mice lacking the transcription factor relb develop t cell-dependent skin lesions similar to human...

0014-2980/00/0808-2323$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

Mice lacking the transcription factor RelB developT cell-dependent skin lesions similar to humanatopic dermatitis

Debra Barton1, Harm HogenEsch2 and Falk Weih1

1 Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, Karlsruhe, Germany2 Department of Veterinary Pathobiology, Purdue University, West Lafayette, USA

Mice with a targeted disruption of the Rel/NF- ‹ B family member RelB develop a complexinflammatory phenotype and hematopoietic abnormalities. RelB-deficient (relB–/–) mice wereclinically normal until 4–10 weeks after birth when thickening of the skin and hair loss devel-oped. Histological and immunohistochemical evaluation of relB–/– skin lesions revealedhyperkeratosis and marked epidermal hyperplasia. Many CD4+ T cells and eosinophilsmixed with lesser numbers of CD8+ T cells and neutrophils were present in the dermis. Therewas a moderate increase of MHC class II-positive dermal dendritic cells and dermal mastcells. Increased expression of Th2 cytokines correlated with increased mRNA levels ofeotaxin and CCR3 in relB–/– skin. The dermatitis did not develop in the offspring of relB–/–

mice crossed with transgenic mice that lack peripheral T cells, demonstrating that the skinlesions were T cell dependent. The dermatitis observed in RelB-deficient mice had manysimilarities with atopic dermatitis in human patients including infiltrating CD4+ T cells andeosinophils in the skin, increased number of eosinophils in the blood and increased serumIgE. Thus, the relB–/– mouse should be a useful model to study the pathogenesis of this com-mon allergic human disease.

Key words: RelB/NF- ‹ B / Atopic dermatitis / T cell / Cytokine / Inflammation

Received 19/4/00Accepted 10/5/00

[I 20796]

Abbreviations: AD: Atopic dermatitis DC: Dendritic cellsDDC: Dermal dendritic cells LC: Langerhans cell PCNA:Proliferating cell nuclear antigen RT: Reverse transcriptionICAM: Intercellular adhesion molecule

1 Introduction

The transcription factor NF- ‹ B has attracted widespreadattention among researchers in many fields based on itsunusual and rapid regulation, the wide range of genes itcontrols, its central role in immunological processes, thecomplexity of its subunits, and its apparent involvementin several diseases. Five members of this family havebeen identified in vertebrates: NF- ‹ B1 (encoding theprecursor molecule p105 and the processed form p50),NF- ‹ B2 (encoding p100 and p52), RelA (p65), RelB, andc-Rel. The DNA-binding activity of Rel/NF- ‹ B com-plexes is regulated by members of the I ‹ B family andseveral distinct I ‹ B molecules with homologies toankyrin repeats have been described. In most cell typesRel/NF- ‹ B proteins are trapped in the cytoplasm bythe I ‹ B inhibitors. A wide range of stimuli activatesthe I ‹ B kinase complex, resulting in the phosphoryla-

tion, ubiquitination and degradation of I ‹ B. Conse-quently, the Rel/NF- ‹ B proteins translocate to thenucleus and bind to so-called ‹ B sequence motifs [1–5].The Rel/NF- ‹ B family member RelB is expressed pre-dominantly in specific regions of lymphoid organs andhigh levels of nuclear RelB are found in professionalantigen-presenting dendritic cells (DC). RelB is notexpressed at significant levels in whole skin and epider-mal Langerhans cells (LC) express only low amounts ofRelB. LC resemble immature DC and both RelB proteinlevels and DNA-binding activity are markedly inducedupon DC maturation [6–8]. RelB-deficient mice display acomplex phenotype including impaired development oflymphoid organs, reduced numbers of mature DC, multi-organ inflammation, and multifocal defects in immuneresponses [9–12].

Atopic dermatitis (AD) is one of the most common skindiseases in patients with an individual or family history ofatopy and is frequently associated with elevated serumIgE levels [13, 14]. AD afflicts 10–15 % of children andadolescents in the Western world [15] and its pathogene-sis is still poorly understood. Similar to other allergicdiseases AD has a strong genetic component, but few

Eur. J. Immunol. 2000. 30: 2323–2332 RelB-deficient mice develop skin lesions similar to atopic dermatitis 2323

Fig. 1. Hyperkeratosis, acanthosis, dermal thickening andinflammation in relB–/– skin. Dorsal skin sections from a 10-week-old wild-type control (A), a 9-week-old minimallyaffected (grade 1) relB–/– mouse (B), and a 13-week-oldmoderately affected (grade 3) relB–/– mouse (C) stained withhematoxylin and eosin are shown.

Fig. 2. Hyperproliferation of basal keratinocytes and alteredkeratin expression in relB–/– skin. Skin from a control mouse(A) with very few PCNA-positive cells (red staining). PCNAstaining of skin from a relB–/– mouse (B) with moderate epi-dermal hyperplasia showing a marked increase of proliferat-ing keratinocytes in the basal layer. Skin section from a con-trol (C) and from a moderately affected mutant relB–/– mouse(D) stained with an Ab specific for keratin 6.

candidate genes have been identified [16, 17]. Recentresults indicate that immunological mechanisms play akey role in the generation of AD. In addition to a mono-nuclear cellular infiltrate that mainly consists of CD4+ Tcells, eosinophils, mast cells, and inflammatory cyto-kines have been involved in the development of AD [18].Although great attention has been focused on the patho-genesis, the exact etiology of AD is still unclear. Estab-lishment of a suitable animal model would greatly help toelucidate the pathogenesis of AD and to develop newapproaches to therapy.

Here, we show that RelB-deficient mice had histopatho-logical changes, inflammatory infiltrates and cytokineexpression patterns in skin similar to patients with AD. Inparticular, the finding that the skin phenotype wasdependent on T cells makes relB–/– mice an interestingmodel for this inflammatory skin disease.

2 Results

2.1 Histopathology of relB–/– skin

Between 4 and 10 weeks of age, approximately 70–80 %of relB–/– mice regardless of genetic backround devel-oped a dermatitis that was characterized clinically bythickened and reddened skin, scaling, itching and loss ofhair. The dermatitis occurred on the trunk and head ofthe mice with occasional involvement of the ears andfootpads. Light microscopic examination of hematoxylinand eosin-stained skin sections from wild-type andRelB-deficient mice of various ages revealed hyperkera-tosis, acanthosis, dermal thickening and mixed cellularinflammation in the dermis of relB–/– skin compared tocontrol animals (Fig. 1). There was marked variation inthe severity of the skin lesions between animals and onlya moderate increase in severity over time. Occasionalmicroabscesses in the stratum corneum and ulcerationwere present, probably the result of scratching and notpart of the primary dermatitis. Prominent blood vesselsin the dermis were suggestive of neovascularization. Afew apoptotic keratinocytes were observed in the epider-mis of some relB–/– mice. Bacterial or fungal infections asthe primary cause for the skin lesions were excluded byspecific stains (data not shown). A summary of the histo-pathological findings is presented in Table 1.

2.2 Keratinocyte hyperoproliferation and up-regulation of keratin 6 in relB–/– skin

To determine whether the epidermal thickening in relB–/–

mice was caused by increased proliferation, skin sec-tions were stained with a mAb specific for the cell cycle

2324 D. Barton et al. Eur. J. Immunol. 2000. 30: 2323–2332

Table 1. Histopathology of relB–/– skin

Phenotype relB–/+ (6–20 weeks) relB–/– (4–10 weeks) relB–/– (12–20 weeks)

Epidermal hyperplasia −a) G2.3 ± 1.2b) (6/7)c) G2.8 ± 0.8 (10/12)

Inflammation − G2.2 ± 1.1 (5/7) G3.0 ± 0.7 (10/12)

Ulceration − G1 (2/7) G2.4 ± 1.3 (5/12)

Mast cell infiltration − G2 (1/7) G2.1 ± 0.6 (7/12)

a) −, not present.b) Histopathological grading; G1, minimal; G2, mild; G3, moderate; G4, marked (± SD).c) Number of animals with pathological changes/number of animals analyzed.

marker proliferating cell nuclear antigen (PCNA) [19].Whereas control mice had very few PCNA-positive cellsin the epidermis, PCNA staining of relB–/– skin revealed amarked increase of proliferating keratinocytes in thebasal layer (Fig. 2 A and B).

Keratin intermediate filaments are considered hallmarkproteins of epithelial cell differentiation and changes inkeratin structure or gene expression are associated withmany skin disorders [20, 21]. In normal skin the keratinpolypeptide pair K6/K16 is only expressed in the outerroot sheaths of hair follicles. K6 and 16 have been calledhyperproliferative keratins because they are also foundin high turnover mucosal epithelia [22, 23]. The keratino-cyte hyperproliferation prompted us to examine relB–/–

skin for altered keratin expression. Immunohistochemi-cal staining of skin sections with a K6-specific Abrevealed a dramatic up-regulation of K6 expression inacanthotic epidermis from RelB-deficient mice com-pared to control animals. Staining of hair follicles wasobserved in both relB–/– and normal skin (Fig. 2 C and D).

2.3 Increased numbers of T cells, MHC class II+

cells, mast cells, neutrophils and eosinophilsin skin from RelB-deficient mice

To characterize the inflammatory cells in the relB–/– der-matitis in more detail, we performed specific histologicalstaining and immunohistochemistry with a panel of mAb.Immunohistochemical staining for infiltrating T lympho-cyte showed numerous CD4+ Th cells nd CD8+ cytotoxicT cells in the skin of relB–/– mice (Fig. 3 A and B), althougha smaller number of CD8+ lymphocytes occurred bothquantitatively and proportionally in the infiltrates. Theskin of control mice showed only few resident CD4+ andCD8+ cells (not shown). In human inflammatory skin dis-orders increased numbers of HLA-DR+ dermal dendriticcells (DDC) are frequently observed beneath the hyper-

plastic epidermis surrounded by T cells, suggesting animportant role of DDC/T cell interaction in the inflamma-tory phenotype [24]. Therefore, we stained skin sectionsfrom control and relB–/– mice with a mAb against I-Ab toidentify MHC class II+ cells. Fig. 3 C shows marked I-Ab

staining in the dermis of mice lacking RelB whereas onlyfew cells were positive in sections from control animals.We also observed an increased number of CD11c+ cells,suggesting that many of the I-Ab-positive cells wereDC (not shown).

Skin sections were stained with toluidine blue to detectmast cells. Increased numbers of intact as well as degra-nulated mast cells were observed in the dermis of RelB-deficient mice (Fig. 3 D and data not shown). Stainingwith the neutrophil-specific mAb 7/4 [25] revealed amoderate number of neutrophils in the dermis and inoccasional intracorneal pustules in the epidermis ofRelB-deficient mice (Fig. 3 E). In addition, skin sectionswere stained for CN-resistant peroxidase activity, a pro-cedure that specifically detects eosinophilic granulo-cytes. Numerous eosinophils infiltrated both the dermisand epidermis of RelB-deficient mice (Fig. 3 F). In con-trast, skin sections from control mice had very few eosin-ophils (not shown).

2.4 RelB-deficient mice develop pulmonaryinflammation

Since human AD is frequently accompanied by respira-tory problems [17], we analyzed RelB-deficient mice witha moderate to marked skin phenotype for pulmonary dis-ease. As shown in Fig. 4 A and B, mice lacking RelB hadseveral features of allergic lung inflammation. In agree-ment with previous results [10], there was a markedaccumulation of inflammatory cells around blood vesselsand airways of the lungs of relB–/– mice. Trichrome stain-ing demonstrated newly synthesized collagen depositedat sites of heaviest inflammation and periodic acid-Schiff


Fig. 3. Increased numbers of T cells, MHC class II+ cells,mast cells, neutrophils and eosinophils in relB–/– skin. Immu-nohistochemical detection of CD4+ (A) and CD8+ T cells (B)in the skin of a relB–/– mouse. (C) Staining or relB–/– skin witha mAb against the MHC class II molecule I-Ab. (D) Toluidineblue (T.blue) staining of skin from a relB–/– mouse showingnumerous mast cells in the dermis. (E) Staining of neutro-phils with the mAb 7/4 in skin from a relB–/– mouse. (F) CN-resistant peroxidase staining of skin from a relB–/– mouseshowing infiltrating eosinophils in dermis and epidermis.

Fig. 4. RelB-deficient mice develop features of allergic lunginflammation. (A) Trichrome staining of a relB–/– lung sectionshowing intense inflammation (dark color) surroundingblood vessels and airways. Newly synthesized collagendeposited at sites of heaviest inflammation is stained in lightblue. Periodic acid-Schiff staining (A, inset) revealed hyper-plasia of mucus-secreting goblet cells (bright pink) within air-ways. (B) CN-resistant peroxidase staining of lung from arelB–/– mouse showing the presence of numerous eosino-phils in the inflammatory infiltrate.

Fig. 5. Increased expression of Th2 and pro-inflammatorycytokines in skin from relB–/– mice. (A) Total skin RNA from acontrol, a minimally affected and a moderately affectedrelB–/– mouse was reverse transcribed and examined byPCR analysis using primers specific for IFN- + , IL-2, IL-4,IL-5, IL-1 g , IL-6, TGF- § , TNF- § , GM-CSF, eotaxin, CCR3and GAPDH. (B) Increased expression of ICAM-1 on kerati-nocytes from RelB-deficient mice. Skin sections from a con-trol and (left) from a relB–/– mouse (right) stained with a mAbspecific for ICAM-1.

Fig. 6. The skin lesions in RelB-deficient mice are T celldependent. Skin sections from RelB-deficient mice lackingT cells (nur77TGrelB–/–) were stained for PCNA+ proliferatingcells (A) and for the stress keratin 6 (B). To detect inflamma-tory granulocytes nur77TGrelB–/– skin sections were stainedwith the neutrophil-specific 7/4 mAb (C) and with CN-resistant peroxidase to stain eosinophils (D). Note thereduced staining for all markers in skin from RelB-deficientmice lacking T cells compared to the results shown in Fig. 3.


staining revealed a marked increase of mucus-secretinggoblet cells within the airways. Similar to the dermatitis,eosinophils were a major constituent of the inflammatoryinfiltrate in the lung. Lung sections from control animals didnot show any pathology (not shown).

2.5 Increased expression of pro-inflammatorycytokines in relB–/– skin

Cytokines orchestrate inflammatory reactions and de-termine the type and outcome of the inflammatoryresponse. Therefore, we examined the expression of var-ious cytokines in control, minimally affected and moder-ately affected skin. Since T cells were a prominent com-ponent of the inflammatory infiltrate in relB–/– skin, westarted to analyze cytokines that are predominantlyexpressed by T cells. Fig. 5 A demonstrates increasedlevels of IFN- + mRNA in moderately affected skin (7-fold), whereas IL-2 mRNA levels were unaltered. In con-trast, expression of both IL-4 (200-fold) and IL-5 (15-fold)was dramatically increased in moderately affected skincompared to wild-type controls. Analysis of the expres-sion of inflammatory cytokines that can also be pro-duced by stimulated monocytes, fibroblasts and kerati-nocytes revealed an induction of IL-1 g (9-fold), IL-6 (10-fold), TGF- § (4-fold), TNF- § (5-fold) and GM-CSF (3-fold)in moderately affected skin compared to a wild-typecontrol.

The observed infiltration of eosinophils and the dramaticup-regulation of IL-4 and IL-5 in moderately affected skinprompted us to examine the expression of the CC che-mokine eotaxin, a potent chemoattractant and activatorof eosinophils and Th2 lymphocytes, and its cognatereceptor, CCR3 [26–28]. As shown in Fig. 5 A, expres-sion of both eotaxin (20-fold) and CCR3 (8-fold) wasmarkedly increased in moderately affected skin com-pared to wild-type controls. Minimally affected skin fromrelB–/– mice also showed increased cytokine/chemokineand CCR3 expression although to a much lesser extent.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)mRNA levels were used for normalization and served asa loading control.

IL-1 g , IFN- + and TNF- § exert multiple effects, includ-ing up-regulation of intercellular adhesion molecule(ICAM)-1 expression [29, 30]. Consistent with theincreased expression of these cytokines, reverse tran-scription (RT)-PCR analysis revealed a marked increasein ICAM-1 mRNA levels in skin samples from RelB-deficient mice compared to control animals (data notshown). Both dermal endothelial cells and epidermalkeratinocytes of psoriatic and AD skin express high lev-els of ICAM-1 [31, 32]. Staining with an ICAM-1-specific

mAb showed marked ICAM-1 expression by keratino-cytes of relB–/– skin (Fig. 5 B). The staining was strongestin the basal layer and the intensity decreased towardsthe stratum corneum. No significant staining of the epi-dermis of wild-type controls was observed.

2.6 The skin lesions in RelB-deficient mice areT cell dependent

To examine whether inflammatory T cells are cruciallyinvolved in the development of the pathological changesin the relB–/– skin, we bred RelB-deficient mice with amouse line that carries the nur77 transgene (nur77TG)under the control of T cell-specific regulatory elements.The constitutive overexpression of the orphan nuclearreceptor Nur77 results in apoptotic death of immaturethymocytes and a dramatic reduction of peripheral Tcells [33, 34]. Immunohistochemical staining of skin fromrelB–/– mice and nur77TGrelB–/– littermates revealed amarked reduction in PCNA-positive proliferating cells inthe epidermis of relB–/– mice lacking T cells (Fig. 6 A).Expression of the hyperproliferative keratin 6 and the celladhesion molecule ICAM-1 was also strongly reduced inskin from nur77TGrelB–/– mice compared to relB–/– litter-mates (Fig. 6 B and data not shown). Thus, keratinocytehyperproliferation, epidermal hyperplasia and inductionof ICAM-1 expression in keratinocytes in relB–/– skin areT cell dependent.

As shown in Fig. 6 C and D, both mAb 7/4+ neutrophilsand CN-resistant peroxidase+ eosinophils were absentfrom the skin of RelB-deficient mice lacking T cells. Thenumber of mast cells and of dermal MHC class II+ cellswas also markedly reduced in skin sections fromnur77TGrelB–/– mice compared to relB–/– littermates (datanot shown). These results demonstrate that the skinlesions in RelB-deficient mice are T cell dependent.

3 Discussion

3.1 Histopathology of relB–/– skin

Mice lacking the Rel/NF- ‹ B family member RelBdevelop a complex phenotype that includes mixedinflammatory infiltrates in several nonlymphoid organs[9, 10]. The present study focused on the skin and dem-onstrates that RelB-deficient mice developed dermatitischaracterized by hyperkeratosis, acanthosis and accu-mulation of T cells, eosinophils, mast cells and MHCclass II+ cells. Almost all relB–/– mice examined had histo-pathological changes in skin although we observed pro-nounced interanimal variation (see Table 1). An infectiouscause of the dermatitis was excluded by special stains of


tissue sections for bacterial or fungal microorganismsand lack of evidence for transmission between litter-mates. AD in humans is characterized by infiltration ofCD4+ T cells and eosinophils in the skin and depositionof eosinophil granule proteins in the dermis, blood eosin-ophilia and increased serum IgE, indicating that imunolo-gical mechanisms are important in the pathogenesis ofAD. In particular, infiltrating CD4+ T cells and T cell-derived cytokines have been suggested to be cruciallyinvolved in the induction of this skin abnormality [35–39].The skin lesions of RelB-deficient mice had histopatho-logical changes similar to AD including infiltrating CD4+

T cells and eosinophils. Moreover, we previously demon-strated that RelB-deficient mice have markedlyincreased serum IgE levels (18-fold) [11] and increasednumbers of eosinophils in blood (9-fold) (data notshown). There is a strong association between AD andasthma in human [40], suggesting that common mecha-nisms may underlie the inflammation in these tissues.RelB-deficient mice with a moderate to marked skin phe-notype also developed several features of pulmonaryinflammation. Similar to the skin, the inflammatory in-filtrates consisted predominantly of eosinophils (seeFig. 4) as well as CD4+ and CD8+ T cells [41].

Recently, several spontaneous mouse mutants witheosinophilic dermatitis have been reported. The NC/Ngamouse develops dermatitis characterized by increasednumbers of eosinophils and T cells, elevated serum IgEand increased numbers of IL-4 and IL-5-positive cells inthe dermis when housed under conventional, but notunder SPF conditions [42]. The NOA mouse is a hairlessmouse that develops an ulcerative dermatitis with accu-mulation of eosinophils and mast cells and elevatedserum IgE [43, 44]. The cpdm/cpdm mouse develops asevere dermatitis that is characterized by eosinophil infil-tration and an increase of mast cells [45, 46]. In contrastto the other mice, cpdm/cpdm mice have a decrease ofserum IgE associated with a general defect in isotypeswitching [47]. The genetic defects in NC/Nga, NOA andcpdm/cpdm mice have not been identified but the phe-notype of these mice is different from the phenotype ofRelB-deficient mice. The study of the dermatitis in spon-taneous mouse mutants and RelB-deficient mice shouldprovide new insights into the mechanisms that underlieeosinophil accumulation in the skin and into the patho-genesis of AD.

3.2 Cytokine and chemokine expression inrelB–/– skin

T cell-dependent immune responses can be segregatedinto Th1 or Th2 categories characterized by IFN- + andIL-2 versus IL-4 and IL-5 production, respectively [48].

There was markedly increased expression of IL-4, IL-5 andIFN- + in the skin lesions of RelB-deficient mice indicatingthat both type 1 and type 2 mechanisms play a role in thepathogenesis of the skin lesions. It is well established thatTh2 cells and Th2 cytokines play a key pathogenic role inAD [49] but recent studies indicate that the development ofskin lesions in patients with AD results from sequentialactivation of Th2 and Th1 T lymphocytes [50]. Althoughcytokines regulate the accumulation of eosinophils in tis-sues at multiple levels [51] the greatly increased expres-sion of IL-4 and IL-5 was most likely responsible for theaccumulation of eosinophils in relB–/– skin.

AD has been associated with the activation of peripheralblood T cells that preferentially secrete Th2 cytokinesfavoring the differentiation of eosinophilic granulocytesand supporting the production of high amounts of IgE[52–56]. IL-4, in combination with IL-5 and IL-13, pro-motes Ig isotype switching to IgE [57], a process that isfunctional in B cells isolated from RelB-deficient mice[58]. IL-5 produced by Th2 cells is also required foreosinophil growth and differentiation [59]. IgE is capturedat the cell surface by Fc 4 RI present on mast cells andeosinophils and Fc 4 RI cross-linking stimulates release ofa variety of toxic products that together elicit atopic dis-ease [60]. The strong expression of Th2 cytokines inrelB–/– skin is consistent with an intracellular cytokineTh2 expression profile in splenic CD4+ T cells from RelB-deficient mice [61]. Similar to AD and other atopic dis-eases, RelB-deficient mice also have eosinophilia, IgEoverproduction and mast cell degranulation, suggestingrelated pathological mechanisms.

The CC chemokine eotaxin selectively binds and acti-vates the chemokine receptor CCR3 [62, 63] andrecently it has been shown that expression of botheotaxin and CCR3 is increased in lesional skin from AD,but not in nonatopic controls [64]. Eotaxin is a potentchemoattractant and activator of human eosinophils,basophils and Th2 lymphocytes and its expression is up-regulated by IL-4 in human dermal fibroblasts [63]. Anal-ysis of mRNA levels in skin from RelB-deficient micerevealed a strong up-regulation of both eotaxin andCCR3 expression even in minimally affected animalswhereas pro-inflammatory cytokines were only markedlyinduced in skin from severely affected mutant mice.Thus, the interaction of eotaxin and CCR3, particular inthe initiation phase, may represent a major mechanismfor promoting tissue damage in AD and relB–/– skin. Itshould be noted, however, that the accumulation ofeosinophils in the lung in a model of airway hypersensi-tivity was not or only transiently reduced in eotaxin-deficient mice [65, 66], suggesting that other chemo-kines such as macrophage inflammatory protein-1 § con-tribute to the infiltration of eosinophils in the lung [67].


3.3 Role of T cells in the relB–/– skin phenotype

Immunohistochemical analysis revealed numerous infil-trating CD4+ and to a lesser extent also CD8+ T cells. Inaddition, we observed increased numbers of MHC classII- and CD11c-positive cells in relB–/– skin. It is importantto note that RelB is not expressed at significant levels inwhole skin [6, 9, 10] and resident or freshly isolated epi-dermal LC express only low levels of RelB [8]. Within thethymus, DC are crucially involved in the negative selec-tion of autoreactive T cells, leading to central T cell toler-ance [68, 69]. Whereas LC are present at normal num-bers in the epidermis of relB–/– mouse ears and showonly slightly reduced antigen presenting function, RelB-deficient mice lack mature DC in the thymic medulla[9, 10, 12, 70]. Consequently, RelB-deficient mice poorlydelete autoreactive thymocytes and have splenocytesthat generate an autoreactive response [61, 71], indicat-ing that impaired clonal deletion of autoreactive T cellseventually results in potentially pathogenic T cells pro-moting inflammatory skin lesions in relB–/– mice. Cyto-kines released from these infiltrating T cells, in turn, mayinduce keratinocyte hyperproliferation and activation ofother inflammatory cells. This model is strongly sup-ported by our results from nur77TGrelB–/– mice that lackperipheral T cells. These animals lack the mixed inflam-matory infiltrates in lung and liver [72] and also devel-oped a much milder form of the skin lesions with mark-edly reduced epidermal hyperplasia, keratinocyte prolif-eration and dramatically reduced inflammatory infil-trates. In addition, while adoptive transfer of relB–/– bonemarrow cells into lethally irradiated wild-type miceinduces minimal, if any, tissue inflammation [9, 61, 73],injection of spleen cells from RelB-deficient mice resultsin the development of skin lesions and inflammation inother tissues [74]. These findings further indicate a cru-cial role of miseducated autoreactive T cells in the devel-opment of the relB–/– phenotype.

Recently, it has been shown that stimulation of relB–/–

fibroblasts with LPS results in persistent up-regulation ofcytokine and chemokine expression correlating withinduced p50-RelA/NF- ‹ B activity due to decreasedI ‹ B § stability [73, 75]. Therefore, a feedback cyclebetween inflammatory T cells and nonlymphoid cellsmay exist, where autoreactive or otherwise defective Tcells activate relB–/– fibroblasts or other resident perma-nent cells to release cytokines and chemokines. Onceactivated, these cells attract more leukocytes into theaffected tissue and enter a prolonged activation state.

Our analysis of skin from RelB-deficient mice suggeststhat NF- ‹ B dysregulation should be studied as a possi-ble factor that determines the genetic susceptibility toAD. This is also supported by the finding that I ‹ B § defi-

ciency results in a sustained NF- ‹ B response and severewidespread dermatitis in mice [76]. Further experimentsare required to analyze whether resident skin cells in ADpatients show altered p50-RelA/NF- ‹ B activity. Theexact etiology of AD is still unclear and suitable animalmodels would greatly help to elucidate the pathogenesisof this common allergic human disease and to developnew approaches to therapy.

4 Materials and methods

4.1 Mice

Generation of relB–/– and T cell-deficient nur77TG mice hasbeen described previously [10, 34]. Analyses were per-formed on mice from a C57BL/6, 129/Sv, and a mixedB6x129 genetic background. All animals were housed andbred under standardized conditions with water and food adlibidum in an SPF mouse facility at the ForschungszentrumKarlsruhe, Institute of Toxicology and Genetics (approved bythe Regierungspräsidium Karlsruhe, Germany).

4.2 Histopathology and specific stains

Histopathological analyses were performed on a minimumof seven animals per age group. Sections were graded forseverity of the phenotype without knowledge of genotype orage as follows: −, none; 1, minimal; 2, mild; 3, moderate; 4,marked. Tissues were either frozen in Polyfreeze (Poly-sciences Inc., Warrington, PA) and stored at − 80 °C for fro-zen sections or immersion-fixed in 10% buffered formalinand processed by routine methods for paraffin sectioning.Paraffin sections, 6 ? m thick, were stained with hematoxylinand eosin, Gram’s stain, periodic acid-Schiff, Gomori’s tri-chrome, methenamine silver and toluidine blue and exam-ined by light microscopy. Frozen sections were stained forCN-resistant peroxidase activity to specifically detect eosin-ophils [77]. The peroxidase activity of other granulocytes,mast cells and macrophages is not CN resistant and thesecells consequently do not stain. Frozen sections were fixedin acetone and incubated with the standard DAB peroxidasereagent kit from Vector with the addition of 4 drops of 10 mMKCN/5 ml of DAB solution for 20 min followed by hematoxy-lin counter staining.

4.3 Immunohistochemistry

All immunohistochemical staining procedures were per-formed with standard avidin/biotin peroxidase complex pro-cedures using products from Vector Laboratories, Burlin-game, CA. Endogenous peroxidase activity was quenchedin 0.3 % hydrogen peroxide and sections were blocked withavidin D/biotin reagents, followed by 0.5 % casein in PBSwith 1.5 % rat, rabbit or mouse serum as required for appro-priate blocking of nonspecific Ab binding. Primary Ab incu-


bation was either at 4 °C overnight or at room temperaturefor 2 h. Appropriate biotinylated secondary anti-mouse, -rat,-hamster or -rabbit Ab were diluted 1:100 and applied for30 min. Either DAB or AEC reagents were used for visualiza-tion of the immunostaining followed by hematoxylin counter-staining and coverslipping. Frozen blocks were cut at8–10 ? m and after air-drying, sections were fixed in coldacetone prior to immunohistochemical staining. Frozen sec-tions were stained for CD4+ and CD8+ T cells with specificmAb from PharMingen, San Diego, CA (clone RM4–5 diluted1:50 and clone 53-6.7 diluted 1:20, respectively), for MHCclass II expression with biotinylated anti-I-Ab mAb (cloneAF6–120.1 from PharMingen, diluted 1:100), for ICAM-1(clone 3E2 from PharMingen, diluted 1:200), and for keratin6 (gift from M. Blessing diluted 1:1000). Paraffin sectionswere cut and stained for neutrophils (clone 7/4 from HarlanBioproducts for Science Inc., Indianapolis, IN, diluted 1:50)and proliferating cellular nuclear antigen (clone PC10 fromDAKO Diagnostika GmbH, Hamburg, Germany, diluted1:200). For negative control slides the primary Ab was sub-stituted with normal mixed serum. All negative control slideswere free of staining. Micrographs were taken with a ZeissAxioskop and Jenoptik ProgRes 3012 digital camera systemat 3.15x (Fig. 4 A), 6.3x (inset Fig. 4 A), 12.6x (Figs. 1, 3 and4 B), and 25.2x (Figs. 2, 5 and 6) original magnifications.

4.4 RT-PCR analysis of cytokine gene expression

RNA was extracted from skin using peqGOLD TriFastTM

reagent according to the manufacturer’s specifications(Peqlab Biotechnologie GmbH, Erlangen, Germany). Twomicrograms of total RNA were oligo-dT primed and reverse-transcribed using SuperScript IITM from Life Technologies,Rockville, MD. The following PCR primers were used: CCR3,5'-TGC TAC TAT CAC CAG TAT CAT TAC C-3' and 5-GCTTGT TCT TTC CAT TTT CTC ACC-3'; eotaxin 5'-AAC TTCCAT CTG TCT CCC TC-3' and 5-CTC AAT AAT CCC ACATCT CCT TTC-3'; GAPDH 5'-ACC ACA GTC CAT GCC ATCAC-3' and 5'-TCC ACC AC CTG TTG CTG TA-3'; GM-CSF5'-CCC ATC ACT GTC ACC CGG CCT TGG-3' and 5-GTCCGT TTC CGG AGT TGG GGG GC-3'; IFN- + 5'-TAC TGCCAC GGC ACA GTC ATT GAA-3' and 5'-GCA GCG ACTCCT TTT CCG CTT CCT-3'; IL-1 g , 5'-ATG GCA ACT GTTCCT GAA CTC AAC T-3' and 5'-CAG GAC AGG TAT AGATTC TTT CCT TT-3'; IL-2, 5'-GTC AAC AGC GCA CCC ACTTCA AGC-3' and 5'-GCT TGT TGA GAT GAT GCT TTG ACA-3'; IL-4, 6'-ACG GAG ATG GAT GTG CCA AAC GTC-3' and5'-CGA GTA ATC CAT TTG CAT GAT GC-3'; IL-5, 5'-AAGGAT GCT TCT GCA CTT GA-3' and 5'-ACA CCA AGG AACTCT TGC A-3'; IL-6 5'-GAC AAA GCC AGA GTC CTT CAGAGA G-3' and 5'-CTA GGT TTG CCG AGT AGA TCT C-3';TGF- § 5'-GGG GAC AAG AGG ACA AAA GAG-3' and 5'-GGG AAA CAA AAC AAA ACA GTG G-3'; and TNF- § , 5'-ATG AGC ACA GAA AGC ATG ATC-3' and 5'-TAC AGG CTTGTC ACT CGA ATT-3'. Amplification conditions using an MJResearch PTC-255 thermal cycler were 94 °C for 1 min,

60 °C for 1 min, 72 °C for 1 min for 25 cycles in the presenceof 1 ? Ci [ § -32P] dCTP. Amplified products were electropho-retically separated in 6 % polyacrylamide gels. The gelswere dried and quantified with a Fujix BAS 1000 Phosphor-imager. Relative mRNA levels were normalized for GAPDHexpression and samples from wild-type mice were set to 1.For negative controls reverse transcriptase was omitted.

Acknowledgements: We gratefully acknowledge BuketYilmaz, Monika Pech, and Claudia Stoll for excellent techni-cal assistance, Michal Malewicz for helpful suggestions andManfred Blessing for providing anti-keratin 6 Ab. We alsothank Martin Hegen and Hubert Schorle for valuable com-ments on this manuscript and all the staff in the animal facil-ity at the Institute of Toxicology and Genetics.

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Correspondence: Falk Weih, Forschungszentrum Karls-ruhe, Institute of Toxicology and Genetics, P.O. Box 3640,D-76021 Karlsruhe, GermanyFax: +49-7247-823354e-mail: falk.weih — itg.fzk.de


mice lacking the transcription factor relb develop t cell-dependent skin lesions similar to human...

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