darier 's disease ( dd ) is an infrequent   autosomal dominantly inherited skin disorder caused by mutations in the atp2a2 gene , which encodes a calcium pump highly expressed in epidermal keratinocytes . dd is histologically characterized by dyskeratosis and acantholysis , due to an altered keratinization and a loss of adhesion between epidermal keratinocytes . this leads to the development of keratotic papular lesions and plaques in the seborrheic areas of the head , neck , and trunk ; hypertrophic and bullous lesions , as well as mucosal involvement are more rare clinical manifestations of the disease . disfiguring , malodorous lesions covering most of the body may also occur and fatal cases have been reported . the disease runs a chronic course ; topical ( emollients , corticosteroids , retinoids , 5-fluorouracil ) , physical ( excision , electrodessication , dermabrasion , ablative laser , photodynamic therapy ) , as well systemic ( oral antibiotics , retinoids ) therapies are among the treatment options , which , however , are often unsuccessful ; some relief may be obtained with systemic retinoids .       the cell - mediated immunity of dd patients was usually found to be normal . there is , however , a predisposition to bacterial , fungal and viral infections , and defects in cell - mediated immunity have been described , although in a few reports . it is generally believed that infections in dd occur due to the compromised skin integrity , leading to skin denudation and superimposed infections . to our knowledge , there are no studies that characterized cells involved in local cutaneous   immune response in dd patients . we herein performed an immunohistochemical analysis and quantified the inflammatory cell infiltrate in skin biopsies of dd patients , aiming at   investigating a possible pathogenic involvement of the local immune response in the disease . for comparison , lesional skin of patients affected by pemphigus vulgaris ( pv ) , as well as cutaneous lichen ruber planus ( lrp ) we analyzed 16 skin biopsies of patients with darier 's disease ( dd ) , ranging in age from 25 to 50 years ( females outnumbered males from 9 to 7 ) . skin biopsies were performed in recently - appeared , uncomplicated lesions , on grouped keratotic papules of the   abdomen and trunk . there was not any personal nor family history of autoimmune disease .   for comparison , 14 patients with pemphigus vulgaris ( pv ) ,   ageing 3055 years , and 10 with lichen ruber planus ( lrp ) ,   ageing 3251 years the diagnosis was previously established by clinical and   histopathological examination , and , in pv group , also by immunofluorescence studies . as negative control , we examined normal skin ( nsk ) excised from the abdomen of 12 healthy subjects   who underwent   aesthetic surgery . informed consent was obtained from all patients , and the study , performed according to local ethical guidelines , was approved by the local ethical committee . 4-m - thick sections , cut from formalin - fixed ,   paraffin - embedded skin biopsies , were investigated immunohistochemically . in all cases , leukocyte subsets were characterized using   a panel of antibodies , including anticd3 ( cd3 polyclonal antibody ; neomarkers ; san diego , ca ; dilution 1  :  1000 ) , anticd4 ( cd4 polyclonal antibody , 412 ; menarini , florence , italy ; dilution 1  :  50 ) , anticd20 ( cd20 monoclonal antibody , clone l26 ; bio - optica ; milan , italy ; dilution 1  :  150 ) , anticd8 ( cd8 monoclonal antibody , cd8 - 144b clone ; dako ; milan ; dilution 1  :  50 ) , antigranzyme b ( granzyme b monoclonal antibody , gzbo1 clone ; bio - optica ; dilution 1  :  100 ) , anticd25 ( cd25 polyclonal antibody ; bio - optica ; dilution 1  :  50 ; trypsin pretreatment ) antifoxp3 ( foxp3 monoclonal antibody , abcam ; dba , milan , italy ; dilution 1  :  50 ; microwave pretreatment ) , anticd1a ( clone ab-5 010 ; biooptica , milan ; dilution 1  :  50 ; edta pretreatment ; ) , anticd68 ( igg , clone pg - m1 ; dako ; dilution 1  :  300 ) , and   anticd123 ( 6h6 , ebioscience , san diego , ca , dilution 1  :  100 ) . antigen retrieval was obtained   either with wcap buffer ( ph 6 , 98c for 40  min ) , using the ultravision detection system antipolyvalent hrp ( labvision , fremont , ca , u.s.a . ; bio - optica ) ; or with microwave pretreatment , using the ultravision lp detection system ap polymer ( labvision ) . sections were then incubated with the antibodies for 60c min at room temperature . for staining development ,   we used either diaminobenzidine or fuchsin ( dab ; dako ) , as chromogens . we also performed a double staining with antibodies against cd25 and foxp3 to identify the cd4+cd25+foxp3 + subset of regulatory t cells ( tregs ) , as previously described , and against human cd68 and cd123 to identify the cd123 + pdcs . negative controls were obtained by replacing the specific antibody with nonimmune serum immunoglobulins at the same concentration as the primary antibody . sections were then counterstained with harris haematoxylin , dehydrated in alcohol , cleared in xylene and coverslipped . in all samples , immunohistochemical staining was quantified visually by two independent observers ( c.m . and g.m . ) in at least 5 randomly chosen high power fields ( hpf ) ( 40 objective and 10 eyepiece ; 0.16  mm per 1 hpf ) . cd4 + , cd8 + , granzyme b+ , cd20 + , cd25+/foxp3 + , and cd68+cd123 +   cells were counted and recorded as a percentage of the total inflammatory infiltrate . cd1a+ lcs were counted separately in the epidermis and dermis , and given as mean number/1 hpf . mean values + sd of immunostained cells were statistically compared among dd , pv , lrp and nsk by the nonparametric kruskal - wallis test . clinics was typical of dd ( figure 1 ) . at histology , dd cases showed suprabasilar   acantholysis , dyskeratosis , with overlying columns of parakeratosis , and a usually mild , mostly lymphocytic , inflammatory infiltrate . blisters , due to a suprabasilar detachment , and a mild inflammatory infiltrate , with admixed lymphocytes and eosinophils , characterized pv lesions . hyperkeratosis , hypergranulosis , acanthosis and a dense , predominantly lymphocytic , band - like , subepidermal inflammatory infiltrate was observed in lrp samples .         in all three diseases cd3 + t lymphocytes outnumbered cd20 + b lymphocytes ( figure 2 ) .   in dd , cd4 + t lymphocytes constituted the large majority of inflammatory cells , with a minor component of cd8 + t lymphocytes ( figure 2 ) ; very few granzyme b+   lymphocytes were observed . viceversa , in pv and in lrp , high numbers of   t cells were of the cd8 phenotype , and many granzyme b+ positive cells were observable . lymphocytes were rarely observed in the nsk .     in dd the epidermal , as well as the dermal cd1a+ lcs were noticeably decreased in lesional skin when compared with pv and lrp lesions , and nsk ( figure 3 ) . lcs were also smaller in size and with fewer and shorter dendrites , when compared to nsk . in the perilesional dd skin , when documentable in the biopsy , cd1a+ lcs reappeared , although less numerous than those in nsk . in pv the cd1a+ lcs appeared as isolated cells or in small clusters in the epidermis as well as in the dermis ; more numerous than those   in dd , they , however , appeared decreased when compared to lrp and nsk and often reduced in size . in lrp , both epidermal and dermal cd1a+ lcs were , instead , detectable in high numbers , with evident , long dendrites , and often grouped together . dermal   plasmacytoid dendritic cells ( pdcs ) were identifiable based on both their plasmacytoid morphology and positivity for cd123 . cd123   was also expressed   by endothelial cells , and therefore they served as an internal positive control for the anticd123 antibody ( figure 4 ) . cd123 + pdcs were observed , at low percentages , usually single or in groups of two ,   in the dermis in dd ; at higher percentages , preferentially in the dermis and inside the bullae , but also in the epidermis in pv ; in greater numbers , single and , more often , in coalescent groups of cells , in the dermis in lrp . cd4 + t lymphocyte percentages significantly differ from cd8 + percentages in each group of lesions , cd4 + percentages being higher than cd8 + in dd , and lower in pv and lrp . percentages of cd4 + , and cd8 + t lymphocytes did not statistically differ between pv and lrp . in dd , instead , cd8 + and cd4 + lymphocytes were , respectively , significantly lower and significantly higher than those   in pv and lrp . granzyme b lymphocyte percentages were significantly lower in dd than those   in pv and lrp ; and in pv than those   in lrp . mean percentages of cd25+foxp3 + tregs were significantly higher in dd than those in pv and lrp ; they did not differ between dd and lrp .     in dd , epidermal and dermal cd1a+ lc mean number , as well as   cd123 + dc percentage   were significantly lower   than those   in pv , lrp and in the nsk . in pv , epidermal and dermal cd1a+ lc mean numbers cd123 + dc mean percentages were significantly lower in dd than those   in pv and lrp ; they were also significantly lower in pv than   those in lrp . comparison with the nsk was limited to cd1a+ lcs , due to the virtual absence of lymphocytes and plasmacytoid cells in controls . a genetic basis corroborate darier 's disease ( dd ) , an autosomal dominant genodermatosis , with sporadic cases reported , in which an aberration localized to the 12q23 - 24.1 region of chromosome 12 has recently been discovered , leading to a defect in a   keratinocyte calcium pump , which , in turn , results in a loss of keratinocyte adhesion and consequent acantholysis , typical of the disease . autoimmunity is , instead , the main cause of pemphigus vulgaris ( pv ) and lichen ruber planus ( lrp ) , in which altered immune response , as well as skin   inflammatory cell composition , functionality and immune profile have been well documented [ 513 ] . dd and pv are both characterized by epidermal suprabasilar acantholysis , due to loss of adhesion between keratinocytes in the former , and caused by autoantibodies in the latter . despite an ascertained predisposition to bacterial , fungal and viral infections , no specific immunological abnormality has been discovered in dd . infective complications are ascribed only to superimposition of secondary infections on the denuded skin areas . the observation that a usually mild inflammatory infiltrate is , however , detectable also in of recent onset , uncomplicated dd skin lesions , prompted us to characterize it by immunohistochemistry , in order to investigate cell composition of the local immune response ; we also made comparison with inflammatory infiltrate of pv and lrp skin lesions . in the latter conditions , our results are in line with the literature [ 513 ] . to our knowledge , there are no investigations , to date , that characterized the inflammatory infiltrate in dd . in all dd skin lesions we found that it was mostly represented by cd4 + t lymphocytes , with a minor component of cd8 + t lymphocytes , and a negligible percentage of cd20 + b lymphocytes . the most striking finding in all dd cases , in our experience , was a loss of epidermal cd1a+ lcs in the affected acantholytic and dyskeratotic skin areas . they reappeared abruptly in the neighbouring , apparently normal epidermis , although at lower number , when compared to the normal skin ( nsk ) . we also observed low percentages of a subset of dermal dendritic cells , the so - called plasmacytoid dcs , identifiable by cd123 positivity . it has been demonstrated that keratinocyte signals induce lc proliferation ; keratinocytes secrete cytokines that promote lc residence and regulate their immune functions , and it has been suggested that   cytokeratin differentiation provide a microenvironment that determines the density of langerhans cells . the profound defect of keratinocyte differentiation in dd , could , therefore , result in epidermal lc loss in the affected epidermis . basically , epidermal lc functions are largely unknown , however , although debated , their cooperation in generating specific apten - induced effector t cells and stimulating t - cell immunity was demonstrated in some studies [ 1821 ] . lcs were found capable of promoting and regulating t cell  mediated immune response [ 2224 ] , and were potent activators of cd8 + t cells in the skin . epidermal lc reduction or loss might contribute to a cd8-mediated effector local immune response impairment , and , consequently , to a poor response to infective agents in dd . furthermore , since keratinocytes seem to function as accessory cells competent to prime naive skin - reactive t cells , their defect in dd , might directly determine an impaired cd8-mediated effector immune response . it is known that uv irradiation exacerbates dd cutaneous lesions [ 2 , 3 ] : this might partly be due to a direct modulation of keratinocytes by uv to produce proinflammatory cytokines such as interleukin ( il)-1 , il-6 , il-8 and tumour necrosis factor ( tnf)-a , leading to skin inflammation . based on the hypothesis of a haploinsufficiency in dd , it was additionally hypothesized a worsening of cutaneous lesions due to a direct downregulation of the intact allele of the atp2a2 gene by cytokines secreted by uv - irradiated keratinocytes , leading to an altered intracellular distribution of calcium , and , consequently , to a further loss of epidermal cell adhesion , which is modulated by the cellular calcium level . on the other hand , it has been ascertained that uv induces immunosuppression [ 28 , 29 ] , probably through more than one mechanisms and targeting various cell types , including keratinocytes , antigen presenting cells , such as epidermal langerhans cells ( lcs ) , and t lymphocytes . uv halts antigen presentation functions , and , furthermore , expands t cell population with regulatory properties on effector immune cells .        although , at least in experimental studies on mice , a dermal subset of lc rather than epidermal lc was critical in the uv - induced immunosuppression , many studies demonstrated numerical , morphological , phenotypical and functional abnormalities of epidermal lcs following uv exposure [ 2933 ] . uv seems to affect lcs directly or indirectly through suppressive cytokines . in dd skin , uv might , therefore , further on reduce lc number , that we observed both in epidermis and dermis , contributing to a local immunosuppression .         in our study , we also found low percentages of   dermal pdcs , as decorated by cd123 antibody ,   which labels interleukin-3 receptor alpha chain protein , that is expressed at high levels on the surface of pdcs ,   allowing for their identification . the dc system consists of two main subsets : myeloid dcs ( mdcs ) and plasmacytoid dcs ( pdcs ) , and at least five distinct dc subsets have been discovered in the skin . epidermal cd1a+ lcs , dermal cd1a+ lcs and cd14 + dcs are the most known mdcs in the skin , dermal cd1a+ dcs bearing intermediate characteristics between the first two subsets . it was hypothesized that the humoral and the cellular arms of adaptive immunity are preferentially regulated by cd14 + dermal dcs , and lcs , respectively . an emerging role of the plasmacytoid subset of dcs has recently been evidenced in several skin diseases , in which they have been found in high numbers [ 35 , 36 ] . in nsk , cd123 + pdcs are capable of producing type i interferon , and there is evidence that pdcs are the main source of this cytokine in skin inflammation . cd123 + pdcs have in fact been found involved in antiviral immunity and in several autoimmune disorders [ 25 , 3537 ] . interferon - alpha is able to induce the production of chemokines , which , in turn , recruite cd8 + t cells and enhance the expression of cytotoxic mediators by nk cells and cytotoxic lymphocytes [ 38 , 39 ] . a low number of pdcs , as we observed in dd , might result in a low production of interferon , and interferon - inducible chemokines , leading to an explosion of infectious agents , because of inadequate cytotoxic local immune response . we can not , however , assess whether the altered number of cd123 + pdcs that we registered might be ascribed to the complex interactions with other inflammatory cells , including cd1a+ lcs , or to other factors .        in our experience , also in pv , we found impaired numbers of both cd1a+ lcs and cd123 + pdcs . we , in fact , found that in pv , cd1a+ lcs were lower than those   in nsk and lrp , and that   cd123 + pdcs were lower than those   in lrp , although both dc subsets were significantly higher than those   in dd . pv is also characterized by acantholysis , overwhelmingly due to autoimmune mechanisms ; it might be hypothesized an additional contribute of damaged keratinocytes to the decrease of some dc subsets . we , furthermore , observed a different pattern of cd123 + dc distribution ( dermal in dd and lrp , intraepidermal and dermal in pv ) , which is supposed to have pathogenic implications in distinct diseases . in line with other studies , we registered significantly lower numbers of   cd25+foxp3 + regulatory t cells ( treg ) in pv and lrp , whereas , in dd , their percentages were within the range normally reported in inflammatory infiltrates . tregs are a phenotypically heterogeneous population of lymphocytes which are critical for the regulatory mechanisms of immune system , being responsible for the induction and maintenance of immune tolerance . their numbers , and , overall , their suppressive functions were found reduced in autoimmune as well as in allergic diseases [ 8 , 40 , 41 ] . foxp3 is considered the best marker for the most important among treg subsets , the cd4+cd25+foxp3 + cells , that constitute approximately 510% of cd4 t cells in man . the scenario of well - represented cd4 + t infiltrates , with cd25+foxp3 + within the normal range , in front of a low number of effector cells in dd skin , although the presence of inflammatory cells itself does not necessary mirror their functionality , suggest a defect of effector rather than an enhancement of regulatory cells being effective in dd . dendritic cell numerical impairment seems the first cause of a defective local immune   response . in conclusion , our present study provides direct evidence for a numerical impairment of epidermal cd1a+ lcs and dermal cd123 + pdcs in skin lesions of dd , which may be pathogenetically determinant for the development of the inflammatory reactions that complicate the disease . to our knowledge , this investigation represents the first description of an altered composition of the cellular   inflammatory infiltrate in this genetical disorder , suggesting that an impaired local immune response may contribute to the progression of the disease . the dramatic loss and/or absence of some skin dc subsets , such those that we observed , might drive the pathogenic events leading to dd skin complications , suggesting novel therapeutic approaches that could open new horizons for the treatment of this notoriously poorly responsive disease and for a better management of patients . further functional studies , as well as expanding investigation on other cells and cytokine mediators of immune response and inflammation are , of course , mandatory to better elucidate the impact of epidermal lc and dermal cd123 + dc loss in dd lesional microenviroment .
cell - mediated immunity is considered to be normal in darier 's disease ( dd ) , an inherited skin disorder complicated by skin infections . to date 
 , there are no investigations on   the local inflammatory infiltrate in dd skin lesions . in this immunohistochemical study we characterized and quantified it , making comparisons with two other inflammatory skin disorders , that is , pemphigus vulgaris ( pv ) and lichen ruber planus ( lrp ) , and with the normal skin ( nsk ) . 
 we found a significant ( p < .05 ) decrease of cd1a+ langerhans cells ( lcs ) in dd , compared to pv , lrp , and nsk , and of cd123 + plasmacytoid dendritic cells ( pdcs ) , compared to pv and lrp . 
 we hypothesize that the genetic damage of keratinocytes might result in a loss of some subsets of dendritic cells and , consequently , in an impaired local immune response , which might worsen the infections that inevitably occur in this disease .