Oral Diseases (2015) 21, 378–385 doi:10.1111/odi.12290 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com

ORIGINAL ARTICLE

Histamine H4 receptor in oral lichen planus A Salem1,2, A Al-Samadi1,2, V Stegajev1, H Stark3, R H€ayrinen-Immonen4, M Ainola1, J Hietanen2,5, YT Konttinen1,6 1

Department of Medicine, University of Helsinki and Helsinki University Central Hospital, Helsinki; 2Department of Oral Pathology, at University of Helsinki, Helsinki, Finland; 3Institut f€ur Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universit€ D€ usseldorf, D€usseldorf, Germany; 4City of Helsinki Health Care Centre, Helsinki; 5HUSLAB, Helsinki University Central Hospital, Helsinki; 6ORTON Orthopedic Hospital of the Invalid Foundation, Helsinki, Finland

OBJECTIVES: Oral lichen planus (OLP) is an autoimmune disease characterized by a band-like T-cell infiltrate below the apoptotic epithelial cells and degenerated basement membrane. We tested the hypothesis that the high-affinity histamine H4 receptors (H4Rs) are downregulated in OLP by high histamine concentrations and proinflammatory T-cell cytokines. MATERIALS AND METHODS: Immunohistochemistry and immunofluorescence staining, image analysis and quantitative real-time polymerase chain reaction of tissue samples and cytokine-stimulated cultured SCC-25 and primary human oral keratinocytes. RESULTS: H4R immunoreactivity was weak in OLP and characterized by mast cell (MC) hyperplasia and degranulation. In contrast to controls, H4R immunostaining and MC counts were negatively correlated in OLP (P = 0.003). H4R agonist at nanomolar levels led to a rapid internalization of H4Rs, whereas high histamine concentration and interferon-c decreased HRH4-gene transcripts. CONCLUSION: Healthy oral epithelial cells are equipped with H4R, which displays a uniform staining pattern in a MC-independent fashion. In contrast, in OLP, increased numbers of activated MCs associate with increasing loss of epithelial H4R. Cell culture experiments suggest a rapid H4R stimulation-dependent receptor internalization and a slow cytokine-driven decrease in H4R synthesis. H4R may be involved in the maintenance of healthy oral mucosa. In OLP, this maintenance might be impaired by MC degranulation and inflammatory cytokines. Oral Diseases (2015) 21, 378–385 Keywords: histamine H4 receptor; oral lichen planus; mast cells; cytokines; oral pathology

Correspondence: Yrj€o T. Konttinen, MD, PhD, Department of Medicine, Institute of Clinical Medicine, PO Box 700 (Haartmaninkatu 8, Biomedicum 1), 00029 HUS Helsinki, Finland. Tel: +358 50 448 4685, E-mail: yrjo.konttinen@helsinki.fi Received 18 February 2014; revised 3 September 2014; accepted 3 September 2014

Introduction Oral lichen planus (OLP) is a mucosal autoimmune disease, which affects 1.27% of the population (McCartan and Healy, 2008). Histologically, the pathognomonic feature of OLP is apoptosis of basal epithelial cells and degenerated basement membrane (BM) associated with a band-like subepithelial T-cell-rich lymphocyte infiltrate (Jungell et al, 1987). This is compatible with a delayed lymphocyte-mediated reaction against damaged basal cells and BM (Sugerman et al, 2002). This interpretation is supported by similar histological findings in graft-versus-host reaction (Sato et al, 2006) and local lichenoid reactions against amalgam filling (McParland and Warnakulasuriya, 2012). Indeed, OLP is characterized by antigen-driven oligoclonal expansion of T cells (Kawamura et al, 2003). The mechanisms responsible for the maintenance of healthy human oral epithelium and its pathologic changes in OLP are unclear. Histamine concentration in mast cell (MC) granules is ~100 mM (Harvima, 2008). Due to the increased numbers of resting and degranulated mast cells in OLP (Sugerman et al, 2002), it was thought that burst released 10–50 lM histamine, able to stimulate the lowaffinity histamine 1 receptor (H1R) and histamine 2 receptor (H2R), could damage basal epithelial cells. However, H1R (‘antihistamines’) and H2R antagonists were not helpful in the treatment of OLP (Lodi et al, 2012), and speculations on the potential role of histamine in the pathogenesis of OLP were abandoned. The recent discovery of high expression of histamine H4 receptor (H4R) (Oda et al, 2000) in human skin keratinocytes (Yamaura et al, 2013) together with the recognition of these cells as nonprofessional (Table 1) histamine producing cells (Inami et al, 2013) have revived interest in the potential role of histamine in healthy and OLP mucosa. H4R-equipped cells are highly sensitive to histamine. The pKi value of H4R is 8.3 compared to 4.2 for H1R and 4.3 for H2R viz. over 10 000-fold difference (Walter and Stark, 2012). Further, a certain basal level H4R activity is maintained due to its relatively high intrinsic activity even in the absence of any ligand. Half-maximal stimulation is achieved already at

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Table 1 Main characteristics of professional and non-professional histamine producing cells (modified from Konttinen et al, 2013) Characteristic Type of utilized HDC Histamine synthesis Histamine storage Stimulus requirement Histamine release Histamine concentration Main functional receptors Examples

Professional Low mw (53 KD) Fast Yes (in secretory granules) Yes

Non-professional

Active High (micromolar)

High mw (74 KD) Slow No (transient release to the cytoplasm) No (flows along the concentration gradient) Passive Low (nanomolar)

H1R and H2R

H3R and H4R

Mast cells, basophils, enterochromaffin-like cells

T-lymphocytes, dendritic cells, epithelial cells

HDC, histidine decarboxylase; mw, molecular weight.

10 nM histamine. This led to the hypothesis that low nanomolar histamine produced and continuously released by non-professional cells and acting on H4R could participate in the maintenance of healthy oral epithelial cells. This homeostasis could be disturbed by high histamine concentrations burst released by activated MCs in OLP, which primarily act on conventional low-affinity histamine receptors but which due to oversaturation of H4Rs might downregulate their expression. Due to immuneinflammation typical for OLP, also the effects of two key pro-inflammatory cytokines, tumor necrosis factor-a (TNFa) and interferon-c (IFN-c) on H4R were also studied.

Materials and methods Patients and controls The Ethics Committee of the Hospital District of Helsinki and Uusimaa approved the study (42/13/03/01/2013). All subjects gave their informed consent before entering the study. All patients (11 females, seven males, mean age 56 years, range 22–84) were clinically and histologically diagnosed with OLP according to the WHO recommendation and its 2009 modification (Kramer et al, 1978; Rad et al, 2009). Control oral mucosal samples were collected from healthy individuals undergoing wisdom tooth extraction (10 controls, mean age 26 years, range 12–51). In addition, two more samples of healthy oral mucosa were collected from two patients for MC stimulation studies. OLP biopsies were taken from active lesion areas by a specialized oral surgeon under local anesthesia (20 mg ml
1 xylocaine and 12.5 lg ml
1 adrenaline) and fixed in 10% neutral buffered formalin (NBF), dehydrated in an increasing ethanol series, cleared in xylene, embedded in paraffin, and stored as paraffin blocks at room temperature (RT). Cell culture and stimulations Human oral SCC-25 epithelial cell line isolated from a 70-year-old male patient (ATCC, Rockville, MD, USA) and primary human oral keratinocytes HOKs (ScienCell, Uppsala, Sweden) were cultured in Dulbecco’s Modified Eagle Medium with Nutrient Mixture F-12 (DMEM/F-12) medium (Life Technologies, Grand Island, NY, USA) and

oral keratinocyte medium OKM (ScienCell, Uppsala, Sweden), respectively, containing 10% fetal bovine serum and antibiotics. Tumorigenic cell line (SCC-25) was selected because of the fast growth of SCCs in cultures, abundant supply, and unlimited storability in liquid nitrogen (Masters, 2000; Uehara et al, 2005, 2007). Confluent oral epithelial cells were detached and plated in serum-free medium in 24-multiwell cell culture plates (BD Falcon, Lawrence, KS, USA). To avoid possible cross-binding of histamine with its other receptors, oral epithelial cells were for internalization studies incubated without and with 50 nM H4R-specific agonist HST-10 [N-(3-(1H-imidazol-4yl) propyl)-2-cyclohexylacetamide] for 10, 20, 30, and 60 min at +37°C. The affinity of HST-10 to human H4R is 117 nM in agonist mode (EC50 value) and 45 nM in antagonist mode (Ki value). Its intrinsic activity is 0.85 and it is considered as the most potent and selective ligand to human H4R (Kottke et al, 2011). For cytokine stimulation studies, oral epithelial cells were incubated without and with 50 lM histamine (Sigma-Aldrich, St Louis, MO, USA), 10 ng ml
1 human recombinant (IFN-c) or 10 ng ml
1 human recombinant (TNF-a) (both from R&D Systems, Minneapolis, MN, USA) for 24 h.

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Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Total RNA was isolated using the RNeasy Mini Kit (Qiagen, D€usseldorf, Germany). Hundred nanograms of total RNA was reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) containing a blend of oligo(dT) primers and random hexamers and RNase H+ iScript MMLV-derived reverse transcriptase. Primer sequences were as follows: for human H4R, forward 50 -TGGAAGCGTGATCATCTCAG- 30 and reverse 50 -ATATGGAGCCCAGCAAACAG- 30 ; GAPDH housekeeping gene, forward 50 -CGCTCTCTGCTCCT CCTGTT- 30 and reverse 50 -CCATGGTGTCTGAGC GATGT- 30 . Quantitative RT-PCR was run using 2 ll first strand cDNA and 250 nM primers in iQ SYBR Green supermix in iQTM5 Multicolour RT-PCR Detection System (Bio-Rad Laboratories Inc.). Immunostaining Five-lm-thick tissue sections from patient with OLP and control samples were deparaffinized in xylene and rehydrated in a decreasing ethanol series and finally in dH2O. Antigen retrieval was performed in 10 mM sodium citrate buffer, pH 6.0, in a Micromed T/T Mega Microwave processing Lab station for Histology (HACKER Instruments & Industries Inc., Winnsboro, SC, USA) for 25 min at +95°C, followed by cooling at +22°C for 20 min. Tissue sections were rinsed in tap water and incubated at RT if not otherwise indicated in 1) 1% H2O2 for 10 min and washed 3 9 5 min in 10 mM phosphate buffered 140 nM saline, pH 7.4 (PBS); 2) 10% normal goat or horse serum (Vector Laboratories, Burlingame, CA, USA) for blocking of nonspecific staining for 1 h; 3) 1 lg ml
1 polyclonal peptide affinity-purified rabbit anti-human H4R IgG (MBL International, Woburn, MA, USA), 0.05 lg ml
1 monoclonal peptide affinity-purified mouse anti-human MC tryptase IgG1 (AbD Serotec, Oxford, UK) overnight at +4°C and washed Oral Diseases

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3 9 5 min in PBS; 4) appropriate biotin-conjugated secondary antibodies against IgG (Vector Laboratories; 1:200 in 1.25% bovine serum albumin (BSA)-PBS) for 1 h and washed 3 9 5 min in PBS; 5) avidin–biotin– peroxidase complex (ABC complex, 1:200 in H2O, Vector Laboratories) for 1 h and washed 3 9 5 min in PBS; and 6) 0.006% H2O2 and 0.023% 3,30 -diaminobenzidine tetrahydrochloride for 5–10 min and washed 3 9 5 min in dH2O. Slides were counterstained using hematoxylin or fast-red, dehydrated and mounted in Mountex (HistoLab, Gothenburg, Sweden). Non-immune normal rabbit IgG and non-relevant mouse IgG1 (R&D Systems) were used for negative staining controls. To confirm that MC tryptase staining enables the recognition of MC degranulation, tissue explants from healthy oral mucosa were cut into small pieces of ~2–3 mm3 and washed in PBS. For the activation of MCs, cells were transferred into 2 ml of DMEM containing rabbit IgG antibody against human IgE (150 lg ml
1). Non-specific rabbit IgG (150 lg ml
1) (DAKO, Carpinteria, CA, USA) was used as a negative control. Cultures were incubated at +37°C in 5% CO2 in air for 24 h (Juurikivi et al, 2005) followed by fixation in 10% NBF and embedding in paraffin. Tissue sections were prepared and stained for MC tryptase as described above. Cells cultured on coverslips were (1) fixed in 4% paraformaldehyde in PBS at RT, after which the cover slips were washed 3 9 10 min in PBS; (2) permeabilized with 0.5% Triton X-100 in PBS for 10 min at RT and washed 3 9 10 min in PBS; (3) blocked with 10% normal goat serum (Vector Laboratories) for 1 h at RT; (4) incubated in 1 lg ml
1 polyclonal peptide affinity-purified rabbit antihuman H4R IgG (MBL International, Woburn, MA, USA) overnight at +4°C and washed 3 9 10 min in PBS; (5) incubated in the Alexa Fluor 568-conjugated secondary goat anti-rabbit IgG (1:200 in 1.25% BSA-PBS; Invitrogen, Carlsbad, CA, USA) for 1 h at RT and washed 3 9 10 min in PBS; (6) incubated for nuclear staining in 5 mg ml
1 DAPI (diamidino-2-phenylindole; Sigma, Steinheim, Germany) in dH2O for 5 min; and (7) mounted in VectorFluorescent Mounting Medium (Vector Laboratories). Microscopy and image analysis The intensity of staining in both control and patient samples was measured using Image J software (v. 1.43u National Institute of Health, Bethesda, MD, USA) to determine the mean gray value. The hue value used was between 0 and 161. The relative staining intensity was measured by applying the inverse mean gray value. Statistical analysis Two-tailed Student’s t-test was used to determine the statistical significance of differences between the groups. Linear regression analysis was used to assess the correlation between two variables. P values