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ORIGINAL ARTICLE Differential expression in chronic GVHD of the conjunctiva

H Westekemper1,4, S Meller2,4, S Citak1, C Schulte3, K-P Steuhl1, B Homey2 and D Meller1

1Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany; 2Department of Dermatology, University Hospital Du¨sseldorf, Du¨sseldorf, Germany and 3Department of Bone Marrow Transplantation, University of Duisburg-Essen, Essen, Germany

In chronic GVHD after BMT, the conjunctiva represents about 30–50%.1,2 The current understanding of chronic a target organ. GVHD can lead to severe inflammation GVHD defines it as an overlap syndrome with features of and dry-eye syndrome (sicca syndrome). The molecular both the acute and chronic form, but also includes the mechanisms are largely unknown. We examined the classic, time-dependent definition of chronic GVHD.3 In expression of in the conjunctiva in cases of contrast to the acute GVHD, the chronic form is rather a chronic GVHD. In this study, we included 10 patients with disseminated multi-organ disease in which the ocular chronic GVHD and 10 healthy controls. Clinical data surface has been recognised as a target organ.4–6 were collected and tear film analysis and conjunctival Ocular surface effects in chronic GVHD most often cytology were carried out. Conjunctival biopsies were result in a severe dry-eye syndrome (sicca syndrome) with taken from all participants. expression profiles of loss of the ocular surface integrity. The symptoms and chemokines and their corresponding receptors were complications can reduce the quality of life dramatically evaluated by means of quantitative real-time PCR. and even threaten vision.7–11 Ocular involvement seems to Chemokine protein expression was analysed by immuno- correlate to the severity of the GVHD.4,5 histochemical analyses. Expressions of the Th1-associated Activated donor T cells have been identified as the major chemokines, chemokine (C-X-C motif) ligand (CXCL) 9 effectors in the pathogenesis of the GVHD.1,12 T cells (Mig), CXCL10 (IP-10), and their receptor chemokine maintain and amplify the conjunctival inflammation (C-X-C motif) receptor 3 (CXCR3) were significantly through the release of effector , such as IFN-g13 increased in GVHD patients. Immunohistochemical ana- and subsequently lead to tissue destruction,1,14 here on the lysis confirmed marked expression of the inflammatory ocular surface. CXCR3 ligands. A total of six patients had a moderate or Chemokines are small, secreted proteins that have been severe sicca syndrome. Impression cytology revealed a shown to mediate directed migration and critically mild keratinisation, moderate keratinisation or severe regulate organ-specific homing of leukocytes.15–18 In ocular squamous metaplasia in three patients, respectively. surface diseases, chemokines have an important role in Chronic GVHD of the conjunctiva is characterised by inflammatory processes occurring in vernal keratoconjuncti- the expression of Th1-associated chemokines. Taken vitis19,20 and atopic conjunctivitis.21,22 Moreover, studies have together, our results confirm that the conjunctiva is a demonstrated that corneal epithelial and stromal cells are target organ in this T cell-mediated process and add to capable of producing various chemokines in herpetic stromal molecular understanding of conjunctival GVHD. keratitis and inflamed corneas after perforating injuries.23,24 Bone Marrow Transplantation (2010) 45, 1340–1346; It has been demonstrated that members of the chemokine doi:10.1038/bmt.2009.346; published online 8 February 2010 superfamily are involved in immunological and autoimmu- Keywords: chemokine; CXCL9; CXCL10; CXCR3; nological diseases.25–27 The upregulation of several chemo- conjunctiva; GVHD kines is associated with acute GVHD.28,29 However, the pathogenic role of chemokines in chronic conjunctival GVHD remains elusive to a large extent. Until now there have been few data to show the involvement of chemokines and their receptors in the Introduction pathogenesis of conjunctival chronic GVHD. In this study, we analysed the chemokine gene and protein expression Despite HLA typing, the incidence of GVHD after allo- pattern in the conjunctiva of patients suffering from geneic haematopoietic stem cell transplantation (HSCT) is chronic GVHD. Using quantitative real-time PCR, we found significantly elevated levels of pro-inflammatory chemokines in the conjunctiva of patients with chronic Correspondence: Dr D Meller, Department of Ophthalmology, University GVHD compared with healthy controls. These findings of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany. were confirmed by immunohistochemistry using monoclo- E-mail: [email protected] 4These authors contributed equally to this work. nal antibodies against different chemokines and their Received 23 February 2009; revised 1 July 2009; accepted 29 September respective receptors in conjunctival biopsies of patients 2009; published online 8 February 2010 with chronic GVHD compared with healthy controls. Conjunctival chemokine expression in GVHD H Westekemper et al 1341 Materials and methods pathological changes. These tests specify the extent of the fluid- or lipid-deficiency syndrome and together with the Patients results of the IPC they sum up to the grade of the sicca The study has been approved by the appropriate local syndrome. ethics committee and followed the tenets of the Declaration of Helsinki. In this study, 10 patients who suffered from chronic GVHD after HSCT were investigated. The grading Quantitative real-time PCR analysis of GVHD followed the ‘Consensus Conference in Acute Quantitative real-time PCR analyses were performed as GVHD Grading’30 and the grading system of Gratwohl previously described.27,36.Conjunctival biopsy specimens et al.31 for chronic GVHD. As a control group, we included were homogenised in liquid nitrogen using a Micro- 10 patients who visited our clinic for cataract or strabismus Dismembrator U (Braun Biotech, San Diego, CA, USA), surgery. All patients signed an informed consent form and and RNA was extracted with RNAzol according to the underwent clinical examination to detect dry-eye-associated manufacturer’s protocol (Tel-Test, Friedensburg, TX, ocular surface changes. A conjunctival biopsy was USA). A total of 4 mg of RNA was treated with DNase I performed under local anaesthesia from all patients from (Boehringer, Mannheim, Germany) and reverse transcribed the bulbar conjunctiva, quickly frozen in liquid nitrogen with oligo(dT)14–18 (Gibco BRL, Gaithersburg, MD, and stored at À80 1C until analysis. The biopsies were used USA) and random hexamer primers (Promega, Madison, to perform quantitative real-time PCR analysis and WI, USA), using standard protocols. A total of 25 ng of immunohistochemistry. complementary DNA was amplified in the presence of 12.5 ml of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 0.625 ml of gene- Clinical examination specific TaqMan probe, 0.5 ml of gene-specific forward and Clinical examination routinely included best-corrected reverse primers, and 0.5 ml of water. Chemokine ligand- and visual acuity, intraocular pressure, slit-lamp examination -specific primers and target-specific of the anterior segment and funduscopy of the retina. Dry- probes were obtained from Applied Biosystems. In the eye syndrome related examination included Schirmer test first step, a chemokine-expression screening was performed (basal tear secretion), fluorescein clearance test, vital on the following selected chemokines: chemokine (C-C staining with fluorescein (50 ml fluorescein-Na 1%) and motif) ligand (CCL) 1 (I-309), CCL2 (MCP-1), CCL17 Rose Bengal (50 ml Rose Bengal 0.5%), tear film break-up (TARC), CCL18 (PARC), CCL27 (CTACK), chemokine time, Meibomian gland status and impression cytology (C-X-C motif) ligand (CXCL) 9, CXCL10, CXCL11 and (IPC) of the conjunctiva. the receptors chemokine (C-C motif) receptor (CCR) 2, Vital staining of the corneal surface reveals epithelial chemokine (C-X-C motif) receptor (CXCR) 3 and CXCR4. defects, including erosion or punctuate erosions and late This was done to identify interesting target chemokines fluorescein staining in case of vascularisation of the cornea. using only a set of five probes from patients and controls. For the fluorescein clearance test, 10 ml of fluorescein Na Afterwards CXCL9, CXCL10 and CXCL11, as well as 1% was applied to the conjunctival surface. The fluorescein their receptor CXCR3 were analysed in all probes. Gene- clearance test gives information about the basal and specific PCR products were measured with an ABI PRISM stimulated aqueous tear secretion and about the fluorescein 7700 or 5700 Sequence Detection System (Applied Bio- clearance from the ocular surface. A continuous staining systems), continuously during 40 cycles. Target gene reflected a ‘delayed tear clearance’.32,33 expression was normalised between different samples on The Meibomian gland status was tested by transpalpeb- the basis of the expression values of the internal positive ral illumination and graded into three groups. Grade 1 control (18S RNA) or .27,36 reflected a Meibomian gland atrophy in less than one-third of the eye lid, grade 2 an atrophy of no more than two- thirds and grade 3 a Meibomian gland atrophy of more Immunohistochemistry than two-thirds of the eye lid.34 For immunohistochemical analysis of the expression of The IPC was performed using 4 Â 7 mm filtration paper chemokines and chemokine receptors, conjunctival sections that was pressed gently on the conjunctival surface under were fixed with acetone and pre-processed with H2O2 local anaesthesia. The collected specimen were fixated in followed by an avidin–biotin blocking step (Avidin/Biotin formalin (3.7%) and stained with periodic acid-Schiff– Blocking Kit; Vector, Burlingame, CA, USA). Sections haematoxylin staining. The IPC was used to detect dry-eye were stained with monoclonal antibodies against CXCR3 associated morphological changes in the conjunctival (mouse IgG1; R&D Systems, Minneapolis, MN, USA), epithelium, such as loss of goblet cells, cellular desquama- CXCR4 (mouse IgG2b; R&D Systems), CXCL9 (goat IgG; tion resulting in squamous metaplasia. We applied the R&D Systems), CXCL10 (goat IgG; R&D Systems) and grading system according to Koch et al.35 CXCL12 (mouse IgG2a, K15C; Unite0 d’Immunologie A pathological tear film break-up time points to a lipid Virale, Institute Pasteur, Paris, France). CXCL12 served as tear deficiency on the ocular surface, whereas a patholo- positive control as it is a homeostatic chemokine and was gical Schirmer test (basal tear secretion) points to an therefore expected to be present in patients as well as aqueous tear deficiency. Often, both occur. The meibomian controls. For each antibody, an appropriate isotype control gland affection, the vital staining and the clearance (all R&D Systems) was stained. Development of the reduction were graded in relation to the intensity of the staining was performed using a Vectastain ABC kit and

Bone Marrow Transplantation Conjunctival chemokine expression in GVHD H Westekemper et al 1342 a Vectastain AEC kit (both from Vector). Sections were included TBI (10 Gy) and high-dose CY treatment in eight counterstained with haematoxylin. cases, TBI and fludarabin treatment in one case, and BU combined with high-dose CY treatment in one case. The GVHD prophylaxis was performed by MTX and CYA in Statistical analysis eight cases, CYA alone in one case and CYA with Statistical analysis was performed using Microsoft Excel mycophenolate mofetil in another. The analysis of the 2000 and Stat View for Windows (Abacus Concepts, donor–host relation data revealed nine HLA-identical Berkeley, CA, USA, Version 4.55). When calculating matches, one partially matched pair and eight pairs were correlations, a P-value 0.05 was considered statistically p gender matched. In six cases, the donor was unrelated to significant. Spearman correlation and unpaired t-test were the recipient, in two cases the sister was the donor, in one performed to test associations between chemokine gene case each the brother and the cousin. At the time of the expression and clinical findings or ocular complications. study examination, all patients were in complete remission. Three patients did not receive any systemic immunosup- pression at the time of examination. Two of them had Results received a transplant from an HLA-identical unrelated donor and one from a related donor (cousin). All three Demographic data donors were male. The mean age at the time of SCT was 42.07±9.49 years (median: 41.3, range: 30.9–59.8 years). The mean age at the time of study examination was 46.0±10.4 years in the Ocular surface examinations patients’ group (median: 43.2, range: 34.8–63.6 years) and Mean time between SCT and the onset of dry-eye 70.8±13.7 years in the control group (median: 75.4, range: symptoms was 10.9±3. 97 months (median: 10.3, range: 39.0–88.4 years). Interval-time between HSCT and the 9.0–18.3 months). The results of ocular surface diagnostics study examination was 48.28±31.61 months (median: 44.4, and gradings are provided in Table 1. A total of seven range: 9.3–109.0 months). All patients underwent allo- patients had a ‘Delayed Tear Clearance’ in the fluorescein geneic PBSCT. In four cases, the patients received PBSCT clearance test. The results of the IPC are presented in because of AML, in four cases because of CML. One Table 2. Ocular surface complications during follow-up patient suffered from ALL and another from a myelodys- included corneal ulcers in four cases (two perfora- plastic syndrome. The conditioning regimen before SCT ting ulcers), limbal stem cell deficiency with ingrowths of

Table 1 Results of the ocular surface diagnostics

Patient Break-up Tear MG-affection Delayed tear Vital staining Lipid tear Aqueous tear Sicca time secretion clearance (RB and Fluo) deficiency deficiency grade

1 Path Path Delayed clearance Punctuate erosion Moderate Moderate Mild 2 Norm Norm Moderate atrophy Delayed clearance Mild staining Moderate Moderate Moderate 3 Path Norm Total atrophy Delayed clearance Corneal erosion Severe Severe Severe 4 Punctuate erosion Moderate Moderate Severe 5 Path Path Mild atrophy Delayed clearance Mild staining Mild Mild Mild 6 Path Path moderate atrophy Delayed clearance Corneal erosion Severe Severe Severe 7 Path Path Mild atrophy Normal clearance Punctuate erosion Moderate Moderate Moderate 8 Path Path Normal MG Delayed clearance Punctuate erosion Moderate Moderate Mild 9 Path Path Normal clearance Punctuate erosion Moderate Moderate Moderate 10 Norm Path Mild atrophy Delayed clearance Mild staining Mild Mild Mild

Abbreviations: Fluo ¼ fluorescein; IPC ¼ impression cytology; MG ¼ Meibomian gland; norm ¼ normal; path ¼ pathological; RB ¼ Rose Bengal.

Table 2 Impression cytology results with grading of the conjunctival squamous metaplasia

Patient Cellular enlargement Nuclear changes Desquamation Goblet cell density Grade

1 None None Present Normal Normal 2 Mild None Present Little decreased Mild keratinising 3 Mild Moderate Present Markedly decreased Moderate keratinising 4 Severe Moderate Present Markedly decreased Severe squamous metaplasia 5 Mild Mild None Little decreased Mild keratinising 6 Severe Moderate Present Complete loss Severe squamous metaplasia 7 Severe Mild Present Complete loss Severe squamous metaplasia 8 Moderate None None Little decreased Mild keratinising 9 Moderate None Present Complete loss Moderate keratinising 10 Mild Mild None Complete loss Moderate keratinising

Grading is according to Koch et al.35

Bone Marrow Transplantation Conjunctival chemokine expression in GVHD H Westekemper et al 1343 conjunctival tissue in the cornea in four cases and Immunohistochemistry of chemokines CXCL9, CXCL10, conjunctival scarring in two cases. Such complications CXCL11 and CXCR3 result from the dry-eye syndrome and inflammation of the To identify the cellular origin and obtain insights into ocular surface. In the control group, no patient presented the anatomic location of CXCL9, CXCL10 and CXCL11 clinically a dry-eye syndrome or morphological changes and their receptor CXCR3 within the conjunctiva, we indicating squamous metaplasia in the IPC. performed immunohistochemical analyses (Figures 2, 3). A statistically weak correlation existed between the grade CXCL9, CXCL10 and CXCR3 were expressed to a higher of the dry-eye syndrome and the grade of squamous extent in the patient group than in the controls. Within the metaplasia (R2 ¼ 0.44, P-value ¼ 0.058). No correlation was conjunctiva, cells with dendritic morphology found between the grade of dry-eye syndrome or squamous as well as endothelial cells of the superficial stromal metaplasia and the characteristics of the PBSCT (gender of conjunctiva were identified as abundant sources of CXCL9 donor, related donor). expression (Figures 2a and b). In contrast, CXCL10 showed marked expression in all layers of the conjunctival epithelium in the patient’s group but was almost Chemokine negative in the controls (Figures 2c and d). Their receptor, To investigate whether chemokines are involved in lym- CXCR3, demonstrated strong expression on conjunctiva- phocyte recruitment in conjunctival chronic GVHD, we infiltrating leukocytes in perivascular, stromal locations performed quantitative real-time PCR analysis and demon- of GVHD patients (Figures 3a and b). Endothelial strated that the Th1-associated chemokines CXCL9, CXCL10, cells also showed strong staining for CXCR3 (Figures 3a CXCL11 and their shared receptor CXCR3 were markedly and b). The homeostatic chemokine CXCL12 was positive upregulated in conjunctival chronic GVHD compared with in both groups and was observed in the basal epithelium, healthy controls. The levels of CXCL9 (P ¼ 0.007) and in endothelial cells and conjunctiva-infiltrating leuko- CXCL10 (P ¼ 0.007), as well as the corresponding CXCR3 cytes (Figures 3c and d). The corresponding receptor, (P ¼ 0.049) showed a statistically significant difference between CXCR4, was expressed by conjunctiva-infiltrating leuko- both groups (Figure 1). We found no significant difference in cytes (Figures 3e and f). Taken together, our immuno- the expression of the chemokines CCL1, CCL2, CXCL12 and histological findings confirm the results obtained by the receptor CXCR4. Although a trend toward increased quantitative real-time PCR. The findings demonstrate expression of CCL17, CCL18 and CCL27, as well as CCR2 was the expression of chemokine ligands in close anato- observed, statistical analysis did not demonstrate a significant mic proximity with cells expressing their respective up regulation of these in conjunctival chronic GVHD. receptors.

30000 25000 P=0.007 25000 22500 P=0.007 20000 20000 17500 15000 Units 15000 12500 Units 10000 10000 7183.8 7500 7398.8 ± 5898.1 2266.8 ± 895.3 range: 5000 5000 498-29378 2500 1997.7 ± 862.9 0 0 CXCL9GvHD CXCL10 Control 1600 120 1400 P=0.064 P=0.049 100 1200

1000 80 146.1 ± 94.5 800 60 196.9 ± 43.6

Units 871.5

600 Units range: 40 400 15.5-1574 143.4 ± 64.1 200 20 0 0 CXCL11 CXCR3-A Figure 1 Chemokine expression of the CXC (C-X-C motif) chemokines chemokine (C-X-C motif) ligand (CXCL) 9–11 and isoform A of their chemokine (C-X-C motif) receptor (CXCR) 3. P-values are given from the Mann–Whitney U Test. In addition, mean and s.d. values are inserted in or above the boxplots. The horizontal line within a boxplot marks the median. The box itself spans from the 25th percentile (lower end) to the 75th percentile (upper end). The T-lines below and above the box mark the 10th percentile (below) and the 90th percentile (above). CXCL11 expression revealed a trend towards a higher expression in the GVHD group, although the statistical result was not quite significant.

Bone Marrow Transplantation Conjunctival chemokine expression in GVHD H Westekemper et al 1344 Controls GvHD Controls GvHD CXCR3 CXCL9 CXCL12 CXCL10

Figure 2 Immunohistochemistry of conjunctival tissue (magnification  200). (a and c) Controls; (b and d) GVHD patients. (a and b) chemokine

(C-X-C motif) ligand (CXCL) 9 (MIG); (c and d) CXCL10 (IP-10). CXCL9 CXCR4 is predominantly expressed in the conjunctival stroma (arrow), and CXCL10 in the conjunctival epithelium (arrow). Both control sections show a lower expression than the sections of the GVHD group.

Figure 3 Immunohistochemistry of the chemokine (C-X-C motif) recep- tor (CXCR) 3, chemokine (C-X-C motif) ligand (CXCL) 12 (SDF-1a and Association of chemokine gene expression to clinical CXCR4 expression in the conjunctival tissue (magnification  200). (a, c findings and ocular complications and e) Controls; (b, d and f) GVHD patients. (a and b) CXCR3; (c and d) Statistical analysis did not reveal a correlation between any CXCL12; (e and f) CXCR4. CXCR3 is expressed in the conjunctival stroma and perivascular endothelium (arrows). CXCL12 is positively of the chemokines CXCL9–11 and their receptor CXCR3 expressed in both groups and is markedly shown in the basal epithelium-, and the following factors: sicca grade, basal tear secretion, endothelium- and conjunctiva-infiltrating leucocytes (arrows). Conjuncti- tear film break-up time, vital staining of ocular surface, tear va-infiltrating leucocytes also express CXCR4 (arrows). clearance reduction, Meibomian gland effects, IPC, ocular complications, age at HSCT, gender of donor, relation donor to recipient, HLA match, conditioning regime, GVHD their receptors may be a valuable molecular method to prophylaxis and interval from HSCT to occurrence of dry- monitor and diagnose chronic GVHD of the conjunctiva. eye syndrome. The P-values of Spearman’s correlation and Testing on other chemokines (CCL1, CCL2, CCL17, unpaired t-test lay between P ¼ 0.08 and P ¼ 0.93 (data not CCL18 and CCL27) and chemokine receptors (CCR2 and shown). CXCR4) revealed no differential expression between patients with chronic GVHD and controls. In this pilot study, healthy persons were chosen as controls. In a limited cohort as we present in this study, we preferred the Discussion comparison with normal tissue rather than with tissue that might be affected by non-immunological factors, such as Chemokines represent a superfamily of small -like myeloablative therapy before HSCT in patients that proteins that mediate directional migration in vitro and received HSCT and did not develop GVHD. The difference control leukocyte trafficking in vivo.18 in the mean age between patients and controls was accepted In this study, we demonstrate the induction of a distinct because the functional analysis of the ocular surface did not panel of genes and proteins in the conjunctiva of patients reveal any pathological changes in the control group. suffering from chronic GVHD. Notably, the Th1-asso- A hallmark of chronic GVHD is the expression of IFN- ciated chemokines CXCL9, CXCL10 and CXCL11, as well g, even though the pathogenetic importance is controver- as their receptor CXCR3 were the most upregulated sial.37 -g is a potent inducer for the expression of chemokine ligands and receptors in chronic conjunctival the CXCR3 ligands, CXCL9, CXCL10 and CXCL11 GVHD compared with healthy controls. Although these (I-TAC).38,39 Among the memory T cell subsets, CXCR3 chemokines are not specific for chronic GVHD, quantita- is predominantly expressed on the surface of IFN-g- tive monitoring of the gene expression of chemokines and producing Th1 cells.18 The findings in this study demon-

Bone Marrow Transplantation Conjunctival chemokine expression in GVHD H Westekemper et al 1345 strate that the majority of cells infiltrating the conjunctiva Conflict of interest display CXCR3 on their cell surface. Recent studies have shown by gene expression profiling The authors declare no conflict of interest. that pro-inflammatory chemokines are upregulated in different target organs of GVHD.28,29,40 In addition, the influence of chemokines on the recruitment of activated CD4 þ T cells into the skin of GVHD patients has been References 41 elucidated. Supporting these data, another study found 1 Dickinson A, Charron D. Non-HLA immunogenetics in an elevated expression of CXCL10 in the epidermis and hematopoietic stem cell transplantation. Curr Opin Immunol also higher levels of its receptor CXCR3 in dermal 2005; 17: 517–525. inflammatory infiltrates of patients suffering from 2 Fraser C, Scott Baker K. The management and outcome of GVHD.14 Moreover, Zhou et al.42 demonstrated that chronic graft-versus-host disease. Br J Haematol 2007; 138: CXCL9, CXCL10 and CXCL11 are upregulated in the 131–145. skin of an experimental murine model with scleroderma- 3 Filipovich A, Weisdorf D, Pavletic S, Socie G, Wingard J, Lee tous GVHD. Our findings are in line with these reports S et al. National Institutes of Health consensus development and indicate that CXCL9 and CXCL10 have a central project on criteria for clinical trials in chronic graft-versus-host role in the pathogenesis of conjunctival chronic GVHD by disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005; 11: 945–956. the recruitment of CXCR3 þ T cells to the sites of 4 Jabs D, Wingard J, Green W, Farmer E, Vogelsang G, Saral inflammation. R. The eye in bone marrow transplantation. III. Conjunctival Synergistic cooperation between inflammatory and graft-vs-host disease. 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