Christina Orsmark1, Tiina Skoog1, Leila Jeskanen3, Juha Kere1,4 and Ulpu Saarialho-Kere1,2,5

1Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, Departments of 2Dermatology and 3Pathology, Helsinki University Central Hospital, and Biomedicum Helsinki, University of Helsinki, Helsinki, Finland, 4Department of Medical Genetics, Biomedicum, University of
Helsinki, Helsinki, Finland, and 5Department of Dermatology, Karolinska Institutet, Stockholm Söder Hospital, Sweden

Allograft inflammatory factor-1 (AIF-1) is an evolutionarily conserved, inflammatory protein produced by activated macrophages during chronic transplant rejection and in inflammatory brain lesions. Since T-cell-mediated inflammation is common to various dermatoses and nothing is known about AIF-1 in skin, we studied its protein expression at the tissue level and regulation in monocytic cell lines by various agents. Using immunohistochemistry, we found that AIF-1 is expressed at low levels in normal skin, but is highly upregulated in various inflammatory skin disorders, such as psoriasis, lichen planus, graft-versus-host disease and mycosis fungoides. The main cell types expressing AIF-1 in affected skin are macrophages and Langerhans’ cells. We also show by real-time PCR that AIF-1 mRNA levels in monocytic THP-1 and U937 cell lines are significantly upregulated by retinoic acid as well as a number of cytokines. We conclude that AIF-1 may mediate survival and pro-inflammatory properties of macrophages in skin diseases. Key words: CD-163; Langerhans’ cell; macrophage; immunohistochemistry; retinoic acid.

(Accepted November 14, 2006.)

Acta Derm Venerol 2007; 87: 223–227.

Christina Orsmark, Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 57 Huddinge, Sweden. E-mail: christina.orsmark@biosci.ki.se

Allograft inflammatory factor-1 (AIF-1) was originally cloned from activated macrophages in rat atherosclerotic allogenic heart grafts undergoing chronic transplant rejection (1). The full-length AIF-1 is a 147 amino-acid (17 kDa) interferon-g (IFN-g) inducible Ca2+-binding protein. AIF-1 expression has been associated with inflammatory lesions of the central nervous system, stimulation of smooth muscle cell proliferation and the pathogenesis of insulin-dependent diabetes mellitus (2). However, it is not only associated with allograft inflammation, but could also function in cell-mediated autoimmune responses.

We have previously studied the α-helical coiled-coil rod homolog (HCR) as a psoriasis candidate gene located in the PSORS1 locus in the major histocompatibility complex (MHC) region on chromosome 6p21.3 (3). In the process of identifying genes co-regulated with HCR (see http://microarray.genomecenter.columbia.edu/cgi-bin/find-links.cgi), we found a set of genes to be consistently co-expressed with HCR, among them AIF-1, which mapped to the same MHC region on
chromosome 6 known for clusters of genes involved in the inflammatory response. Since T-cell-mediated inflammation is one of the features of psoriasis, and since no data exist on AIF-1 in cutaneous biology, we decided to investigate the relevance of AIF-1 in skin.

We show here that AIF-1 is expressed at low levels in normal skin, but is highly upregulated in various inflammatory skin disorders. The main cell types expressing AIF-1 in affected skin are macrophages and Langerhans’ cells. We also show that AIF-1 mRNA levels in monocytic cell lines are strongly upregulated by retinoic acid as well as a number of cytokines.

MATERIALS AND METHODS

Clinical specimens

Formalin-fixed paraffin-embedded specimens of psoriasis, non-lesional and lesional (n = 10), X-linked ichthyosis (n = 3), prurigo nodularis (n = 3), atopic dermatitis (n = 2), mycosis fungoides (n = 3), lichen ruber planus (n = 2), pityriasis rubra pilaris (n = 4), chronic wounds (n = 5), normally healing timed wounds (n = 3), graft-versus-host reaction (n = 4) and normal skin (n = 4) were obtained from the Department of Dermatopathology, University of Helsinki, Finland. The diagnoses were confirmed by an experienced dermatopathologist (LJ). The use of the archival human samples was approved by the relevant ethics committee and followed the Declaration of Helsinki Guidelines.

Immunohistochemistry

Immunostaining was performed using the avidin-biotin-peroxidase complex technique (Vectastain ABC kit; Vector laboratories, Inc. Burlingame, CA, USA). Diaminobenzidine (DAB) was used as chromogenic substrate, Mayer’s solution as counter-stain, and goat polyclonal antibodies diluted 1:600 to target the AIF-1 protein (ab5076, Abcam, Cambridge, UK). Sections were pre-treated by 30 min incubation in 1× Target Retrieval Solution buffer (DAKOCytomation) in a 95°C water bath. As negative controls, sections were treated with phosphate-buffered saline (PBS) containing 3% bovine serum albumin (BSA). Serial sections were stained for Langerhans' cells (CD1a; MTB1, 1:20, Novocastra Laboratories, Newcastle Upon Tyne, UK), macrophages (CD163; 10D6, 1:100, Novocastra Laboratories) and T cells (CD3; A0452, 1:75, Dako A/S, Glostrup, Denmark). Double-immunostaining for AIF-1 and CD163 or CD1a was performed with peroxidase-anti-peroxidase technique combining Vector mouse and goat kits. After pre-treatment in 95ºC citrate buffer, pH 6.0, CD163 antibodies (1:100) or CD1a antibodies (1:15) were incubated on sections for 1 h at 37ºC and detected using aminoethylcarbazole (AEC) (red) as chromogenic substrate. After the protein blocking solution treatment, sections were incubated with AIF-1 antibodies (1:600) overnight at 4ºC and the immuno-signal was detected using DAB with nickel enhancement (brown-black) as a chromogenic substrate. Semi-quantitative immunoreactivity grading for AIF-1 (Table I) was based on the number of positive cells, which was assessed as follows, using 200× magnification of a light-field microscope: + a few AIF-1 positive cells; ++ moderate number of positive cells; +++ a large number of positive cells. The slides were analysed independently by two experienced investigators (LJ and US-K).

Cell cultures and stimulations

Expression of AIF-1 mRNA was studied in two monocytic cell lines, THP-1 and U937. The cells were grown in RPMI 1640 medium supplemented with 10% foetal calf serum (FCS) (GIBCO Invitrogen Life Technologies, Paisley, UK),
1 mM sodium pyruvate, 10 mM HEPES, 100 U penicillin and 100 µg/ml streptomycin (Sigma, St Louis, MO, USA, and Roche Molecular Biochemicals, Indianapolis, IN, USA). The medium for THP-1 cells was further supplemented with
0.05 mM β-mercaptoethanol. Medium was changed three times a week and cell density kept at 0.2–1×106 cells/ml. To study the regulation of AIF-1, 1.4×106 monocytes in 2 ml cell culture were plated on 6-well plates and grown overnight. Cells were depleted of serum overnight prior to 24 h of stimulation with tumour necrosis factor-alpha (TNF-α, 10 ng/ml, Sigma), basic fibroblast growth factor (bFGF, 10 ng/ml, Sigma), epidermal growth factor (EGF, 10 ng/ml, Sigma), insulin-like growth factor-1 (IGF-1 100 ng/ml, Sigma), vascular endothelial growth factor (VEGF, 10 ng/ml, Sigma), interleukin-1 beta (IL-1β,
5 U/ml, Roche Molecular Biochemicals), IFN-γ, 10 ng/ml, (Sigma), dexamethasone (Dxm, 1×10–5M, Sigma), all-trans retinoic acid (RA, 1×10–6 M, Sigma) or lipopolysaccharide (LPS, 10 ng/ml, Sigma). As controls, cells incubated in fresh serum-free medium for 24 h were used. Total cellular RNA was extracted from the cells using the RNeasy miniprep-Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s instructions. 250 ng of total RNA was reverse transcribed into cDNA using the SuperScript™III First-Strand kit (Invitrogen, Carlsbad, CA, USA) with oligo dT, and used as a template for reverse transcript PCR and quantitative real-time PCR (qRT-PCR). All cell stimulations were carried out three times and each sample was run in triplicate in the qRT-PCR.

Reverse transcript PCR and quantitative real-time PCR (TaqMan RT-PCR)

AIF-1 primers used for reverse transcript PCR amplification were: forward 5’-GACCTTAATGGAAATGGCGATA-3’ and reverse 5’-ATCTCTTGCCCAGCATCATC-3’. GAPDH primers were used as an endogenous control (4). PCR was carried out in 25 µl reactions containing 2 µl cDNA, 1× reaction buffer for HotStarTaq polymerase, 200 nM of each primer, 200 µM dNTPs and 0.75 U of HotStarTaq polymerase (Qiagen). PCR was carried out as follows: 95°C for 15 min, (95°C for 20 s, 56°C for 30 s and 72°C for 60 s) times 40 cycles for AIF-1 and 25 cycles for GAPDH, followed by 72°C for 3 min. For qRT-PCR, commercially available gene expression assays were used (TaqMan®Gene Expression Assays, Applied Biosystems, Foster City, CA, USA). Two assays for AIF-1 (assay identification and order numbers: Hs00741549g1 and Hs00610419g1) labelled with FAM™ reporter dye, and as an endogenous control GAPDH (4310884E) labelled with VIC™ reporter dye was used. The reaction was performed in 10 µl volume mix containing 2 µl cDNA, 1× pre-formulated assay mix and 1× TaqMan™Universal Master Mix and run as follows: 50°C for 2 min, 95°C for 10 min (95°C for 15 s and 60°C for 1 min) times 40. The reaction was performed with the Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems).

Statistics

The statistical significance of the differences in mRNA expression levels in TaqMan analysis was calculated using Student’s unpaired, two-tailed t-test.

RESULTS

AIF-1 was detected by immunohistochemistry in several different cell types in samples representing various inflammatory dermatoses (Table I). It was expressed by macrophages particularly in psoriasis, lichen planus, mycosis fungoides, and graft-versus-host disease (Fig. 1A–C, G, H). AIF-1 was also frequently detected in Langerhans’ cells, particularly in psoriasis, lichen planus and mycosis fungoides (Fig. 2A, B). Subsets of positive lymphocytes were seen in, e.g. mycosis fungoides (Fig 2C, D) and chronic wounds. More rarely, positive neutrophils were detected, e.g. in wounds or prurigo nodularis (Fig. 2E). The degree of inflammation clearly correlated positively with the number of AIF-1 positive cells (Fig. 1A, B versus D). In normal skin, scattered macrophages and dendritic cells expressed AIF-1 (Fig. 1D–F). AIF-1 protein was particularly upregulated in psoriasis, lichen planus, graft-versus-host disease and mycosis fungoides (Table I). However, keratinocytes were never positive in any of the samples examined.