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  • 8/12/2019 allergen skin challenges.pdf


    Clinical StudyCirculating Conventional and Plasmacytoid Dendritic CellSubsets Display Distinct Kinetics duringIn VivoRepeated

    Allergen Skin Challenges in Atopic Subjects

    Stelios Vittorakis,1,2 Konstantinos Samitas,1,2 Sofia Tousa,2

    Eleftherios Zervas,1 Maria Aggelakopoulou,2 Maria Semitekolou,2

    Vily Panoutsakopoulou,2 Georgina Xanthou,2 and Mina Gaga1

    th Respiratory Department and Asthma Centre, Athens Chest Hospital, Athens , Greece Cellular Immunology Laboratory, Division o Cell Biology, Center or Basic Research,

    Foundation or Biomedical Research o the Academy o Athens, Athens , Greece

    Correspondence should be addressed to Konstantinos Samitas;

    Received February ; Accepted April ; Published April

    Academic Editor: Petros Bakakos

    Copyright Stelios Vittorakis et al. Tis is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

    Upon allergen challenge, DC subsets are recruited to target sites under the inuence o chemotactic agents; however, detailspertinent to their trafficking remain largely unknown. We investigated the kinetic proles o blood and skin-inltrating DC subsetsin twelve atopic subjects receiving six weekly intradermal allergen and diluent injections. Te role o activin-A, a cytokine inducedin allergic and tissue repair processes, on the chemotactic proles o DC subsets was also examined. Plasmacytoid (pDCs) andconventional DCs (cDCs) were evaluated at various time-points in the blood and skin.In situactivin-A expression was assessed inthe skin and its effects on chemokine receptor expression o isolated cDCs were investigated. Blood pDCs were reduced h aferchallenge, while cDCs decreased gradually within h. Skin cDCs increased signicantly h afer the rst challenge, inverselycorrelating with blood cDCs. Activin-A in the skin increased h afer the rst allergen challenge and correlated with inltratingcDCs. Activin-A increased the CCR/CCR expression ratio in cultured human cDCs. DC subsets demonstrate distinct kineticproles in the blood and skin especially during acute allergic inammation, pointing to disparate roles depending on each phaseo the inammatory response. Te effects o activin-A on modulating the chemotactic prole o cDCs suggest it may be a plausibletherapeutic target or allergic diseases.

    1. Introduction

    Allergic diseases, such as asthma and atopic dermatitis, areon the rise in western societies and pose a signicant burdenor patients and health care systems. Allergic inammationrelates to excessive helper type (H2) cell-mediatedresponses against innocuous environmental allergens. Den-dritic cells (DCs) are known sentinels o the immune sys-tem entrusted with the chie task o antigen recognition,presentation, and cell activation. Different DC subsetswith diverse unctionalities have been described, dependingon the level o expression o specic surace markers, theiractivation status, and anatomic location. Tere are two major

    human DC subsets: myeloid or conventional DCs (cDCs),generally considered immunogenic [,], and plasmacytoidDCs (pDCs), which can exhibit suppressive unctions onallergen-driven H2cell-mediated responses []. Pertinent tothe skin, cDCs constitute the major resident DC populationin normal human dermis and are characterized by CDcexpression (also known as blood dendritic cell antigen-(BDCA)-) [,]. Plasmacytoid DCs are present in the skinand can be readily identied by expression o CD, alsoknown as BDCA- [,].

    Increased numbers o DC subsets have been previouslyreported in the blood, nasal, and/or lung mucosa o sub-

    jects with atopy, allergic rhinitis, and/or asthma [],

    Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 231036, 14 pages
  • 8/12/2019 allergen skin challenges.pdf


    BioMed Research International

    suggesting that specic DC subgroups are induced inresponse to allergic inammation. Nevertheless, ew clinicalstudies have addressed DC kinetics in allergic responses,and these involve mostly allergen inhalation or segmentalbronchial allergen challenge []. Understanding o thetrafficking o human DCs upon allergen challenge in vivois

    essential or controlling the balance between immunity andtolerance.DC migration is guided by the rapid upregulation o

    adhesion molecules and chemokine receptors in responseto chemokine and cytokine gradients generated by tissue-resident and inltrating immune cells. Activin-A is apleiotropic cytokine-member o the GF- superamily oproteins. It is produced by inammatory and structuraltissue cells and acts as an important regulator o allergicinammation [] and skin repair processes []. Inact, recent studies have revealed potent anti-inammatoryeffects o activin-A in vivoduring allergen-induced cutaneoussensitization []. Activin-A is produced by different DCsubsets, promotes DC differentiation, and affects the abilityo mature DCs to take up antigens []. In addition, activin-A is involved in the differentiation and migration o humanLangerhans dendritic cells, mostly through the regulation ochemokines and chemokine receptor networks [,]. Still,the effects o activin-A on shaping the chemotactic responseso human DC subsets during exposure to allergen in vitroremain elusive.

    In the present study, we hypothesized that circulatingcDCs and pDCs exhibit different kinetic proles in vivo uponrepeated skin allergen challenges, reecting their distinctroles during acute and established chronic inammation.o explore this, we used a well-established human in vivomodel o repeated skin allergen challenges. Furthermore,we examined activin-A expression in the inamed skin andexplored possible correlations with DC subset inltrationollowing allergen challenge. Finally, the effects o activin-A on the chemokine receptor prole o allergen stimulatedhuman CDc+ DCs were also investigated.

    2. Methods

    .. Study Population and Design. Healthy nonsmoking vol-unteers between the ages o and were initially screenedand subjected to skin prick testing (SP) to a panel o common aeroallergens (HAL Allergy, Benelux). otal blood

    counts, serum biochemistry, total IgE levels measurements,and spirometry were perormed. A total o twelve subjectswith strong positive SP reaction to Dermatophagoides

    pteronyssinus, the European house dust mite, were enrolledin the study. Subject characteristics are summarized inable . All subjects signed an inormed consent orm beoreenrolment. Te study was approved by the Sotiria HospitalResearch Ethics Committee and the Greek National Orga-nization or Medicines and was conducted according to theDeclaration o Helsinki principles.

    All participants were ree o atopic symptoms or at leastone month beore and during the study. Participants hadno history o inection and had not received any treatment

    30 BU intradermal allergen with equal volume diluent controlSkin measurements perormed at 15 min and1 hour

    Days: 30 10 2 8 15 22 29 36 37

    Screening period

    Allergen and diluent skin biopsy

    K3EDTA collected whole blood sampleFACS-DC acquisition

    F : Study design. Flow diagram showing the time-pointswhen the skin challenges with allergen and diluent were perormedand when the samples were taken. Te dose oDermatophagoidespteronyssinusadministered was BU at the allergen site with equalvolume o diluent at the opposing site every week (solid arrows).A screening period o weeks was introduced to clinically veriythat subjects did not exhibit seasonal allergic symptoms or anupper/lower respiratory inection.

    with oral/inhaled corticosteroids, antihistamines, anti-IgE,or antileukotrienes or one month beore and during thestudy. Each subject received a total o six weekly intradermalinjections o allergen, to which they were sensitive, anddiluent on the extensor aspect o the lef and right orearms,respectively. Skin biopsies were obtained h afer the rstchallenge and h afer the last challenge at both allergenand diluent sites. Whole blood was taken beore the rstchallenge, h afer the rst challenge, and just beore eachbiopsy acquisition. Te study design is depicted inFigure .

    .. In Vivo Allergen Challenges. A -gauge needle wasused to deliver L ( BU) o allergen aqueous solution

    (Dermatophagoides pteronyssinus, Allergopharma JoachimGanzer KG, Germany) intradermally at the same site onthe extensor aspect o the lef orearm. Te same diluent

    volume (.% sodium chloride) was administered in thesame manner on the extensor aspect o the right orearm.In this way, each patient served as his/her own control.Te challenge tests were perormed at the same time eachday. Measurements o skin reactions were perormed at min, h, and h, as previously described [], by a singleinvestigator throughout the study.

    .. Preparation o Skin Biopsies. Skin specimens wereobtained rom the centre o both allergen and diluent sites

    in each subject using a mm punch biopsy tool (StieelLaboratories). Local anaesthesia was induced by injectingsubcutaneously . mL o % lidocaine hydrochloride. issuesamples rom the same arm afer the last challenge weretaken . cm apart. Afer appropriate orientation and han-dling, specimens were cut in hal and one o the resultingtissue samples was embedded in OC medium and snap-rozen in isopentane (BDH Chemicals) precooled in liquidnitrogen, while the other was xed in ormalin overnight andembedded in paraffin. Frozen specimens were stored at C.

    .. Immunohistochemistry. Paraffin sections - mmthick were deparaffinised in xylol, rehydrated in graded

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