CD4+ T Cell-Associated Pathophysiology Critically Depends on CD18 Gene Dose Effects in a Murine Model of Psoriasis

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Effects in a Murine Model of Psoriasis Critically Depends on CD18 Gene Dose CD4+ T Cell-Associated Pathophysiology

Scharffetter-KochanekWerner Müller, Thomas Krieg and Karin Stephan Grabbe, Roswitha Nischt, Cord Sunderkötter,Wickenhauser, Samir Tawadros, Karin Loser, Georg Varga, Daniel Kess, Thorsten Peters, Jan Zamek, Claudia

http://www.jimmunol.org/content/171/11/56972003; 171:5697-5706; ;J Immunol 

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CD4� T Cell-Associated Pathophysiology Critically Dependson CD18 Gene Dose Effects in a Murine Model of Psoriasis1

Daniel Kess,*‡� Thorsten Peters,*‡� Jan Zamek,*‡ Claudia Wickenhauser,† Samir Tawadros,*Karin Loser, § Georg Varga,§ Stephan Grabbe,§ Roswitha Nischt,* Cord Sunderkotter,§�

Werner Muller, ¶ Thomas Krieg,*‡ and Karin Scharffetter-Kochanek2*‡�

In a CD18 hypomorphic polygenic PL/J mouse model, the severe reduction of CD18 (�2 integrin) to 2–16% of wild-type levelsleads to the development of a psoriasiform skin disease. In this study, we analyzed the influence of reduced CD18 gene expressionon T cell function, and its contribution to the pathogenesis of this disease. Both CD4� and CD8� T cells were significantlyincreased in the skin of affected CD18 hypomorphic mice. But only depletion of CD4� T cells, and not the removal of CD8� T cells,resulted in a complete clearance of the psoriasiform dermatitis. This indicates a central role of CD4� T cells in the pathogenesisof this disorder, further supported by the detection of several Th1-like cytokines released predominantly by CD4� T cells. Incontrast to the CD18 hypomorphic mice, CD18 null mutants of the same strain did not develop the psoriasiform dermatitis. Thisis in part due to a lack of T cell emigration from dermal blood vessels, as experimental allergic contact dermatitis could be inducedin CD18 hypomorphic and wild-type mice, but not in CD18 null mutants. Hence, 2–16% of CD18 gene expression is obviouslysufficient for T cell emigration driving the inflammatory phenotype in CD18 hypomorphic mice. Our data suggest that thepathogenic involvement of CD4� T cells depends on a gene dose effect with a reduced expression of the CD18 protein in PL/J mice.This murine inflammatory skin model may also have relevance for human polygenic inflammatory diseases.The Journal ofImmunology, 2003, 171: 5697–5706.

� 2 integrins (CD11/CD18) are leukocyte adhesion mole-cules exclusively expressed on hemopoietic cells and re-sponsible for cell-cell contacts in a variety of inflamma-tory interactions (1, 2). The common �-chain (CD18)

associates with four different � subunits, �L, �M, �X, and �D,forming distinct functional heterodimers termed LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18), orCD11d/CD18 (2–4). These interact with �20 ligands, of whichthe most prominent belong to the family of ICAM (5, 6).

Absence of CD18 leads to leukocyte adhesion deficiency type 1(LAD1)3 (7–9). The severity of this disease correlates with thedegree of loss of CD18 (10, 11). In the absence of CD18, severedefects in cell-cell cooperation occur, leading to a lack of homo-typic lymphocyte adhesion (12–14) and impaired T cell activation(15–18) accompanied by a reduced IL-2 release (18, 19). Recently,in a murine model for LAD1 with a CD18 null mutation

(CD18null), we were able to show that lack of the �2 integrin sub-unit markedly impairs T cell extravasation (20).

Introduction of an insertion mutation in the murine CD18 generesulting in a duplication of exons 2 and 3 yielded a mouse modelwith a severe reduction of CD18 expression with only 2–16% ofwild-type levels (21). Due to this hypomorphic (CD18hypo) muta-tion, a skin disease develops in PL/J mice, which strongly resem-bles human psoriasis clinically, histologically, and in its responseto therapy (22). Affected mice present erythema, alopecia, crusts,and scaling as well as abnormal keratinocyte proliferation/differ-entiation, subcorneal microabscesses, and an increased inflamma-tory infiltrate. As in patients treated for severe psoriasis (23–25),the psoriasiform dermatitis in the underlying mouse model can besuppressed by corticosteroids (dexamethasone), suggesting the in-volvement of an autoimmune or otherwise inflammatory process(22). Psoriasiform dermatitis occurred only when the CD18hypo

mutation was backcrossed on the PL/J, but not on the C57BL/6J or129/Sv inbred mouse strains. Homozygous mutant mice on a (PL/J � C57BL/6J) F1 background did not develop the disease, despitethe CD18hypo mutation. Backcross analysis suggests that, in addi-tion to CD18, a small number of other genes determines suscep-tibility to the disease (22).

Different cell types have been suspected to be the primary trig-gers in the pathogenesis of psoriasis (26). Increasing evidence ledto the current view that T cells are the main actors responsible forits initiation (27). Thus, recent treatment strategies focused onblocking T cell function in psoriatic lesions (27) such as Abs di-rected against CD2 (28), CD4 (29–31), IL-2R (32–36) on T cells,and B7 molecules on APCs (37) have already been approved inclinical trials. In addition, a humanized anti-CD11a mAb has alreadybeen successfully tested in clinical trials, leading to impressive clinicaland histologic improvement in psoriasis patients (38, 39).

The pathogenic role of �2 integrins in human psoriasis is poorlyunderstood. CD11b expression has been reported to be reduced on

Departments of *Dermatology and †Pathology, and ‡Center for Molecular Medicine,University of Cologne, Cologne, Germany; §Department of Dermatology and Instituteof Experimental Dermatology, University of Munster, Munster, Germany; ¶GermanResearch Center for Biotechnology, Braunschweig, Germany; and �Department ofDermatology and Allergy, University of Ulm, Ulm, Germany

Received for publication February 28, 2003. Accepted for publication September22, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants to K.S.-K. and T.K. from the Center for Mo-lecular Medicine, University of Cologne (TV60, BMBF 01 KS 9502), and to C.S.from the Interdisciplinary Center of Clinical Research (IZKF-C2/D15), University ofMunster.2 Address correspondence and reprint requests to Dr. Karin Scharffetter-Kochanek,Department of Dermatology and Allergy, University of Ulm, Maienweg 12, D-89081Ulm, Germany. E-mail address: [email protected] Abbreviations used in this paper: LAD, leukocyte adhesion deficiency; CBA, cy-tometric bead array; m, mouse; PASI, psoriasis activity and severity index.

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peripheral blood leukocytes derived from psoriasis patients (40,41). Further circumstantial evidence indicating that reduced CD18expression may causally be involved in the development of a pso-riasiform dermatitis comes from the clinical observation that someLAD1 patients with only moderately reduced CD18 expressionlevels develop a psoriasiform dermatitis (42). Linkage analysis ofpsoriasis families has identified a region on chromosome 17, in-cluding, among other gene loci, ICAM-2, an important ligand ofCD18 heterodimers (43). Vice versa, polymorphisms in the CD18gene have been found predisposing to autoimmune disease eitherby leading to a higher ligand affinity or by increasing expression ofthe CD18 protein (44, 45).

Because to date only correlative evidence exists, we have set outto study the causal role of a stepwise reduction in CD18 expressionon distinct T cell populations in the generation and maintenance ofthe psoriasiform dermatitis in PL/J mice. In contrast to CD18hypo

mice, CD18null mutants did not develop the psoriasiform derma-titis due to a deficient emigration of T cells into the skin. However,the residual expression of CD18 in CD18hypo mice was sufficientfor T cells to extravasate. We furthermore demonstrate that thepsoriasiform skin disorder in CD18hypo mice resembles humanpsoriasis, revealing key features such as causal involvement of Tlymphocytes and a prevalence of Th1 cells. Our data point to acentral role of CD4� T cells in the pathogenesis of the psoria-siform dermatitis, as depletion of CD4�, but not of CD8� T cellsresulted in its complete resolution. And only CD4� T cells isolatedfrom draining lymph nodes of CD18hypo mice were found to beactivated, and to produce distinct Th1-type cytokines. In conclu-sion, our data suggest that the skin disorder of CD18hypo PL/J micedepends on a gene dose effect with reduced, but not completelyabsent, expression of the CD18 protein.

Materials and MethodsMice

Mice with a hypomorphic mutation of the CD18 gene (CD18hypo) on thePL/J inbred strain were examined (22). Additionally, mice with a CD18null mutation (CD18null) (17) were backcrossed seven generations onto thePL/J strain. CD18�/� littermates (CD18wt) resulting from heterozygotecrosses served as wild-type controls. Unless otherwise stated, all CD18hypo

mice showed a strong psoriasiform phenotype. All mice were kept underspecific pathogen-free conditions, and were used for experiments at an ageolder than 6 wk. All experiments were done in compliance with the Ger-man Law for Welfare of Laboratory Animals.

Monoclonal Abs

The following mAbs were purchased from BD PharMingen or BD Bio-sciences (both in Heidelberg, Germany), respectively: mouse (m)CD4(GK1.5, unconjugated, NA/LE), mCD8a (53-6.7, unconjugated, NA/LE),mIgG2a (R35-95, NA/LE), mIgG2b (A95-1, NA/LE), mCD3 (17A2),mCD28 (37.51); CD11a FITC (M17/4), mCD11b APC (M1/70), mCD11cAPC (HL3), mCD18 PE (C71/16), mCD25 FITC (7D4), mCD44 PE(IM7), mCD4 PE (H129.19), mCD4 APC (L3T4), mCD8b.2 PE (53-5.8),mCD3 CyChrome (145-2C11), rat IgG2a FITC, PE, APC (RG7/1.30), ratIgG2b FITC, PE, APC (RG7/11.1), IL-2 PE (JES6-5H4), IL-4 PE (11B11),IL-6 PE (MP5-20F3), IL-10 PE (JES5-16E3), IL-12 PE (C15.6), IFN-� PE(XMG 1.2), and TNF-� PE (MP6-XT22). mCD90 MACS (Thy-1.2) mag-netic microbeads were from Miltenyi Biotec (Bergisch Gladbach,Germany).

Immunohistochemical analysis

Cryosections were prepared and stained immunohistochemically using anindirect immunoperoxidase assay, as described elsewhere (46). For detec-tion of unbiotinylated Abs, goat F(ab�)2 anti-rat IgG and goat F(ab�)2 anti-rabbit IgG conjugated with peroxidase (Dianova, Hamburg, Germany)were used as secondary Abs, and 3-amino-9-ethyl-carbazol served as chro-mogen. In cases in which assay sensitivity needed to be increased, a bio-tinylated secondary rabbit anti-rat Ig mAb and the StreptABComplex/APkit were used in combination with the fast red substrate system (all DAKO,

Glostrup, Denmark). Primary, secondary, and isotype control Abs werediluted in PBS with 1% BSA to maximal concentration of 1.5 �g IgG/ml.

Microscopic evaluation was performed by counting cells in the dermis,and by relating the number of positively stained cells to the total numberof cells per area, defined by a grid ocular. To quantify cells in the epider-mis, the number of positively stained cells/cm of epidermis was deter-mined. For all measurements, the median of 30 evaluations (n � 3) ispresented. The statistical significance was calculated using the Mann-Whit-ney U test. All countings were done by two independent observers.

In vivo depletion of T cells

To deplete T cells, mice were injected i.p. with 100–150 �g mCD4(GK1.5) or mCD8 (53-6.7) mAb for 3 consecutive days. Subsequently,injections were performed every 3 days for a period of 45 or 33 days,respectively. Control mice suffering from similarly severe psoriasiformdermatitis were treated with isotype IgG mAb in an identical time schedule.Depletion efficiency was monitored by FACS analysis of PBMC at differ-ent time points during treatment, and by immunostainings of skin biopsiesobtained at the end of the treatment period. Before and after treatment,disease severity was determined by measurement of ear thicknesses as arough indication for skin inflammation, by assessment of the clinical pic-ture using an adapted psoriasis activity and severity index (PASI) score,and recorded by photography (Dental-Eye II; Yashica, Hamburg,Germany).

FACS analysis

A total of 150–400 �l peripheral blood collected from the tail vein of micewas mixed with heparin (Liquemin N 25000; Hoffmann-LaRoche, Gren-zach-Wyhlen, Germany) to prevent coagulation. Alternatively, cells wereobtained from spleens of mice. RBC were removed using ammonium chlo-ride potassium chloride lysis buffer (0.15 M NH4Cl, 1.0 M KHCO3, 0.1 MNa2EDTA, pH 7.2). The remaining PBMC cell fraction was adjusted to1 � 106 cells/50 �l. Subsequently, 50 �l of the cell suspension was stainedwith �1 �l of the fluorochrome-conjugated mAbs for 30 min at 4°C. Afterfixation of cells in 2% formaldehyde (Merck, West Point, PA), stainedPBMC were analyzed using a FACSCalibur (BD Biosciences).

To determine cytokine production by T cells, intracellular cytokinestaining was performed. CD90� T cells were isolated from draining lymphnodes of CD18hypo and CD18wt mice by magnetic cell sorting (MiniMACScolumns; Miltenyi Biotec) and cultured in RPMI 1640 medium (Life Tech-nologies, Paisley, Scotland) supplemented with 10% FCS and 4 �g/mlCiprobay 2000 (Bayer, Leverkusen, Germany), in the presence of 3 ng/mlPMA and 300 ng/ml ionomycin (both Sigma-Aldrich, Taufkirchen, Ger-many), at 37°C and 5% CO2, either overnight or for 7 days. To inhibitsecretion of cytokines by the cells, 1 �g/ml brefeldin A (Sigma-Aldrich)was added and cells were incubated for 4 h at 37°C, 5% CO2. A total of 1 �106 cells was used per staining. Cells were washed twice with 1 ml PBS(1% FCS). Cells were resuspended in 50 �l PBS (1% FCS), and surfacestaining was performed with the indicated Abs for 30 min at 4°C. After-ward, cells were washed twice with 1 ml PBS (1% FCS), and subsequentlyfixed with 1% paraformaldehyde (Merck) for 15 min at room temperature.Cells were washed twice and then permeabilized in 50 �l 1% saponin(Sigma-Aldrich) for 5 min at room temperature, before indicated cytokineAbs (1:10) were added to the samples (30 min, 4°C). After two morewashes with 1 ml PBS (1% FCS), cells were resuspended in 500 �lPBS/1% paraformaldehyde. Subsequently, cells were analyzed by flow cy-tometry using a FACSCalibur.

To determine cell surface expression of the IL-2R� on T cells for assess-ment of cellular activation, CD90� cells were isolated and cultured overnight,as described above. A total of 1 � 106 T cells per mouse was then analyzedby FACS using triple fluorescence staining (mCD25 FITC/mCD4 PE/mCD3CyChrome) with �1 �l of each fluorochrome-conjugated mAb.

Cytokine release

To determine release of cytokines by T cells, CD90� cells were isolated,as described above. Cells (2 � 105 per well) were then activated by incu-bation on immobilized anti-CD3 mAb and anti-CD28 mAb at the indicatedconcentrations in 96-well plates. After incubation for 24 h at 37°C, thesupernatants were collected and frozen down at �20°C. Cytokine concen-trations of IFN-� and IL-4 were measured using Quantikine M ELISA(R&D Systems, Wiesbaden-Nordenstadt, Germany), according to the dis-tributed protocols.

Alternatively, the indicated cytokines were determined in the superna-tants of CD90� T cells cultured in RPMI 1640 medium supplemented with10% FCS and 4 �g/ml Ciprobay 2000, in the presence of 3 ng/ml PMA and300 ng/ml ionomycin, at 37°C and 5% CO2 overnight, using the CytokineBead Array technique (BD Mouse Inflammation CBA, BD Mouse Th1/Th2

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CBA; BD Biosciences). All staining and analysis were done without mod-ification, according to the manufacturer’s instructions.

Allergic contact dermatitis

Allergic contact dermatitis experiments were performed, as described pre-viously (47, 48). Briefly, mice were sensitized by painting 100 �l of 2%oxazalone (Sigma-Aldrich) in acetone/olive oil (4:1) onto the shaved ab-domen. After 5 days, 10 �l of 0.5% oxazalone was applied to both sides ofthe ears. Ear thickness was measured before, and 3, 6, 30, and 42 h afterchallenge using a calibrated caliper (The Dyer Company, Lancaster, PA).Ear swelling was calculated by subtracting the ear thickness before chal-lenge from ear thickness after challenge.

ResultsFACS profiles of �2 integrin � subunits sharing the mutated �2

subunit (CD18hypo) differ quantitatively from those of CD18wt

leukocytes

To study whether the reduced expression of the CD18hypo proteinon the cell surface of leukocytes in the CD18hypo psoriasiformmouse model may result in differential capacity of CD18 to asso-ciate with the four known �2-associated � subunits, FACS analysisusing CD11 (�-chain) subunit-specific mAbs was performed forCD4� T cells and overall PBMC isolated from CD18hypo andCD18wt mice. The cell surface expression of the three major �subunits, CD11a, CD11b, and CD11c, revealed a similar relativedistribution as compared with the relative distribution of the�-chains on CD18wt leukocytes. However, the protein quantities ofthe three different �-chains were substantially reduced on the cellsurfaces of CD18hypo leukocytes compared with CD18wt leuko-cytes. This was in parallel to the well-established reduction ofCD18 molecules underlining that the �2 integrin deficiency, pri-marily originating from the �2 subunit (CD18), leads to a second-ary reduction of the three major �-chains on CD4� T cells (Fig.1B) as well as on overall PBMC (Fig. 1A). Evidence for a defectiveassociation between �2 integrin �- and �2-chains potentiallycaused by a qualitative alteration in the CD18hypo molecule wasnot supported by our data, as a deficiency in dimerization willnormally lead to internalization of either subunit and, thereby, to aloss of cell surface expression (49, 50).

T cells are highly increased in psoriatic lesions ofCD18hypo mice

To study the composition of the inflammatory infiltrate of the pso-riasiform dermatitis, 30 skin sections taken from CD18hypo andCD18wt mice (n � 3) were immunostained with anti-CD4 andanti-CD8 mAbs (Fig. 2). Compared with CD18wt skin with only afew CD4� T cells in the dermis (Fig. 2A), the number of CD4�

and CD8� T cells was highly increased, both in the epidermis anddermis of CD18hypo mice (Fig. 2B). Statistic evaluation revealedthat this increase was highly significant for both cell types ( p �0.0001, Fig. 2C).

Depletion of CD4� T cells results in resolution ofpsoriatic lesions

To analyze the potential role of T cells in the pathogenesis of thepsoriasiform dermatitis, both CD4� and CD8� T cells were re-moved in vivo in CD18hypo mice using depleting mAbs. Depletionof CD4� T cells in CD18hypo mice with a severe psoriasis phe-notype including extensive scaling and alopecia (n � 3, Fig. 3A)led to a complete resolution of the psoriasiform dermatitis after 6wk of treatment (Fig. 3B), while skin lesions in mice treated withthe isotype control mAb remained unchanged (data not shown).Mice treated with an isotype control mAb showed a prominentCD4� T cell population (Fig. 3C). By contrast, i.p. administrationof the anti-CD4 mAb resulted in an almost complete removal ofCD4� T cells from the blood circulation (Fig. 3D). Depletion of

CD4� T cells was further confirmed by immunohistochemistry ofthe skin. Administration of an isotype-matched control mAb didnot reveal any effect on numbers of CD4� T cells in the psoria-siform skin lesions (Fig. 3E). By contrast, removal of CD4� Tcells from the skin was almost complete when anti-CD4 mAbswere injected (Fig. 3F).

The effect of the CD4� T cell-depleting mAbs was evaluated bythe severity of clinical symptoms using an adapted PASI score asused for assessment of the severity of human psoriasis elsewhere.For CD18hypo mice, the PASI score was modified accordingly: 0 �

FIGURE 1. Expression of the three major �2 integrin �-chains (CD11a,CD11b, CD11c) in proportion to the �2-chain (CD18). PBMCs were iso-lated from spleens of CD18hypo (dark gray areas) and CD18wt mice (blacklines), as splenic T cells have been found to show less activation andinflammatory bias of integrin expression patterns than T cells taken fromnodes of CD18hypo mice (data not shown), and FACS staining was per-formed with the indicated mAbs. Isotype control mAbs are also shown(light gray areas). In addition to CD11 and CD18 mAbs, staining for mCD4and mCD3 was done. A, Histograms demonstrate the reduction of the meanfluorescence intensities for CD11/CD18 on overall PBMCs of CD18hypo

and CD18wt mice. B, Mean fluorescence intensities of mCD18 and CD11aassembling the �2 integrin LFA-1, which is to date described as the only�2 integrin expressed on CD4� T cells, are shown for cells gated forCD4�CD3� positivity, and were also significantly reduced.

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no symptoms; 1 � slight erythema of the ears; 2 � strong ery-thema of the ears; 3 � slight hair loss at the head; 4 � extensivehair loss including the trunk; 5 � slight hair loss, isolated scaling;6 � extensive hair loss, isolated scaling; 7 � extensive hair loss,widespread slight scaling; 8 � moderate scaling at a large area ofthe body; 9 � widespread hair loss, strong scaling at few, smallerareas; 10 � extensive hair loss, extensive scaling at a large area ofthe body. A complete resolution of the psoriasiform disease wasobserved in two (10 to 1), and an almost complete resolution in oneanimal (8 to 2) treated with anti-CD4 mAbs. This clearance of thedermatitis persisted for an observation period of 6 mo after the lastanti-CD4 mAb injection. For the three CD18hypo mice treated withisotype IgG mAbs, no significant changes in the PASI scores wereevident (Fig. 4A).

Similar results were obtained by the determination of ear thick-nesses before and after treatment as a clinical assessment of theseverity of skin inflammation. In mice treated with anti-CD4mAbs, ear thicknesses substantially decreased, suggesting a re-duced inflammation, while ear thicknesses remained unchanged inthe control mice (data not shown).

Because CD4� T cells can exert their effects via activation ofcytotoxic effector functions of CD8� T cells, CD18hypo mice werealso treated with CD8� T cell-depleting mAbs in an analogousexperimental setting. As monitored by the PASI score, treatmentwith CD8� T cell-depleting mAbs, or isotype-matched control

mAbs, did not result in any improvement of the psoriasiform der-matitis (Fig. 4B). This was accompanied by an ongoing increase inear thicknesses before and after treatment, indicating the persis-tence of skin inflammation (data not shown). Even though no clin-ical improvements were observed, immunohistochemistry showedthat CD8� T cells had been successfully depleted from the skin ofmice, confirming that sufficient amounts of CD8-depleting mAbshad been administered during the time of treatment. This was fur-ther supported by FACS analysis, showing that CD8� T cells weredrastically reduced in the peripheral blood of these mice as com-pared with mice treated with isotype-matched mAbs. The latterstill had a prominent CD8� T cell population (data not shown).These results suggest that CD8� T cells are not crucial for thepathogenesis of the dermatitis of this psoriatic mouse model.

Activated T cells with a bias toward Th1 cytokines prevail inCD18hypo mice

To determine the ex vivo activation state of T cells, CD90� T cellswere isolated from draining lymph nodes of CD18hypo and CD18wt

mice. A significant increase in the expression of CD25 (IL-2R�) asa measure of T cellular activation was detected on CD4�, but noton CD8� T cells obtained from CD18hypo mice (Fig. 5). Becausethe mean expression of CD25 on CD4� T cells was twice as highin CD18hypo as in CD18wt mice, this suggested an increased stateof activation in CD4� T cells from CD18hypo mice.

FIGURE 2. Distribution of T cells in the skin ofCD18hypo and CD18wt mice. Immunohistochemistrywith mAbs directed against CD4 or CD8 was per-formed on cryosections from skin derived fromCD18wt (A) and CD18hypo mice (B). A peroxidasedetection system with 3-amino-9-ethyl-carbazol aschromogen was used. Cell nuclei were counterstainedwith hematoxylin (original magnification, �400). C,Quantitative analysis of T cells in the skin ofCD18hypo and CD18wt mice. To quantify cells in thedermis, the percentage of positively stained cells inrelation to total cell number was calculated. To quan-tify cells in the epidermis, the number of positivelystained cells/cm of epidermis was determined. For allmeasurements, the median of 30 countings (n � 3) ispresented. f, Represent CD18hypo; �, CD18wt mice.Differences were found to be statistically highly sig-nificant (p � 0.0001). e, epidermis; d, dermis.

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To determine whether T cells obtained from draining lymphnodes had been primed to secrete either Th1- or Th2-type cyto-kines, we measured the release of IFN-� or IL-4 after culturingwith different concentrations of immobilized anti-CD3 and anti-CD28 mAbs. CD90� T cells from CD18hypo mice released up to40-fold higher concentrations of IFN-� compared with CD18wt

mice (Fig. 6A), while no IL-4 could be detected in the supernatantsof activated T cells of either group (data not shown).

Because these initial results pointed at a prevalance of Th1 cellsin CD18hypo mice, a wider range of Th1/Th2 key cytokines re-leased by T cells that had been isolated ex vivo from draininglymph nodes was screened. Cytometric bead arrays (CBA) wereused to detect cytokines in the supernatants of overnight-culturedT cells, and flow cytometry was used to measure intracellular cy-tokines. As shown in Fig. 6B, highly increased concentrations ofthe Th1-type cytokine IFN-� were measured in the supernatants ofCD18hypo T cells using the CBA, whereas also with this method,the Th2 cytokine IL-4 was near the lower detection limit, therebyconfirming our previous results obtained by ELISA. BesidesIFN-�, very high levels of the Th1 key cytokine IL-2 also weredetected in the supernatants of CD18hypo T cells. However, theTh2 cytokine IL-10 also was slightly increased. Still, these dataclearly demonstrate the prevalence of Th1 cytokines in T cellsisolated from the skin lesion-draining lymph nodes of CD18hypo

mice. In comparison, supernatants of CD18wt T cells neither

showed increased amounts of the Th1- nor of the Th2-type cyto-kines tested.

These results were further supported by intracellular cytokinestaining of the isolated T cells. For all Th1-type cytokines mea-sured, the increase in cytokine-producing CD4� T cells fromCD18hypo mice was �2-fold, when compared with the wild type(Fig. 7A). In contrast to CBA analyses of the supernatants, a pro-nounced increase in intracellular IL-12 was found. T cells produc-ing Th2-like cytokines were only slightly increased (Fig. 7B).

CD18null mice do not reveal any psoriasiform phenotype

To investigate whether total absence of CD18 may equally lead tothe development of a psoriasiform phenotype in mice of the PL/Jstrain, a PL/J mouse line with complete CD18 deficiency(CD18null) was generated. Interestingly, PL/J CD18null mutants(n � 200) did not develop any psoriasiform skin disease during anobservation period of more than 2 years.

An allergic contact dermatitis can be induced in CD18wt andCD18hypo, but not in CD18null mice

Because Ag-specific T cells are most likely to play a central rolein the pathogenesis of psoriasis, but the responsible Ag could notyet be identified, it is difficult to study the emigration kinetics ofAg-specific T cells. Therefore, we have used a model, which al-lows inducing of an allergic contact dermatitis, a T cell-mediated

FIGURE 3. In vivo depletion of CD4� T cellsin CD18hypo mice. To monitor the clinical effect ofCD4� T cell depletion, neutralizing Abs were in-jected i.p. at a dose of 100–150 �g twice weekly.A, Depicts a CD18hypo mouse with a severe psori-asiform dermatitis. B, Shows the same mouse 6 wkafter treatment with CD4� T cell-depleting mAbs.The depletion efficiency was evaluated by FACSanalysis of peripheral blood cells from CD18hypo

mice treated with the isotype control mAbs (C) orwith CD4� T cell-depleting mAbs (D). Mouse anti-rat (MAR) IgG2b FITC mAbs were applied for de-tection of residual rat anti-mCD4 mAbs, which hadpreviously been used for depletion of CD4� Tcells. The red circle highlights the CD4� T cellpopulation. Skin sections from CD18hypo micetreated with isotype control mAbs (E) or withCD4� T cell-depleting mAbs (F) were immuno-stained with mCD4 mAbs (original magnification,�400). Arrows indicate the murine full thicknessepidermis form cornified to basal layer.

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hypersensitivity reaction of type IV, as classified by Coombs andGell, under standardized conditions using oxazolone as a definedAg. Because the absence of a psoriasiform phenotype in CD18null

mutants may be due to a lack of T cell emigration from bloodvessels (20), we compared the T cell-dependent inflammatory re-sponse of CD18wt, CD18hypo, and CD18null mice on the PL/J strainafter induction of an allergic contact dermatitis. To clearly differ-entiate between the induced allergic contact dermatitis and thespontaneously developing psoriasiform dermatitis in CD18hypo

PL/J mice, we only analyzed clinically healthy CD18hypo mice inwhich the lateron evolving psoriasiform dermatitis was not yetpresent, and compared these mice with equally treated CD18wt andfully CD18-deficient (CD18null) mice as controls. Upon repeatedoxazalone challenge, allergic contact dermatitis could be inducedin CD18hypo and CD18wt mice, but not in CD18null mutants (Fig.8). No significant difference in ear swelling was detectable be-tween CD18hypo and CD18wt mice after 30 h ( p � 0.132) or 42 h( p � 0.1, Mann-Whitney U test). However, onset of ear swellingwas delayed in CD18hypo mice, and ear thicknesses were furtherincreased after 42 h. H&E stainings (Fig. 9A) displayed a promi-nent perivascular and diffuse infiltration of inflammatory cells aswell as spongiosis of the epidermis in CD18hypo and CD18wt mice,indicating a strong allergic response to oxazalone. By contrast,only a slight edema without any inflammatory cells was detected inCD18null mutants. To determine whether failure of T cells to em-igrate from the vessels into the tissue causally contributed to theunresponsiveness of CD18null mutants to oxazalone challenge, im-munostainings with mAbs against CD4 (Fig. 9B) and CD8 (Fig.9C) were performed of sections derived from oxazalone-chal-lenged ears. Both CD18wt and CD18hypo mice showed a clear in-crease in CD4� and CD8� T cells in the tissue, while virtually no

T cells were observed in the oxazalone-challenged ears ofCD18null mice. Hence, 2–16% of CD18 gene expression appar-ently was sufficient for oxazalone-specific T cells to emigrate fromblood vessels driving the allergic contact dermatitis. Given the factthat in psoriasis vulgaris specific T cells also are directed againsta to date unidentified epidermal Ag (51, 52), our finding that Tcells cannot efficiently extravasate into the skin in CD18null mu-tants may explain the absence of a psoriasiform phenotype in thesemice. Our findings support the conclusion that the pathogenic in-volvement of CD4� T cells in the skin disorder of the CD18hypo

PL/J mice depends on a gene dose effect of CD18 expression.

DiscussionIn this study, we demonstrate that for the murine psoriasiformdermatitis found in CD18hypo PL/J mice CD4� T cells, but notCD8� T cells, are mandatory. We further reveal that: 1) CD4� Tcells from skin-draining lymph nodes reveal an altered activationpattern and an increased production of the Th1 key cytokines IL-2,IL-12, and IFN-�; 2) the potential of CD4� T cells to elicit murinepsoriasis needs to be linked to the hypomorphic expression of theCD18 gene between 2 and 16% of normal expression levels; and 3) at

FIGURE 4. Effect of T cell depletion on the clinical state of CD18hypo

mice. To assess the severity of the psoriasiform phenotype, an adaptedPASI score was used for CD18hypo mice before and after treatment with Tcell-depleting mAbs (open symbols), or isotype IgG control mAbs (filledsymbols). A, Demonstrates the PASI score before and after depletion ofCD4� T cells; B, depicts the PASI score before and after depletion ofCD8� T cells.

FIGURE 5. Ex vivo activation status of T cells from CD18hypo andCD18wt mice. As a measure for T cell activation, the relative fluorescenceintensities for CD25 FITC on T cells isolated from skin-draining lymphnodes of CD18hypo and CD18wt mice were determined by FACS analysis.A, Shows a representative staining for CD25 for one of three CD18hypo

(filled area) or CD18wt (filled line) mice, respectively. CD90� T cells weregated for CD4 (upper histogram) or CD8 (lower histogram) positivity. B,Mean fluorescence intensities for CD25 FITC on CD4� and CD8� T cellsof CD18hypo (filled bars) and CD18wt mice (open bars) were also calculated(n � 3). Differences were found to be statistically significant (p � 0.001)for CD4� T cell activation derived from CD18hypo compared with CD18wt

mice, and not significant (p � 0.3180) for CD8� T cell activation in lymphnodes of CD18hypo compared with CD18wt mice.

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least one other allele present in PL/J mice, but not in C57BL/6 mice,is required in addition to the CD18 hypomorphic mutation (22).

We thus evolved two major principles for murine psoriasis thathave direct relevance for human psoraisis: first, the necessity ofCD4�, but not CD8� T cells is consistent with those concepts on

human psoriasis, which assign a mandatory role to CD4� cells (53,54) and Th1 cells (55, 56), while our findings do not support con-cepts, which include CD8� T cells among the decisive constituentsfor psoriasis (57, 58). Second, the polygenic nature of murine pso-riasis including reduced gene dose for CD18 is consistent withobservations that reduced CD18 expression is one feature in hu-man psoriasis (40–42). Thus, our murine psoriasis model is thefirst polygenic model for psoriasis. It has already partially unrav-eled gene dose effects of CD18 and it will allow identification offurther involved genes.

In accordance with the current view that psoriasis is a T cell-mediated immunological disease (27), T cells are crucial for thegeneration and maintenance of the skin disease in this mousemodel. We show that highly increased numbers of CD4� andCD8� T cells reside in the skin of CD18hypo PL/J mice, a hallmarkalso in human psoriasis (53, 59). The higher number of CD4�

compared with CD8� T cells in the skin of CD18hypo mice withsevere psoriasiform phenotype provides first evidence that CD4�

FIGURE 7. Detection of intracellular Th1/Th2cytokine expression patterns in T cells fromCD18hypo and CD18wt mice. CD90� T cells wereprepared as described for the CBA cytokine detec-tion (n � 2). This time, T cells were cultured in thepresence of 3 ng/ml PMA and 300 ng/ml ionomy-cin for 7 days. Subsequently, after removal of su-pernatants from the cultured cells, the obtained Tcell samples were subjected to intracellular fluo-rescence staining of the indicated Th1 (A)- and Th2(B)-type cytokines. The percentages of CD4� Tcells expressing the indicated cytokines (upperright quadrants) are given.

FIGURE 6. Release of Th1- or Th2-type cytokines by T cells fromCD18hypo (filled bars) and CD18wt (open bars) mice. A, Concentration ofIFN-� in supernatants of CD90� T cells isolated from skin-draining lymphnodes of CD18hypo and CD18wt mice 24 h after stimulation with differentconcentrations of mCD3 and mCD28 mAbs (n � 3), as detected by ELISA.�, p � 0.05 for CD18hypo vs CD18wt T cells. B, Simultaneous measurementof the indicated cytokines in supernatant CD90� T cells by CBA (n � 2).T cells were cultured in the presence of 3 ng/ml PMA and 300 ng/mlionomycin overnight before supernatants were obtained for CBA.

FIGURE 8. Induction of an allergic contact dermatitis in CD18null,CD18hypo, and CD18wt mice after oxazalone challenge. Mice were used atan early age of 6–12 wk, in which no clinical phenotype of the later evolv-ing psoriasiform dermatitis was yet obvious in CD18hypo mice. All micewere sensitized with 2% oxazalone, and challenged with 0.5% oxazaloneafter 5 days to induce an allergic contact dermatitis. Mean ear swelling ofCD18wt (filled bars), CD18hypo (striped bars), and CD18null mice (openbars) was measured at the indicated time points after oxazalone challenge(n � 3). Ear swelling was calculated by subtracting the thicknesses of theear before and after challenge. ��, p � 0.01; �, p � 0.05; p � not signif-icant for all other groups.

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T cells are important in the generation and maintenance of the skindisease in this psoriasis model. This is in analogy to affected skinof psoriasis patients in which CD4� T cells prevail (29, 54, 59). InCD18hypo PL/J mice, depletion of CD4� T cells, but not CD8� Tcells, results in the complete resolution of the skin disease. This isalso consistent with human psoriasis, as treatment with Abs di-rected against CD4 cures psoriatic lesions or significantly de-creases the PASI score (29–31). In psoriatic CD18hypo PL/J mice,only CD4�, but not CD8� T cells from skin-draining lymph nodespresent signs of activation, such as enhanced IL-2R� expression inCD4�, but not in CD8� T cells. Thus, our results point at a centralrole of CD4� T cells in the pathogenesis of the psoriasiform skindisorder of CD18hypo PL/J mice.

This is in line with the hypotheses of Thivolet and Nicolas (54)and Valdimarsson et al. (53), who consider CD4� T cells to be thekey players in the pathogenesis of psoriasis, while Prinz (57) andNickoloff (58) favor a cooperation between CD4� and CD8� Tcells responsible for the development of psoriatic plaques. Ourfindings show that only CD4� and not a cooperation betweenCD4� and CD8� T cells is required to sustain the psoriasiformdisease.

The type of T cell effectors identified in the murine CD18hypo

PL/J model corresponds to that of human psoriasis, which is a Th1disease (55, 56). Compared with CD18wt mice, stimulated T cellsfrom affected CD18hypo mice released up to 40-fold higher con-centrations of the Th1 cytokine IFN-�, and also produced consid-erable amounts of IL-2 and IL-12, whereas characteristic Th2 cy-tokines were not detected, apart from low levels of IL-10. Thispoints to a prevalence of Th1 cells in the psoriasiform dermatitis ofCD18hypo mice. The skewing of the cytokine pattern from theTh1 to the Th2 type recently proved to be an effective thera-peutic strategy in psoriasis patients. In fact, treatment with Th2cytokines such as IL-4, IL-10, or IL-11 or blocking of Th1cytokines by anti-IFN-� mAbs has been successfully tested inclinical trials (60 – 63).

Our major finding, however, is that this T cell-mediated psori-atic skin disease in PL/J mice most likely depends on the CD18gene dose and subsequent expression levels of CD18 protein, withno inflammatory disease in CD18null and CD18wt mice. This con-

clusion is at least partly supported by our findings that the expres-sion levels of all four possible � subunits differed only in quantity,according to the reduction in total levels of �2 integrin het-erodimers, when compared with CD18 wild-type leukocytes; andalso by previously published results derived from studies on pa-tients suffering from LAD1 (49, 50, 64, 65). These authors statedthat deficiency in �2-integrin heterodimers appears to be quanti-tative rather than qualitative, with two patients expressing �0.5%and one patient 5% of normal amounts of CD18. The latter patientshad �/� complexes on the cell surface of their leukocytes, as de-tectable by immunoprecipitation with reduced absolute numbers,but similar ratios compared with healthy controls. However, ourdata do not allow us to completely exclude the possibility that theCD18hypo gene product may exhibit qualitative differences toCD18 wild-type proteins.

Results from our studies of allergic contact dermatitis show thatresidual CD18 expression is distinctly required for the extravasa-tion of reactive T cells, while in CD18null PL/J mutants, Ag-spe-cific T cells cannot emigrate from the blood vessels. Psoriasis isregarded to be an autoimmune disease (53, 57, 59). This implicatesthat T cells recognize self Ags in the skin, leading to the initiationof an inflammatory response. If T cells are severely impaired toenter the skin, as is the case in the CD18null mutation, they do nothave access to specific self Ags in great numbers, and may sub-sequently fail to initiate a relevant inflammatory response. In fact,after induction of an allergic contact dermatitis, no T cells wereobserved in the ears of CD18null mice, pointing to a lack of T cellemigration from blood vessels. This has already been shown inCD18null mutants of a different genetic background (129/Sv �C57BL/6J), suggesting that the failure of T cell emigration ratherdepends on the CD18 deficiency and not on the genetic back-ground (20). This may explain at least in part the absence of apsoriasiform phenotype in PL/J CD18null mutants. By contrast, inCD18hypo PL/J mice, highly increased numbers of CD4� andCD8� T cells, comparable to CD18wt littermates, were present inthe skin after induction of an allergic contact dermatitis indicatingthat a CD18 rest expression of 2–16% is sufficient for the emigra-tion of T cells from skin vessels into the tissue. Our findings are inline with results of a study on 31 psoriasis patients who had been

FIGURE 9. T cells in oxazalone-challengedears of CD18null, CD18hypo, and CD18wt mice.A, H&E histology (H&E staining, original mag-nification, �200), and immunostainings of earsections with mAbs directed against CD4� (B)and CD8� T cells (C). Ears were taken fromsensitized CD18wt, CD18hypo, and CD18null

mice 30 h after challenge with 0.5% oxazalone.An alkaline phophatase detection system wasused (red staining). Cell nuclei were counter-stained with hematoxylin (original magnifica-tion, �400).

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treated with different doses of a humanized mAb against CD11a, apart of the heterodimeric receptor LFA-1 (CD11a/CD18). Clinicalresolution of psoriasis could only be achieved when saturatingconcentrations of the mAb against CD11a were applied, whilenonsaturating concentrations were ineffective (39). The authors didnot give any explanation for the observed dose-dependent effect. Inthis study, we provide direct evidence that the complete preventionof T cell emigration requires a complete structural or functionalabsence of CD18 heterodimers.

Interestingly, in CD18wt mice with unaffected T cell emigration,no psoriasiform dermatitis develops, suggesting that apart from theability of proper emigration of T cells, additional pathogenic fac-tors depending on reduced CD18 expression are mandatory for thedevelopment of the murine psoriasis. Among several possibilities,reduced CD18 expression may cause the generation and persis-tence of autoreactive T cells, e.g., by impairing normal deletion inthe thymus.

In fact, �2 integrins have been reported to be involved in thymicT cell development and selection. LFA-1 (CD11a/CD18) mediatesdifferentiation from CD4�CD8� to CD4�CD8� thymocytes (66)and plays a central role in the regulation of apoptosis during neg-ative selection of autoreactive thymocytes (67, 68). The role ofCD18 in lymphocyte activation has been characterized in second-ary lymphoid organs. During immune responses, �2 integrins arecrucial for the adjustment of Ag-dependent activation thresholds.In the absence of CD18, a 100-fold increase in Ag concentrationsis required for efficient T cell activation (16). This may in part becaused by defects in structures, such as immunologic synapses inCD18null mice.4 Similar structural defects may also account forfunctional deficiencies in primary lymphoid organs such as thethymus, if CD18 is reduced or absent.

Other animal models with similarity to psoriasis have been de-scribed and include the mouse mutations flaky skin, chronic pro-liferative dermatitis, transgenic HLA-B27 rats (69), graft-vs-hostdisease due to differences in the minor histocompatibility Ags (70),epidermal dysregulation of NF-�B-mediated signaling (71), trans-genic �1 integrin overexpression in the murine suprabasal epider-mis (72), and transplantation of human psoriasis-affected skin ontoSCID mice (73). All these models reveal some similarities to hu-man psoriasis both in terms of the clinical and histological pictureand various aspects of its pathogenesis. Two models even mimicthe autoreactive nature of T cells in psoriasis (70, 73). By contrastto all models, the disease model described in this work is of par-ticular interest in that one relevant mutation resulting in reducedCD18 expression is known, and it is feasible to identify majormodifier genes and their impact on thymic selection or other tol-erance-maintaining processes. In this study, we provide first directevidence that reduced CD18 expression is distinctly involved inthe pathogenesis of a psoriasiform skin disease. Gene dose effectsin other cell surface receptors crucial in the control of immune cellinteraction such as CD40 (74) and CD19 (75) have recently beendescribed to promote the development of autoimmune disorders. Inconclusion, the CD18hypo PL/J mouse model represents a valuabletool for future investigations in the pathogenesis of psoriasis, andshould help to clarify the role of a CD18 rest expression of 2–16% inthe development and maintenance of the psoriasiform phenotype.

AcknowledgmentsWe are grateful to Eva Nattkamper, Stefan Seeliger, and Karin Fischer forexcellent technical help.

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