells in the cornea. Prog Mol Biol Transl Sci appeared rounder.22 We noted that A8-reactive and A9-reactive 2015;134:25–41. stromal DCs were uniform in appearance compared with those 4 Hamrah P, Zhang Q, Liu Y, et al. Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells. Invest lacking expression of these proteins that were located within the Ophthalmol Vis Sci 2002;43:639–46. epithelium (figures 1–3). One hypothesis is that the expression 5 Yamagami S, Yokoo S, Usui T, et al. Distinct populations of dendritic cells in the of these proteins defines maturity of DC subpopulations.18 normal human donor corneal epithelium. Invest Ophthalmol Vis Sci Interleukin 10 (IL-10) is an immunosuppressive that 2005;46:4489–94. 6 Knickelbein JE, Watkins SC, McMenamin PG, et al. Stratification of promotes tolerogenic properties in DC. IL-10-treated human antigen-presenting cells within the normal cornea. Ophthalmol Eye Dis DCs derived from peripheral blood lose their typical dendrite 2009;1:45–54. morphology, exhibit reduced T cell stimulatory function and 7 Donato R, Cannon BR, Sorci G, et al. Functions of S100 proteins. Curr Mol Med express high levels of the A8 and A9 .18 Results from 2013;13:24–57. 8 Hiroshima Y, Hsu K, Tedla N, et al. S100A8 induces IL-10 and protects against those studies suggested that A8 and A9 contribute to inhibition – 18 acute lung injury. J Immunol 2014;192:2800 11. of DC maturation. Our extensive studies show that IL-10 is 9 Hsu K, Champaiboon C, Guenther BD, et al. Anti-infective protective 9 essential for A8 induction in several cell types. Moreover, properties of S100 calgranulins. Antiinflam Antiallergy Agents Med Chem A8 and A9 expression levels are strongly enhanced by glucocor- 2009;8:290–305. ticosteroids, and mechanisms for their gene induction strongly 10 Lim SY, Raftery MJ, Geczy CL. Oxidative modifications of DAMPs suppress 9 inflammation: the case for S100A8 and S100A9. Antioxid Redox Signal support immunomodulatory roles. Furthermore, in the lung, 2011;15:2235–48. A8 strongly induces IL-10 in airway epithelial cells, indicating 11 Ikemoto M, Murayama H, Itoh H, et al. Intrinsic function of S100A8/A9 complex as an important feedback loop8 that could potentially influence an anti-inflammatory protein in liver injury induced by lipopolysaccharide in rats. DC cell functions. Alternatively, as in atherosclerosis24 one DC Clin Chim Acta 2007;376:197–204. population may originate from a monocyte precursor that is 12 Clark HL, Jhingran A, Sun Y, et al. Zinc and manganese chelation by S100A8/A9 () limits extracellular Aspergillus fumigatus hyphal growth likely to express A8 and A9, and another may mature/differenti- and corneal infection. J Immunol 2016;196:336–44. ate from a precursor of different origin that does not express 13 Tong L, Lan W, Lim RR, et al. S100A Proteins as molecular targets in the ocular the S100s. Others have proposed that A9 reduces DC differenti- surface inflammatory diseases. Ocul Surf 2014;12:23–31. ation from haematopoietic progenitor cells to favour accumula- 14 Li C, Zhang F, Wang Y. S100A proteins in the pathogenesis of experimental corneal 18 neovascularization. Mol Vis 2010;16:2225–35. tion of myeloid-derived suppressor cells. 15 Nubile M, Lanzini M, Calienno R, et al. S100 A and B expression in normal and Our results on examining the functions of A8 led us to inflamed human limbus. Mol Vis 2013;19:146–52. propose important modulatory roles that may contribute to 16 Lood C, Stenström M, Tydén H, et al. Protein synthesis of the pro-inflammatory immune homeostasis. In particular, this protein is an efficient S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on scavenger of hypohalous acid oxidants typically generated by leukocyte subpopulations in systemic lupus erythematosus. Arthritis Res Therapy 10 2011;13:R60. activated phagocytes during infection. A8 also suppresses 17 Kumar A, Steinkasserer A, Berchtold S. Interleukin-10 influences the expression of Toll-like receptor 4 activation by endotoxin and has profound MRP8 and MRP14 in human dendritic cells. Int Arch Allergy Immunol immunomodulatory effects on production of chemokines and 2003;132:40–7. that modulate acute inflammation in murine lung and 18 Cheng P, Corzo CA, Luetteke N, et al. Inhibition of dendritic cell differentiation and 8 accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 is principally expressed in M2 macrophages. Moreover, the A8/ protein. J Exp Med 2008;205:2235–49. A9 calprotectin complex is important in antimicrobial defence 19 McCormick MM, Rahimi F, Bobryshev YV, et al. S100A8 and S100A9 in human and invasiveness,9 and A8 and A9 neutralisation on the ocular arterial wall. Implications for atherogenesis. J Biol Chem 2005;280:41521–9. surface increases infection by Pseudomonas aeruginosa, resulting 20 Streilein JW. Anterior chamber associated immune deviation: the privilege of – in severe keratitis.25 The data presented herein suggest that immunity in the eye. Surv Ophthalmol 1990;35:67 73. 21 Hamrah P, Liu Y, Zhang Q, et al. The corneal stroma is endowed with a significant A8-expressing and A9-expressing DCs within the cornea con- number of resident dendritic cells. Invest Ophthalmol Vis Sci 2003;44:581–9. tribute to the modulation of innate immune homeostasis in the 22 Knickelbein JE, Buela KA, Hendricks RL. Antigen-presenting cells are stratified within normal human ocular surface. normal human corneas and are rapidly mobilized during ex vivo viral infection. Invest Ophthalmol Vis Sci 2014;55:1118–23. Contributors AW and NK contributed equally to the article. AW: collection, 23 Klechevsky E. Functional diversity of human dendritic cells. Adv Exp Med Biol assembly and analysis of data, manuscript writing. NK: collection, assembly and 2015;850:43–54. analysis of data. CG: analysis, interpretation of data, manuscript writing, financial 24 Koltsova EK, Ley K. How dendritic cells shape atherosclerosis. Trends Immunol support. NDG: collection, assembly and analysis of data, manuscript writing, 2011;32:540–7. financial support, final approval of manuscript. 25 Gao N, Sang Yoon G, Liu X, et al. Genome-wide transcriptional analysis of differentially expressed genes in flagellin-pretreated mouse corneal epithelial cells in Funding University of New South Wales. response to Pseudomonas aeruginosa: involvement of S100A8/A9. Mucosal Competing interests None declared. Immunol 2013;6:993–1005.

1308 Wilkinson A, et al. Br J Ophthalmol 2016;100:1304–1308. doi:10.1136/bjophthalmol-2016-308827 Downloaded from http://bjo.bmj.com/ on August 30, 2016 - Published by group.bmj.com

S100A8 and S100A9 proteins are expressed by human corneal stromal dendritic cells

Adam Wilkinson, Naomi Kawaguchi, Carolyn Geczy and Nick Di Girolamo

Br J Ophthalmol 2016 100: 1304-1308 originally published online July 7, 2016 doi: 10.1136/bjophthalmol-2016-308827

Updated information and services can be found at: http://bjo.bmj.com/content/100/9/1304

These include: References This article cites 25 articles, 9 of which you can access for free at: http://bjo.bmj.com/content/100/9/1304#BIBL Email alerting Receive free email alerts when new articles cite this article. Sign up in the service box at the top right corner of the online article.

Notes

To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions

To order reprints go to: http://journals.bmj.com/cgi/reprintform

To subscribe to BMJ go to: http://group.bmj.com/subscribe/