5700549 A

HLA-C Expression Pattern Is Spatially Differentbetween Psoriasis and Eczema Skin LesionsLina Carlen1, Kazuko Sakuraba1, Mona Stahle1 and Fabio Sanchez1

Interactions between genetic and environmental factors underlie the immune dysregulation and keratinocyteabnormalities that characterize psoriasis. Among known psoriasis susceptibility loci (PSORS), PSORS1 onchromosome 6 has the strongest association to disease. Altered expression of some PSORS1 candidate geneshas been reported but little is known about HLA-C expression in psoriasis. This study compared expression ofmajor histocompatibility complex class Ia and HLA-C in psoriasis, allergic contact eczema, and normal skin.Although HLA-C was abundant in protein extracts from both eczema and psoriasis, a consistent and intriguingdifference in the expression pattern was observed; strong immunoreactivity in the basal cell layer, polarizedtowards the basement membrane in psoriasis, whereas in eczema lesions HLA-C immunostaining was presentmostly in suprabasal cells. Inflammatory cells in the dermis were strongly stained in both diseases. Normal skinepithelium showed less intense but similar HLA-C staining as eczema lesions. HLA class Ia expression overallresembled that of HLA-C in all samples. The distinct HLA-C expression patterns in psoriasis and eczema suggesta functional role in the specific psoriasis immune response and not only a general feature of inflammation.

Journal of Investigative Dermatology (2007) 127, 342348. doi:10.1038/sj.jid.5700549; published online 28 September 2006

INTRODUCTIONAssociation between psoriasis and the major histocompat-ibility complex (MHC) region is firmly established (McMi-chael et al., 1978; Laurentaci et al., 1982; Henseler andChristophers, 1985). A minimal genomic segment definingthe psoriasis susceptibility locus 1 (PSORS1) on chromosome6 (Jenisch et al., 1998; Balendran et al., 1999; Oka et al.,1999; Nair et al., 2000) contains a number of proposedcandidate genes, for example HLA-C, CDSN (corneodesmo-sin), CCHCR1 (coiled-coil alpha-helical rod protein 1), SEEK,and PSORS1C3 (Bowcock and Krueger, 2005). All of themhave been investigated for strength of association to psoriasisin genetic studies but only some have been studied forexpression in skin (Holm et al., 2003; Suomela et al., 2003;Capon et al., 2004). There are no published studies thatdescribe HLA-C expression in psoriasis despite the fact that itsassociation to disease has been known for several decades.

It is known that under non-inflammatory conditions allnucleated cells express class I molecules, and that one oftheir important functions is to suppress self-reactivity byengaging inhibitory receptors on natural killer (NK), NK-Tand CD8 cells (Martin and Carrington, 2005; Rajagopalan

and Long, 2005). HLA-C but not HLA-A and B is expressed bytrophoblast where it regulates maternal NK cell reactivity(Parham, 2004). HLA-C has recently gained attention intransplantation biology because it has been recognized thatmismatches in the killer immunoglobulin-like receptor (KIR)-HLA class I system can lead to transplant rejection (Petersdorfet al., 1997). This interaction is strongly influenced by theamino acids present at position 80 and 44 of the HLA-C andKIR molecules, respectively (Boyington et al., 2001) and isalso influenced by the glycosylation state of the ligands (Babaet al., 2000). HLA-Cw*0602 has a lysine at position 80 andinteracts mostly with KIRs that possess a methionine atposition 44 (Winter and Long, 1997), such as KIR2DL1(inhibitory) and KIR2DS1 (activating). Upon interaction,ligand and receptor are internalized into the effector cell(Carlin et al., 2001) which is followed by cellular activationor inhibition (Moretta and Moretta, 2004). HLA-C expressionin the skin has not been described yet, although it is generallyassumed to be low based on expression in other tissues(McCutcheon et al., 1995).

Although several inflammatory diseases associate withpolymorphisms at MHC class I genes, for example, ankylos-ing spondylitis with the HLA-B27 allele and subacutethyroiditis with the HLA-B35 allele (Nyulassy et al., 1977;Rubin et al., 1994), psoriasis is the only inflammatory diseasethat associates with HLA-C (HLA-Cw*0602). The role ofMHC class Ia in the pathophysiological mechanisms ofpsoriasis is not well understood; however, it is clear thatinflammation is a marker of disease activity (Krueger andBowcock, 2005) and that environmental factors such asinfections can trigger this disease (Wolf and Ruocco, 1999;Langley et al., 2005), which provides an immunological

ORIGINAL ARTICLE

342 Journal of Investigative Dermatology (2007), Volume 127 & 2006 The Society for Investigative Dermatology

Received 22 February 2006; revised 17 May 2006; accepted 22 May 2006;published online 28 September 2006

1Dermatology and Venereology Unit, Department of Medicine, KarolinskaInstitutet, Stockholm, Sweden

Correspondence: Dr Fabio Sanchez, Molecular Dermatology, CMM L8:02,Karolinska University Hospital, SE-171 76 Stockholm, Sweden.E-mail: [email protected]

Abbreviations: KIR, killer cell immunoglobulin-like receptor; MHC, majorhistocompatibility complex; NK, natural killer; PSORS, psoriasissusceptibility locus

playground for HLA-C. In particular, streptococcal throatinfections are often concomitant with onset of guttatepsoriasis, which is the psoriasis subphenotype with thestrongest association to HLA-Cw*0602 (Mallon et al., 2000;Holm et al., 2005). Whether these associations are circum-stantial or do indeed contribute to pathophysiology remainsto be established. In the late 1970s, Gross et al. (1977, 1983)reported that, in psoriasis, T-cell functions in response to invitro exposure to streptococcal factors are influenced byHLA-C genotypes. No additional reports addressing thisphenomenon are available since then. It is likely thattechnical difficulties arising from the high degree ofhomology between MHC class I genes and from the highdegree of polymorphism within each gene group explain thescarcity of studies on this subject. Nonetheless, the role ofHLA-C in immune surveillance (Boyington and Sun, 2002)and its importance in relation to infection grants a closer lookat its expression pattern in psoriasis.

RESULTSHLA-C is expressed in skin, tonsil, and adult colon epitheliumbut not in fetal colon epithelium

The level and pattern of HLA-C and HLA-ABC expressionwere investigated in normal specimens of skin, tonsil, and

colon using antibodies recognizing HLA-C (L31) andHLA-A,B and C (W6/32). Immunohistochemical analysisshowed that HLA-C staining in tonsil was strong aroundgerminal follicles and in epithelial cells (Figure 1a). Therewas no HLA-C staining in fetal colon epithelium (Figure 1b),although strong staining in neighboring lymphoid tissue onthe same specimen was evident, in contrast, there was verystrong staining in adult colon epithelium (Figure 1c). HLA-Cexpression in normal skin epithelium was predominantlysuprabasal with a membrane-like staining pattern (Figure 1d).Strong immunoreactivity for HLA-ABC was observed in allcells, including epithelial cells, in all tissues and in leukocyte-like cells, especially large dendritic-like cells (data notshown), except in fetal colon epithelium where the stainingwith this antibody was faint.

Psoriasis and allergic contact eczema skin lesions show spatiallydisparate HLA-C expression patterns

Next, we compared the HLA-C expression between plaquepsoriasis, allergic contact eczema, and normal skin. Epider-mal HLA-C expression in normal and eczematous tissue wasstronger than in plaque psoriasis. Immunoreactivity in plaquepsoriasis was consistently stronger in the basal layer,especially at the tips of rete ridges where the signal often

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Figure 1. HLA-C expression detected by immunohistochemistry with mAb L31. (a) Tonsil, (b) adult colon epithelium, (c) fetal colon epithelium, (d) normal

skin, (e) plaque psoriasis, (f) allergic contact eczema (original magnification 20, Bar 100mM, insets: 100, Bar20 mm) (g) LCL721.221 cells expressingHLA-Cw3 before lipopolysaccharide stimulation, and after (h) 16, (i) 48, and (j) 96 hours post-stimulation (original magnification 40, Bar 50 mm).

www.jidonline.org 343

L Carlen et al.HLA-C Expression Pattern in Psoriasis

polarized towards the basement membrane (Figure 1e). Clearpericellular delineation, compatible with membrane staining,was observed in suprabasal keratinocytes of normal andallergic contact eczema (Figure 1f). Not all cell types in skinstained with the L31 anti-HLA-C antibody; fibroblast-likecells were mostly negative. Leukocyte-like cells as well aslarge dendritic-like cells stained strongly with anti-HLA-C inall sample groups.

MHC class Ia expression was intense and had a similardistribution pattern to that of HLA-C, showing less intensestaining in the epithelium of plaque psoriasis compared tonormal and eczema skin but with relatively strongerimmunoreactivity in the basal cell layer. An inverse relation-ship between the number of infiltrating cells and the averagestaining density at the basal layer was observed.

HLA-C expression on transfected LCL721.221 cells is induciblewith E. coli-derived lipopolysaccharide

In order to determine if HLA-C immunoreactivity correlatedwith changes in cellular functional state, LCL721.221 cells,which lack HLA class I expression, were used. LCL721.221cells transfected with HLA-Cw3 and Cw4, as well as, non-transfected cells were stimulated with lipopolysaccharide forup to 96 hours and then tested for reactivity with the L31antibody. There was a time-dependent increase in thefrequency of L31-positive cells in HLA-Cw3 and Cw4transfectants, which reached a peak at 48 hours (Figure1gj). Very faint expression was observed before lipopoly-saccharide stimulation for both constructs. No increase instaining intensity or in the frequency of positive cells wasobserved in non-transfected cells at the set time points.

HLA-C protein levels are increased in lesional allergic contacteczema and plaque psoriasis skin

Using the L31 antibody, a band of approximately 45 kDa,compatible with the denatured form of HLA-C, was detectedby Western blot in protein extracts from lesional allergiccontact eczema, plaque and guttate psoriasis, non-lesionalsamples for these three sample types, and normal skinsamples. The range of HLA-C intensity values among thedifferent samples was between 0.10 and 1.08 expressionunits. Analysis of variance revealed inter-group differencesaccording to sample category (P 0.0039). Allergic contacteczema had the most intense HLA-C signal (averageexpression index 0.665, standard deviation (0.343),being significantly higher than in the other samples exceptfor plaque psoriasis (average expression index0.425,standard deviation0.189) (Figure 2a). Overall HLA class Iexpression assessed by b2-microglobulin staining was alsoincreased in allergic contact eczema compared to the otherskin samples (average expression index0.256, standarddeviation0.151, P0.0002) (Figure 2b). A representativeimage of an L31-stained membrane is presented in Figure 3.

DISCUSSIONThe contribution of the PSORS1 locus to psoriasis suscept-ibility has been a subject of intense research over the past twodecades (Bowcock and Krueger, 2005). Although several

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LE LG LP NPhenotype

Exp

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inde

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Figure 2. HLA-C protein expression levels in total protein extracts from

psoriasis, eczema and normal skin biopsies. (a) Box plot representation of

HLA-C expression according to sample group, measured by densitometry on

Western blot analysis. (b) Box plot representation of HLA class I expression,

measured by densitometry on Western blot analysis and detected by b2-microglobulin staining. LE lesional eczema, LG lesional guttate psoriasis,LP lesional plaque psoriasis, Nnormal, NLE non-lesional eczema,NLGnon-lesional guttate psoriasis, NLPnon-lesional plaque psoriasis.

45 kDa

C NLG LE N LG N LP NLE LP

a

b

Figure 3. Representative Western blot membrane showing total protein

smears and specific HLA-C bands. (a) Ponceau staining of the skin protein

extracts after transfer to a membrane. (b) Specific L31 signal. CControl,LE lesional eczema, LG lesional guttate psoriasis, LP lesional plaquepsoriasis, N normal, NLE non-lesional eczema, NLG non-lesionalguttate psoriasis.

344 Journal of Investigative Dermatology (2007), Volume 127


genes in this locus are expressed in skin, some withdifferential expression between psoriasis and normal skin(HCR, CDSN), there is no conclusive explanation about theirfunctional role in psoriasis pathogenesis (Bowcock, 2005).HLA-C has been considered a susceptibility gene in itself or agenetic marker in linkage disequilibrium with a psoriasisgene that has not yet been identified (Helms et al., 2005). Ifintrinsic defects in this gene are at the base of psoriasispathogenesis then there should be evidence of altered HLA-Cexpression or function, otherwise this molecule mightfacilitate the effect of third party factors such as autoantigens.

Expression analysis has been critical to understanding therole of HLA-C in other contexts than psoriasis but withthe common denominator of immune regulatory functions forthis molecule. For example, soluble MHC class I moleculesseem to regulate lymphocyte survival through apoptosis,tumors can evade immune surveillance by modifyingexpression of MHC class I molecules, and HLA-C specificallycontributes to regulate maternal immune reactivity againstfetal tissues (Le Bouteiller, 2001; Puppo et al., 2002; Garcia-Lora et al., 2003). Global expression studies of RNA andprotein do not indicate differential expression of HLA-C inpsoriasis compared with normal or non-lesional skin (Zhouet al., 2003; Bowcock, 2005; Carlen et al., 2005), incon-gruent with HLA-C being a psoriasis susceptibility gene.However, qualitative as well as quantitative differences areimportant in defining the role of HLA-C in psoriasispathogenesis. Different patterns of expression can emergeeven in the presence of similar levels of total RNA or protein.Our findings indicate that HLA-C expression in psoriasisdepends predominantly on infiltrating inflammatory cells,whereas non-lesional and normal samples express higherlevels of the protein in the epidermis.

It is expected that all nucleated cells express MHC class Iamolecules because they contribute to preserving peripheralself tolerance under normal circumstances, that is, noinflammation, no infection (Zimmer et al., 2005). However,it is also expected that the constitutive expression of HLA-Cshould be low (Neisig et al., 1998). The present studydemonstrated intense staining on infiltrating inflammatorycells and variable expression levels in the epidermis.

The most striking feature of the HLA-C staining pattern inthis study was the polarization of the signal towards thebasement membrane and the reduced expression on supra-basal keratinocytes in lesional psoriasis samples. It is knownthat overexpression of MHC class I molecules can down-regulate effector immune cell functions (Parham, 2004);therefore, reduced expression in psoriasis epithelium mightcontribute to maintaining inflammation owing to inefficientdownregulation of activated lymphocytes.

Why would HLA-C be restricted to the basal layer inpsoriasis epidermis? MHC class I clustering into lipid rafts hasbeen reported as a requirement to binding T-cell receptors(Lebedeva et al., 2004) and lymphocytes are known to hometowards the skin during psoriasis plaque formation (Davisonet al., 2001), but how these cells interact with the epitheliumis less well documented. HLA-C polarization in psoriasisepidermis might lead to a directional flow of inflammatory

cells towards the tip of papillae, above which microabscessesare typically formed (Kaneko et al., 1991). A relevantobservation regarding this possibility is that the inflammatoryinfiltrate in psoriasis does not seem to affect the structure ofthe basal cell layer, except at the uppermost section ofpapillae where HLA-C expression seems lower. In contrast,the basal cell layer in eczema is characterized by infiltratinginflammatory cells throughout the epidermis. We attemptedto measure the HLA-C signal intensity at the basal layer ofpsoriasis papillae and counted the number of infiltrating cellsthrough this layer; we observed an inverse relationshipbetween these variables (data not shown). Since some classI molecules, including HLA-C, are strong inhibitors of T andNK cells, the reduced epithelial HLA-C expression might leadto increased vulnerability to inflammation (Carosella et al.,2001; Le Discorde et al., 2002; Trowsdale and Betz, 2006),therefore the polarized HLA-C expression might be acompensatory mechanism to reduce lymphocyte infiltration.Support for this view comes from reproduction researchindicating that HLA-C and KIR mismatches appear to fail atinhibiting NK/NK-T cells in pre-eclampsia, (Hiby et al., 2004;Parham, 2004). Also in idiopathic bronchiectasis, combina-tions of HLA-C and KIR associate with the progressiveinflammatory damage to the lungs (Boyton et al., 2006). Inaddition, viral regulation of class I gene expression plays arole in viral persistence; for example, the UL18 protein fromcytomegalovirus can induce HLA-C expression and inhibitHLA-A and B, which can inhibit antiviral NK/NK-T cellresponses (Gewurz et al., 2001).

There is mounting evidence indicating that immunesurveillance of HLA-C by KIR-positive cells might be acontributing factor to psoriasis pathogenesis (Martin et al.,2002; Luszczek et al., 2004; Holm et al., 2005), butfunctional studies on this subject are still lacking. However,it is important to remember that an antigen-driven theory ofpsoriasis has been extensively discussed, where HLA-Cw6 isregarded as a cofactor in the initiation and/or maintenanceof inflammation (Nickoloff et al., 2000; Lin et al., 2001;Johnston et a.l, 2004).

What would be particular about HLA-Cw*0602 comparedto other HLA-C alleles in this scenario? It is known that HLA-C expression varies in relation to the identity of the codingalleles, but it is not yet known if HLA-Cw*0602 has uniquefeatures that would affect its protein expression. Although thisstudy does not specifically target the psoriasis-associatedallele HLA-Cw*0602, it shows that HLA-C expression innormal tissues varies depending on developmental stage andhistological location and reveals that skin epitheliumexpresses HLA-C.

The distinct HLA-C expression patterns in psoriasis andeczema suggest a functional role in the specific psoriasisimmune response and not only a general feature ofinflammation. It will be important to obtain a detaileddescription of coding and non-coding sequences of all allelesin order to identify differences at regulatory sites and designfunctional studies that compare HLA-Cw*0602 to otheralleles. This will increase our understanding of the role ofHLA-C in psoriasis pathogenesis.



MATERIALS AND METHODSTissue samples

Details regarding patients and controls were reported previously

(Carlen et al., 2005). All study subjects provided written consent and

all research procedures were performed in accordance with the

Declaration of Helsinki Principles and with permission from the

Ethics Committee of the Karolinska Institutet. Biopsies were taken

from lesional and non-lesional skin from guttate and plaque psoriasis

patients, lesional and non-lesional skin from nickel-induced contact

eczema patients, and normal skin of healthy individuals. For

immunohistochemistry, five normal, 12 plaque psoriasis, three

non-lesional plaque psoriasis, five lesional eczema, and five non-

lesional eczema samples were analyzed. In addition, one tonsil

sample from a guttate psoriasis patient affected by recurrent

streptococcal throat infections, two normal adult colon and one

fetal colon samples were used for immunohistochemistry. For

Western blot analysis, 10 normal, 12 plaque psoriasis, three non-

lesional plaque psoriasis, seven guttate psoriasis, four non-lesional

guttate, five lesional eczema, and four non-lesional eczema samples

were considered.

Antibodies

The following antibodies were used: (1) Murine mAb L31 which

binds linear a1 domain epitope at position 67 of human HLA-Calleles Cw1 through Cw8 and HLA-B8 and B51 (Setini et al., 1996).

Alleles from Cw9 to Cw17, and B8 and B51 do not associate with

psoriasis, therefore, under-or overestimation of expression levels is

expected to cancel out between patients and controls. (2) Murine

mAb W6/32 (Serotec, Oxford, UK) (Brodsky et al., 1979) which

binds MHC class I heavy chain associated with b-2-microglobulin.(3) Murine mAb MCA2243 (NKVFS1) (Serotec) (Spaggiari et al.,

2002) which recognizes a common epitope on CD158a and

CD158b (KIR2DL1, KIR2DL2), and (4) horse radish peroxidase-

conjugated rabbit polyclonal P0174 (DakoCytomation, Glostrup,

Denmark) which binds human b2m. As secondary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz,

sc-2005) was used.

Sample preparation

Skin biopsies were prepared as described previously (Carlen et al.,

2005). In brief, the biopsies were kept frozen in liquid nitrogen and

mechanically homogenized using a mortar and pestle. The samples

were then solubilized in a buffer containing 7 M urea, 2 M thiourea,

4% SDS, reducing agents, and protease inhibitors. Protein concen-

tration was determined using the Bradford method (BioRad Protein

Assay, BioRad, Stockholm, Sweden) (Bradford, 1976).

Western blot

Total protein extracts were separated, adding 7mg of each sample to12% SDS-PAGE gels, and then electroblotted onto nitrocellulose

membranes (Whatman Schleicher & Schuell, Kent, UK). The

membranes were then stained with 1% Ponceau S (Sigma, St Louis,

MO) and scanned to check for loading and gel run quality. After

blocking with 2% goat serum (Vector Laboratories, Burlingame, CA)

and 1% BSA (Sigma, St Louis, MO) in Tris-buffered saline for 1 hour,

the membranes were incubated separately overnight with L31, W6/

32, MCA2243, or b2m antibody diluted 1:500 in TBS with 1%Tween 20. The membranes were then incubated with horse radish

peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechno-

logy, Santa Cruz, CA) (except for b2m) diluted 1:2,000 in blockingbuffer followed by development using enhanced chemilumines-

cence (Amersham Pharmacia Biotech, Sunnyvale, CA) and the

image acquired with a CCD camera (Fujifilm, Sendai, Japan). The

immunoreactivity of the HLA-C and b2m antibodies was testedagainst recombinant HLA-C and b2-microglobulin. Unspecificbinding of the secondary antibody was prevented by pre-incubation

in normal goat serum.

Immunohistochemistry

All biopsies were snap-frozen and stored at 701C until furtherprocessed. Skin samples from lesional and non-lesional eczema and

plaque and guttate psoriasis, and healthy skin were included in the

same experiments. In short, 67-mm thick cryostat sections wereincubated with L31, W6/32, and MCA2243 antibody at 1:200,

1:20,000, and 1:200 dilutions, respectively. All sections were

stained using the avidin: biotinylated enzyme complex technique

(Vector Laboratories, Burlingame, CA) following the manufacturers

instructions, and counterstained with Gills III hematoxylin solution.

As controls, tissue sections were processed in parallel without

adding primary antibody. No reactivity for secondary antibodies or

signal developing reagents was observed on control histological

sections.

Cell culture

LCL721.221 cells were maintained in DMEM (Gibco-BRL Life

Technologies, Paisley, UK) supplemented with 10% FCS (fetal calf

serum; Hy-Clone, Boule Nordic AB Huddinge, Sweden) and

antibiotics (PEST _ penicillin 50 U/l and streptomycin 50 mg/ml;

Gibco-BRL) at 5% CO2 at 371C. HLA-Cw3 and Cw4 transfectantswere kept under G418 (GibcoBRL) selection. During stimulation

experiments, about 105 cells/well were grown in 12-well plates

without selection with G418 for 2 days and then exposed to 1mg/mlE. coli-derived lipopolysaccharide. HLA-C expression was detected

with the L31 antibody at time 0 and 16, 48 and 96 hours after

stimulation. The cells were then transferred to slides, fixed with

ethanol, and further processed as described in the immunohisto-

chemistry section above.

Data analysis

Image analysis was carried out using Science Lab 99 L Process

software, version 1.95 (Fujifilm, Sendai, Japan). Intensity measure-

ments of specific signals were corrected by the total amount of

protein in their corresponding lanes as derived from measurement of

Ponceau staining intensities of the membranes. Variations in

luminosity between images were adjusted by using an HLA-C-

positive cell line as internal control on every membrane and

the density values were adjusted using the following formulae:

Expression Index (corrected sample reading)/(corrected Ponceaureading).

Corrected sample reading (sample density (Strongest intergelcontrol/samples gel control))

Corrected Ponceau reading reading of control lane/sum ofreadings from all lanes in gel

Statistical analysis was performed using the R language and

statistics package (Ihaka and Gentleman, 1996). Mean expression

indexes from different sample groups were compared by fitting a



one-way analysis of variance model to the data, assuming a

significance level of 0.05. Then a paired t-test was performed in

order to identify comparisons with significant differences.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSWe thank Dr Eniko Sonkoly for critically reviewing this manuscript, Anna-Lena Kastman for skilful technical assistance with immunohistochemistry,Dr Gunther Weber for providing HLA-C-positive cell lines and for assistancewith immunoblotting, Dr Alberto Beretta for providing us with the L31HLA-C antibody, Dr Jack Strominger, and Dr Hugh Reyburn for providingHLA-C constructs, and Dr Kalle Malmberg and Dr Hans Gustav Ljunggren forHLA-C transfected LCLs. This study was supported by funds from theSwedish Research Council, the Swedish Heart and Lung Foundation, theSwedish Psoriasis Association, Ake-Wibergs foundation, Serono, Biogen,Schering-Plough, the National Psoriasis Foundation, Karolinska UniversityHospital, and Karolinska Institutet.

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