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Expression of Epidermal CAMP Changes in Parallel with Permeability Barrier Status

  • Author Footnotes
    7 These three authors contributed equally to this work.
    Marina Rodriguez-Martin
    Footnotes
    7 These three authors contributed equally to this work.
    Affiliations
    Dermatology Service, Hospital Universitario de Canarias, University of La Laguna, Tenerife, Spain

    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Author Footnotes
    7 These three authors contributed equally to this work.
    Gemma Martin-Ezquerra
    Footnotes
    7 These three authors contributed equally to this work.
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA

    Department of Dermatology, Hospital del Mar-IMIM, Universitat Autonoma de Barcelona, Barcelona, Spain
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  • Author Footnotes
    7 These three authors contributed equally to this work.
    Mao-Qiang Man
    Footnotes
    7 These three authors contributed equally to this work.
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Melanie Hupe
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Jong-Kyung Youm
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Donald S. Mackenzie
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Soyun Cho
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA

    Department of Dermatology, Seoul National University Boramae Hospital, Seoul, South Korea
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  • Carles Trullas
    Affiliations
    ISDIN, Barcelona, Spain
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  • Walter M. Holleran
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Katherine A. Radek
    Affiliations
    Department of Surgery, Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, Illinois, USA
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  • Peter M. Elias
    Correspondence
    Dermatology Service, Department of Veterans Affairs Medical Center, UCSF, 4150 Clement Street, San Francisco, California, USA
    Affiliations
    Dermatology Service, Department of Veterans Affairs Medical Center, and Department of Dermatology, University of California, San Francisco, San Francisco, California, USA
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  • Author Footnotes
    7 These three authors contributed equally to this work.
      Two critical defensive functions of the outer epidermis, the permeability barrier and antimicrobial defense, share certain structural and biochemical features. Moreover, three antimicrobial peptides (AMPs), i.e., mouse β-defensin 3 (mBD3), mouse cathelicidin antimicrobial peptide (mCAMP), and the neuroendocrine peptide, catestatin (Cst), all localize to the outer epidermis, and both mBD3 and mCAMP are secreted from the epidermal lamellar bodies with other organelle contents that subserve the permeability barrier. These three AMPs are upregulated in response to acute permeability barrier disruption, whereas conversely, mCAMP-/- mice (unable to combat Gram-positive pathogens) also display abnormal barrier homeostasis. To determine further whether these two functions are co-regulated, we investigated changes in immunostaining for these three AMPs in skin samples in which the permeability barrier function in mice had been either compromised or enhanced. Compromised or enhanced barrier function correlated with reduced or enhanced immunohistochemical expression of mCAMP, respectively, but conversely with Cst expression, likely due to the role of this AMP as an endogenous inhibitor of cathelicidin expression. mBD3 expression correlated with experimental barrier perturbations, but poorly with developmental changes in barrier function. These studies show that changes in cathelicidin and Cst expression parallel changes in permeability barrier status, with a less clear relationship with mBD3 expression.

      Abbreviations

      AD
      atopic dermatitis
      AMP
      antimicrobial peptide
      Cst
      catestatin
      IMQ
      imiquimod
      mCAMP
      mouse cathelicidin antimicrobial peptide
      PS
      psychological stress
      SC
      stratum corneum

      Introduction

      The stratum corneum (SC) of mammalian epidermis mediates several critical protective functions (
      • Elias P.M.
      Stratum corneum defensive functions: an integrated view.
      ), two of which, maintenance of permeability barrier homeostasis and cutaneous antimicrobial defense (distal innate immunity), exhibit certain physical–chemical and biochemical features that contribute simultaneously to both functions (Supplementary Table S1 online) (reviewed in
      • Elias P.M.
      The skin barrier as an innate immune element.
      ,
      • Elias P.M.
      • Steinhoff M.
      “Outside-to-inside” (and now back to “outside”) pathogenic mechanisms in atopic dermatitis.
      , and
      • Elias P.M.
      • Schmuth M.
      Abnormal skin barrier in the etiopathogenesis of atopic dermatitis.
      ). For example, the low pH of the SC creates an ecological milieu that is hostile to microbial pathogens, while simultaneously favoring growth of the normal flora (
      • Aly R.
      • Shirley C.
      • Cunico B.
      • et al.
      Effect of prolonged occlusion on the microbial flora, pH, carbon dioxide and transepidermal water loss on human skin.
      ;
      • Korting H.C.
      • Hubner K.
      • Greiner K.
      • et al.
      Differences in the skin surface pH and bacterial microflora due to the long-term application of synthetic detergent preparations of pH 5.5 and pH 7.0. Results of a crossover trial in healthy volunteers.
      ). Moreover, the highly cohesive and anhydrous characteristics of the normal SC comprise a formidable physical barrier to invading microorganisms, whereas, conversely, pathogens invade between dyshesive corneocytes when the permeability barrier is compromised (
      • Miller S.J.
      • Aly R.
      • Shinefeld H.R.
      • et al.
      In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids.
      ;
      • Elias P.M.
      The skin barrier as an innate immune element.
      ). Furthermore, certain lipids that are required for the permeability barrier, such as free fatty acids of both epidermal (
      • Miller S.J.
      • Aly R.
      • Shinefeld H.R.
      • et al.
      In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids.
      ;
      • Drake D.R.
      • Brogden K.A.
      • Dawson D.V.
      • et al.
      Thematic review series: skin lipids. Antimicrobial lipids at the skin surface.
      ) and sebaceous (
      • Bibel D.J.
      • Miller S.J.
      • Brown B.E.
      • et al.
      Antimicrobial activity of stratum corneum lipids from normal and essential fatty acid-deficient mice.
      ;
      • Georgel P.
      • Crozat K.
      • Lauth X.
      • et al.
      A toll-like receptor 2-responsive lipid effector pathway protects mammals against skin infections with Gram-positive bacteria.
      ) origin, as well as the sphingoid bases of ceramides, also exhibit potent antimicrobial activity. Thus, the increased residence of Staphylococcus aureus and other pathogens on lesions of atopic dermatitis (AD) could be explicable not only by alterations in barrier function and innate immunity (
      • Radek K.
      • Gallo R.
      Antimicrobial peptides: natural effectors of the innate immune system.
      ), but also by the (i) high pH (cited in
      • Hatano Y.
      • Man M.Q.
      • Uchida Y.
      • et al.
      Maintenance of an acidic stratum corneum prevents emergence of murine atopic dermatitis.
      ); (ii) lipid-depleted extracellular matrix (
      • Chamlin S.L.
      • Kao J.
      • Frieden I.J.
      • et al.
      Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: changes in barrier function provide a sensitive indicator of disease activity.
      ); (iii) reduced free fatty acid/sphingosine content (
      • Arikawa J.
      • Ishibashi M.
      • Kawashima M.
      • et al.
      Decreased levels of sphingosine, a natural antimicrobial agent, may be associated with vulnerability of the stratum corneum from patients with atopic dermatitis to colonization by Staphylococcus aureus.
      ;
      • Proksch E.
      • Jensen J.M.
      • Elias P.M.
      Skin lipids and epidermal differentiation in atopic dermatitis.
      ); and (iv) poor cohesion (
      • Cork M.J.
      • Robinson D.A.
      • Vasilopoulos Y.
      • et al.
      New perspectives on epidermal barrier dysfunction in atopic dermatitis: gene–environment interactions.
      ) of the SC in lesional AD. Notably, the cathelicidin protein, hCAP18, and its carboxy-terminal peptide, LL-37, also are downregulated in lesional AD, which is explicable by the increased T helper type 2 signaling (
      • Howell M.D.
      The role of human beta defensins and cathelicidins in atopic dermatitis.
      ) and/or excess serine protease activity (
      • Morizane S.
      • Yamasaki K.
      • Kabigting F.D.
      • et al.
      Kallikrein expression and cathelicidin processing are independently controlled in keratinocytes by calcium, vitamin D(3), and retinoic acid.
      ).
      The link between permeability barrier status and antimicrobial defense is shown not only by their shared physical and biochemical characteristics, but also by the fact that acute perturbations in permeability barrier function stimulate metabolic responses that rapidly restore permeability barrier homeostasis in parallel with enhanced antimicrobial peptide (AMP) expression, e.g., mouse cathelicidin AMP (mCAMP), mouse β-defensin 3 (mBD3), catestatin (Cst), RNase 7, and psoriasin production all increase rapidly after acute barrier disruption (
      • Elias P.M.
      • Choi E.H.
      Interactions among stratum corneum defensive functions.
      ;
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ;
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ;
      • Glaser R.
      • Meyer-Hoffert U.
      • Harder J.
      • et al.
      The antimicrobial protein psoriasin (S100A7) is upregulated in atopic dermatitis and after experimental skin barrier disruption.
      ). Conversely, mCAMP knockout mice display abnormal permeability barrier homeostasis, demonstrating that cathelicidins are required for normal permeability barrier function (
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ). Notably, both the lipids that mediate permeability barrier function (
      • Grayson S.
      • Johnson-Winegar A.G.
      • Wintroub B.U.
      • et al.
      Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization.
      ), and at least three AMPs, i.e., mCAMP (LL-37), mBD3(hBD2), and Cst, were expressed in the outer epidermis. Moreover, both mCAMP (LL-37) and mBD3 (hBD2) are cargoes within the epidermal lamellar bodies (
      • Oren A.
      • Ganz T.
      • Liu L.
      • et al.
      In human epidermis, beta-defensin 2 is packaged in lamellar bodies.
      ;
      • Braff M.H.
      • Di Nardo A.
      • Gallo R.L.
      Keratinocytes store the antimicrobial peptide cathelicidin in lamellar bodies.
      ;
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). Hence, their colocalization and presumed co-secretion insures that constituents of both the permeability and antimicrobial barriers are delivered in parallel to SC extracellular domains.
      Our results suggest close, bidirectional changes in mCAMP expression under a variety of conditions where permeability barrier function is either compromised or enhanced, but an apparent, converse relationship with Cst expression, which could reflect its function as a β-muscarinic inhibitor of cathelicidin expression (
      • Radek K.A.
      • Elias P.M.
      • Taupenot L.
      • et al.
      Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
      and cited therein).

      Results

      Permeability barrier status in various models

      In normal mice, acute abrogations of the epidermal permeability barrier function, induced by either organic solvent applications or repeated tape strippings, provoke a transient decline in AMP levels, followed by rapid upregulation of the expression of several AMPs, i.e., mCAMP, mBD3, Cst, and psoriasin, over 2–6 hours in parallel with barrier restoration (
      • Schroder J.M.
      • Harder J.
      Antimicrobial skin peptides and proteins.
      ;
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ;
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ;
      • Glaser R.
      • Meyer-Hoffert U.
      • Harder J.
      • et al.
      The antimicrobial protein psoriasin (S100A7) is upregulated in atopic dermatitis and after experimental skin barrier disruption.
      ). In these studies, we assessed changes in AMP expression in four situations in which permeability barrier homeostasis is subnormal, i.e., after sustained psychological stress (PS) (
      • Denda M.
      • Tsuchiya T.
      • Hosoi J.
      • et al.
      Immobilization-induced and crowded environment-induced stress delay barrier recovery in murine skin.
      ;
      • Choi E.H.
      • Demerjian M.
      • Crumrine D.
      • et al.
      Glucocorticoid blockade reverses psychological stress-induced abnormalities in epidermal structure and function.
      ), in young adult male mice (testosterone replete) (
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      ), after erythemogenic UVB exposure (
      • Haratake A.
      • Uchida Y.
      • Schmuth M.
      • et al.
      UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
      ), and in chronologically (intrinsically)aged epidermis (
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      ). As our previous studies showed that PS downregulates both mCAMP and mBD3 expression (
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ), samples from PS mice served as positive controls for the other models. AMP status also was assessed in a library of paraffin-embedded materials from our previously published studies where permeability barrier homeostasis had been altered either experimentally or developmentally (
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      ) (Table 1) in testosterone-replete and chronologically aged mice. In these studies, erythemogenic UVB induced a dose- and time-dependent abnormality in permeability barrier function (see below), as reported previously (
      • Haratake A.
      • Uchida Y.
      • Schmuth M.
      • et al.
      UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
      ).
      Table 1Changes in barrier function in various mouse models
      Basal barrier functionBarrier recovery kinetics
      Barrier perturbant
       Psychological stressDeclines
      Denda et al. (2000).
      ,
      Choi et al. (2006).
      Delayed
      Denda et al. (2000).
      ,
      Choi et al. (2006).
       Testosterone replete (male)Declines
      Kao et al. (2001).
      Delayed
      Kao et al. (2001).
       Erythemogenic UVB (5–10 MED)See Figure 5b
      Haratake et al. (1997).
      Delayed
      Haratake et al. (1997).
       Intrinsic agingDeclines
      Ghadially et al. (1995).
      ,
      Choi et al. (2007).
      Delayed
      Choi et al. (2007).
      Improved barrier
       Suberythemogenic UVBImproves
      Hong et al. (2008).
      Accelerates
      Hong et al. (2008).
       CalcipotriolImproves
      Bikle et al. (2010).
      N/D
       Endogenous GC blockadeImproves
      Aberg et al. (2007).
      Accelerates
      Denda et al. (2000).
      ,
      Choi et al. (2006).
       ImiquimodImproves
      Barland et al. (2004).
      Accelerates
      Barland et al. (2004).
       Triple lipidsImproves
      Man et al. (1995). Arch Dermatol 131:809–16.
      Accelerates
      Man et al. (1995). Arch Dermatol 131:809–16.
       PetrolatumN/DAccelerates
      Man et al. (1995). Arch Dermatol 131:809–16.
       PPARαNo changesAccelerates
      Man et al. (2004).
       LXRNo changesPartially normalizes
      Komuves et al. (2002). J Invest Dermatol 118:25–34.
       Chinese herbal mixtureNo changesAccelerates
      Man et al. (2011).
       UreaImproves
      Grether-Beck et al. (2011).
      N/D
      Abbreviations: GC, glucocorticoid; LXR, liver X receptor; MED, minimal erythema doses; N/D, not demonstrated; PPAR, PP activated receptor.
      1
      • Denda M.
      • Tsuchiya T.
      • Elias P.M.
      • et al.
      Stress alters cutaneous permeability barrier homeostasis.
      .
      2
      • Choi E.H.
      • Demerjian M.
      • Crumrine D.
      • et al.
      Glucocorticoid blockade reverses psychological stress-induced abnormalities in epidermal structure and function.
      .
      3
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      .
      4
      • Haratake A.
      • Uchida Y.
      • Schmuth M.
      • et al.
      UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
      .
      5
      • Ghadially R.
      • Brown B.E.
      • Sequeira-Martin S.M.
      • et al.
      The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.
      .
      6
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      .
      7
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      .
      8
      • Bikle D.D.
      • Teichert A.
      • Arnold L.A.
      • et al.
      Differential regulation of epidermal function by VDR coactivators.
      .
      9
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      .
      10
      • Barland C.O.
      • Zettersten E.
      • Brown B.S.
      • et al.
      Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis.
      .
      11 Man et al. (1995). Arch Dermatol 131:809–16.
      12
      • Komuves L.G.
      • Schmuth M.
      • Fowler A.J.
      • et al.
      Oxysterol stimulation of epidermal differentiation is mediated by liver X receptor-beta in murine epidermis.
      .
      13 Komuves et al. (2002). J Invest Dermatol 118:25–34.
      14
      • Man M.
      • Hupe M.
      • Mackenzie D.
      • et al.
      A topical Chinese herbal mixture improves epidermal permeability barrier function in normal murine skin.
      .
      15
      • Grether-Beck S.
      • Felsner I.
      • Brenden H.
      • et al.
      Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression.
      .

      Compromised permeability barrier function correlates closely with decreased mCAMP expression

      Psychological stress

      As reported previously, immunostaining for both mCAMP and mBD3 declined following PS (Figure 1a; see also
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). Moreover, we now show further that Cst immunostaining also declines after short-term PS (i.e., 24–36 hours), but Cst instead appears to normalize, or even supernormalize, following exposure to more prolonged periods of PS (4 days of restraint) (Figure 1b).
      Figure thumbnail gr1
      Figure 1Psychological stress (PS) decreases immunostaining for mouse cathelicidin antimicrobial peptide (mCAMP), β-defensin 3 (mBD3), and catestatin (Cst) in both a glucocorticoid (GC)- and a β-adrenergic-dependent manner. Hairless mice (n=4 or 5 each) were exposed to either insomnia-induced PS for 36–48 hours (short-term PS, PS-ST) or restraint-induced stress for 96 hours (long-term PS, PS-LT), while parallel groups of PS mice (n=4 or 5 each) were co-treated with intraperitoneal antalarmin or Ru486 (not shown; see
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ), or topical timolol (T) (0.38% in saline) (see Materials and Methods for further details). In all, 5 μM frozen sections were labeled with primary antibodies against mCAMP, mBD3, or Cst. Propidium iodide was used to counterstain the nuclei. Green immunostaining represents AMP labeling. Bar=40 μm. A, antalarmin; N, normal; red staining, propidium iodide.

      Androgen status (gender)

      Previous studies have shown that testosterone-replete (adult) mice and humans display normal basal barrier function, but delayed permeability barrier recovery (
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      ) (Table 1). Therefore, we next compared epidermal mCAMP, mBD3, and Cst immunostaining in library skin samples from young adult male versus female mice. Although male mice displayed a marked decline in immunostaining for mCAMP, they appeared to display a modest enhancement of immunostaining for mBD3, and a marked increase in Cst expression (Supplementary Figure S1 online). These results suggest that the decline in permeability barrier with testosterone repletion is paralleled by a concomitant reduction in mCAMP, whereas mBD3 and Cst expression instead appear to increase in androgen-replete males.

      Erythemogenic UVB

      Although suberythemogenic doses of UVB have been shown previously to enhance permeability barrier function (
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      ), erythemogenic UVB instead provokes a transient, delayed (by 48–96 hours), and dose-dependent barrier abnormality, as we reported previously (
      • Haratake A.
      • Uchida Y.
      • Schmuth M.
      • et al.
      UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
      ) (Figure 2). Therefore, we next examined whether erythemogenic UVB produces parallel alterations in AMP expression in mice. In these studies, the intensity of AMP immunostaining was quantitated by a blinded observer on multiple, pooled, coded images at each time point. Erythemogenic UVB (5 minimal erythema doses (MED)) resulted in a progressive decline in mCAMP levels, which returned to normal at day 5 (Figure 2; Supplementary Figure S2A online). In contrast, erythemogenic UVB did not alter mBD3 immunostaining (Supplementary Figure S2B online), whereas it simultaneously stimulated a sustained increase in Cst expression immediately after exposure, with immunostaining remaining elevated until day 5, when it began to decline (Figure 2; Supplementary Figure S2C online). Taken together, these results suggest that the transient defect in permeability barrier function, which resulted from erythemogenic UVB irradiation, is paralleled by a marked decline in mCAMP, a minimal decline in mBD3, but a marked enhancement of Cst expression.
      Figure thumbnail gr2
      Figure 2Quantitation of decline in mouse cathelicidin antimicrobial peptide (mCAMP) immunostaining parallels development of a permeability barrier abnormality. (a) UVB-induced changes in permeability barrier function are modified from
      • Haratake A.
      • Uchida Y.
      • Schmuth M.
      • et al.
      UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
      . (b) Micrographs (≥10 each) from mice treated with erythemogenic UVB (n=4, as in ) were coded, randomized, and graded according to the intensity of staining for mCAMP, β-defensin 3 (mBD3), and catestatin (Cst) by a blinded observer. AMP, antimicrobial peptide; MED, minimal erythema doses; TEWL, transepidermal water loss.

      Chronologically aged mouse skin

      Permeability barrier homeostasis progressively declines during chronologic aging (
      • Ghadially R.
      • Brown B.E.
      • Sequeira-Martin S.M.
      • et al.
      The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.
      ;
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      ) (Table 1). Therefore, we next examined age-related abnormalities in AMP expression in library tissue samples from young versus moderately aged mouse epidermis (15–18 months), analogous to human age 50–65 years (
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      ). Under basal conditions, the epidermis of young mice displayed low constituent levels of immunostaining for both mCAMP and mBD3, with a prominent decline in mCAMP immunostaining in chronologically aged mouse epidermis. In contrast, both mBD3 and Cst levels appeared to markedly increase in aged mouse epidermis (Supplementary Figure S3 online).

      Improved permeability barrier function correlates with enhanced mCAMP expression

      Imiquimod and calcipotriol treatment

      Both the immune enhancer, imiquimod (IMQ), and the 1,25(OH)2 vitamin D3 analog, calcipotriol, improve barrier function under a variety of experimental and clinical conditions (
      • Barland C.O.
      • Zettersten E.
      • Brown B.S.
      • et al.
      Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis.
      ). Therefore, we next delineated the effects of repeated applications of topical IMQ or calcipotriol on mCAMP expression in normal mouse epidermis. Untreated murine epidermis again clearly demonstrated low, but readily detectable immunostaining for both mCAMP and mBD3, localized to the outer epidermis (Aberg et al., 2007, 2008). Although both IMQ and calcipotriol treatments appeared to increase immunostaining for mBD-3 and mCAMP in comparison to vehicle alone, the increase in mCAMP appeared to be greater than that achieved in parallel, calcipotriol-treated mice (Supplementary Figure S4A vs. S4B online). The increase in mCAMP and mBD3 in calcipotriol- and IMQ-treated mice displays a linear pattern in the SC, corresponding to membrane domains, and it also further localized to vesicles in the cytosol of stratum granulosum cells (Supplementary Figure S4B online, inset, arrows), consistent with its known localization in epidermal lamellar bodies (
      • Oren A.
      • Ganz T.
      • Liu L.
      • et al.
      In human epidermis, beta-defensin 2 is packaged in lamellar bodies.
      ;
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). Finally, we examined AMP expression after several other unrelated maneuvers previously shown to enhance barrier function. In each of these examples, mCAMP expression inevitably increased, but mBD3 and Cst did not always change in parallel (Table 2). Taken together, these results demonstrate first that IMQ and calcipotriol treatment appear to increase the expression of both mCAMP and mBD3 in the outer epidermis. Second, several other, unrelated approaches that improve barrier function also enhance mCAMP expression, with more variable results for mBD3 and Cst (not shown).
      Table 2Changes in antimicrobial peptide expression in relation to altered barrier function
      AMP expression
      Permeability barrier statusmCAMPmBD3Cst
      Decreased
       PS
       Exogenous GCN/D
       Testosterone repleteNo changeNo change
       Erythemogenic UVB(↓)↑↑
       Aging
      Increased
       PS+Ru486/antalarmin
      Aberg et al. (2007).
       Suberythemogenic UVB
      Hong et al. (2008).
      Hong et al. (2008).
      N/D
       ImiquimodN/D
       Chinese herbal medicineN/D
       CalcipotriolN/D
       UreaN/D
      Abbreviations: AMP, antimicrobial peptide; Cst, catestatin; GC, glucocorticoid; mBD3, mouse β-defensin 3; mCAMP, mouse cathelicidin antimicrobial peptide; N/D, not demonstrated; PS, psychological stress.
      1
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      .
      2
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      .

      Discussion

      We addressed here the hypothesis that permeability barrier function and antimicrobial defense are integrated and co-regulated functions (
      • Elias P.M.
      Stratum corneum defensive functions: an integrated view.
      ), examining whether experimental perturbations or developmental changes that either reduce or enhance permeability barrier status are accompanied by parallel changes in epidermal AMP expression. The impetus for these studies came first from a previous work that showed that these two functions are co-regulated and interdependent in the normal epidermis (
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ;
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      ;
      • Proksch E.
      • Brandner J.M.
      • Jensen J.M.
      The skin: an indispensable barrier.
      ) and that at least one perturbant of the permeability barrier (PS) downregulates mBD3 and mCAMP expression (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). Several studies have already shown that the converse is true; e.g., epidermal AMP expression, including Cst expression (
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ), increased after acute barrier insults in parallel with barrier recovery (
      • Elias P.M.
      • Choi E.H.
      Interactions among stratum corneum defensive functions.
      ;
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ) and after blockade of both glucocorticoid production and action in PS mice (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). We extend these previous observations here by showing not only that short-term PS reduces mCAMP (LL-37) and mBD3 (hBD2) expression, but also that as PS is prolonged, Cst expression also begins to decline.
      Our results provide several additional examples to support a putative relationship between permeability barrier and antimicrobial status, at least for mCAMP. Testosterone depletion by either surgical or medical means improves permeability barrier function, whereas conversely, testosterone-replete mice and humans display diminished permeability barrier function (
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      ). Although we showed here an apparent, parallel decline in mCAMP expression in male versus female mice, immunostaining for both mBD3 and Cst instead appeared to increase in the epidermis of adult male mice. Thus, it is possible that changes in androgen status could impose potentially important variations in cutaneous antimicrobial defense.
      Suberythemogenic doses of UVB have been shown to upregulate permeability barrier function and mBD3/mCAMP expression simultaneously (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ), further supporting a putative relationship between these two functions. Conversely, we now show that erythemogenic doses of UVB that compromise permeability barrier function (
      • Haratake A.
      • Uchida Y.
      • Schmuth M.
      • et al.
      UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
      ) also markedly appear to downregulate mCAMP, but produce only minimal, transient alteration in mBD3 expression. The progressive decline (and recovery) of mCAMP expression parallels the time course over which the permeability barrier defect evolves and then recovers. Yet, Cst expression instead appeared to increase at all time points after erythemogenic UVB irradiation. Our previous studies showed that the UVB-induced permeability barrier abnormality correlates with passage of a band of secretion-incompetent, apoptotic cells through the stratum granulosum–SC interface (
      • Holleran W.M.
      • Uchida Y.
      • Halkier-Sorensen L.
      • et al.
      Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function.
      ). Although such a toxic mechanism could contribute to the observed decline in production of mCAMP, it certainly did not impede Cst expression. Thus, toxicity alone likely cannot account for the selective decline in mCAMP expression after UVB irradiation. The anti-parallel changes in Cst can be explained instead by its role as a muscarinic inhibitor of cathelicidin expression (
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ). Thus, these studies further support a close link between UVB-induced changes in permeability barrier function and cathelicidin expression. Furthermore, together with the work of
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      on suberythemogenic UVB, these studies provide potential clinical implications on how UVB irradiation should be deployed for the treatment of inflammatory dermatoses. Although current recommendations propose “pushing” UVB phototherapy doses upwards into the erythemogenic range, this approach clearly could pose adverse consequences not only for permeability barrier function but also for cutaneous antimicrobial defense.
      Permeability barrier function begins to decline in adult humans above the age of 50 years (
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      ), becoming further compromised above age 75 years (
      • Ghadially R.
      • Brown B.E.
      • Sequeira-Martin S.M.
      • et al.
      The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.
      ), and/or with superimposed photoaging (
      • Reed J.T.
      • Elias P.M.
      • Ghadially R.
      Integrity and permeability barrier function of photoaged human epidermis.
      ). We showed here that 15–18-month-old mice, analogous to humans over age 50 years, displayed reduced mCAMP levels, whereas both mBD3 and Cst immunostaining instead appeared to increase in this age group (Table 2). In attempting to explain these and other divergent results for mCAMP versus mBD3, it should be noted that these families of AMPs are regulated by entirely different mechanisms (
      • Oren A.
      • Ganz T.
      • Liu L.
      • et al.
      In human epidermis, beta-defensin 2 is packaged in lamellar bodies.
      ;
      • Braff M.H.
      • Di Nardo A.
      • Gallo R.L.
      Keratinocytes store the antimicrobial peptide cathelicidin in lamellar bodies.
      ;
      • Choi E.H.
      • Brown B.E.
      • Crumrine D.
      • et al.
      Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity.
      ;
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ;
      • Peric M.
      • Koglin S.
      • Kim S.M.
      • et al.
      IL-17A enhances vitamin D3-induced expression of cathelicidin antimicrobial peptide in human keratinocytes.
      ;
      • Eyerich K.
      • Pennino D.
      • Scarponi C.
      • et al.
      IL-17 in atopic eczema: linking allergen-specific adaptive and microbial-triggered innate immune response.
      ). Although endogenous 1,25(OH)2 vitamin D3 and other VDR ligands regulate cathelicidin expression (
      • Zasloff M.
      Sunlight, vitamin D, and the innate immune defenses of the human skin.
      ;
      • Elias P.M.
      The skin barrier as an innate immune element.
      ;
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ;
      • Drake D.R.
      • Brogden K.A.
      • Dawson D.V.
      • et al.
      Thematic review series: skin lipids. Antimicrobial lipids at the skin surface.
      ;
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      ;
      • Schauber J.
      • Gallo R.L.
      The vitamin D pathway: a new target for control of the skin's immune response?.
      ), a variety of cytokines instead stimulate β-defensin production (
      • Nomura I.
      • Goleva E.
      • Howell M.D.
      • et al.
      Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.
      ;
      • Elias P.M.
      • Choi E.H.
      Interactions among stratum corneum defensive functions.
      ;
      • de Jongh G.J.
      • Zeeuwen P.L.
      • Kucharekova M.
      • et al.
      High expression levels of keratinocyte antimicrobial proteins in psoriasis compared with atopic dermatitis.
      ;
      • Yano S.
      • Banno T.
      • Walsh R.
      • et al.
      Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1.
      ;
      • Kobayashi M.
      • Yoshiki R.
      • Sakabe J.
      • et al.
      Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes.
      ). Accordingly, endogenous vitamin D levels typically decline with age (
      • Holick M.F.
      Photosynthesis of vitamin D in the skin: effect of environmental and life-style variables.
      ), perhaps accounting for the decrease in mCAMP levels that were observed here in aged murine epidermis. In contrast, cytokine levels vary widely during aging (
      • Ye J.
      • Calhoun C.
      • Feingold K.
      • et al.
      Age-related changes in the IL-1 gene family and their receptors before and after barrier abrogation.
      ;
      • Corsini E.
      • Racchi M.
      • Lucchi L.
      • et al.
      Skin immunosenescence: decreased receptor for activated C kinase-1 expression correlates with defective tumour necrosis factor-alpha production in epidermal cells.
      ), but IL-1α levels in particular decline with chronologic aging, and are associated with decreased epidermal lipid production (
      • Ye J.
      • Calhoun C.
      • Feingold K.
      • et al.
      Age-related changes in the IL-1 gene family and their receptors before and after barrier abrogation.
      ;
      • Barland C.O.
      • Elias P.M.
      • Ghadially R.
      The aged epidermal barrier: basis for functional abnormalities.
      ). In contrast, other epidermal cytokines (e.g., TNFα) increase in the aged epidermis (
      • Corsini E.
      • Racchi M.
      • Lucchi L.
      • et al.
      Skin immunosenescence: decreased receptor for activated C kinase-1 expression correlates with defective tumour necrosis factor-alpha production in epidermal cells.
      ), consistent with our observation that mBD3 immunostaining persists or even increases in moderately aged mouse skin. The basis for the apparent, age-related increase in Cst expression is unclear at present, but it could again be related to the role of this neuropeptide as an endogenous inhibitor of cathelicidin production. Whether further abnormalities in antimicrobial defense occur in the moderately aged epidermis and/or with still more-advanced aging and/or photoaging is not yet known. Nevertheless, these age-related differences in AMP expression, which do not strictly parallel changes in permeability barrier status, could also have important clinical implications, as they suggest that cutaneous antimicrobial defense becomes compromised relatively early during the aging process.
      We also examined here the opposite situation, asking whether maneuvers that are known to enhance barrier function also upregulate AMP expression. The immune response modifier, IMQ, acts through two members of the Toll-like receptor family, Toll-like receptor 7 and/or 8, which recognize microbial pathogens or their metabolic products and function as primary sensors of the innate immune system (
      • Ambach A.
      • Bonnekoh B.
      • Nguyen M.
      • et al.
      Imiquimod, a Toll-like receptor-7 agonist, induces perforin in cytotoxic T lymphocytes in vitro.
      ;
      • Sauder D.N.
      Mechanism of action and emerging role of immune response modifier therapy in dermatologic conditions.
      ;
      • Lai Y.
      • Gallo R.L.
      Toll-like receptors in skin infections and inflammatory diseases.
      ). These Toll-like receptors are cell surface receptors that, when activated, stimulate production of epidermis-derived, IFN-α, TNF, and IL-1α (
      • Sauder D.N.
      The role of epidermal cytokines in inflammatory skin diseases.
      ;
      • Barland C.O.
      • Zettersten E.
      • Brown B.S.
      • et al.
      Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis.
      ;
      • McInturff J.E.
      • Modlin R.L.
      • Kim J.
      The role of toll-like receptors in the pathogenesis and treatment of dermatological disease.
      ;
      • Takeuchi O.
      • Akira S.
      Innate immunity to virus infection.
      ). We have shown that topical IMQ enhances barrier function in normal and aged epidermis, by stimulating IL-1α production, which in turn stimulates epidermal lipid synthesis (
      • Ye J.
      • Calhoun C.
      • Feingold K.
      • et al.
      Age-related changes in the IL-1 gene family and their receptors before and after barrier abrogation.
      ;
      • Barland C.O.
      • Zettersten E.
      • Brown B.S.
      • et al.
      Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis.
      ). As human β-defensins are upregulated by multiple cytokines, it is highly likely that hBD2 (mBD3) upregulation by topical IMQ is signalled by epidermal production of cytokines. Yet, although the apparent increase in mBD3 immunostaining after calcipotriol treatment was unexpected, it could be linked to the well-known effects of VDR ligands on epidermal differentiation (
      • Bikle D.D.
      • Teichert A.
      • Arnold L.A.
      • et al.
      Differential regulation of epidermal function by VDR coactivators.
      ). Finally, we examined changes in AMP expression in two other unrelated situations where barrier function is enhanced, i.e., after treatment with topical 5–20% urea (
      • Grether-Beck S.
      • Felsner I.
      • Brenden H.
      • et al.
      Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression.
      ) and after topical applications of Chinese herbal medications to normal mouse skin (
      • Man M.
      • Hupe M.
      • Mackenzie D.
      • et al.
      A topical Chinese herbal mixture improves epidermal permeability barrier function in normal murine skin.
      ). In both of these situations, mCAMP expression increased in parallel with enhanced permeability barrier function (Figure 3 and Table 2).
      Figure thumbnail gr3
      Figure 3Summary of results—maneuvers that alter barrier functions are paralleled by bidirectional changes in cathelicidin expression.*Parentheses indicate changes in CAMP, but not other AMPs. AMP, antimicrobial peptide; CAMP, cathelicidin antimicrobial peptide.

      Materials and Methods

      Models with compromised permeability function

      Psychological stress

      Our previous studies have shown that both sustained PS and exogenous glucocorticoids downregulate barrier function (
      • Denda M.
      • Tsuchiya T.
      • Elias P.M.
      • et al.
      Stress alters cutaneous permeability barrier homeostasis.
      ;
      • Choi E.H.
      • Brown B.E.
      • Crumrine D.
      • et al.
      Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity.
      ) in parallel with reduced expression of mCAMP and mBD3 (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ). Hence, library biopsy samples from PS (short-term) served as positive controls for the additional conditions studied here, where barrier function also is compromised.
      Male hairless mice (Skh1/hr) were purchased from Charles River Laboratories (Wilmington, MA). To assess the effects of more long-term PS, animals were placed in motion-restricted environments for 12 hours once daily during nighttime for 72–96 hours. Food and water were restricted in parallel in a control non-motion-restricted group. A plastic container (4.0 (W) × 3.0 (H) × 11.5 (L) cm3) with mesh walls on the top was used for PS environments, of which the inner space was minimized to allow animals to rotate their bodies. All animals were studied between 8 and 10 weeks of age. The animal experiments described in this study were conducted in accordance with accepted standards of humane animal care, under the protocols approved by the local institutional animal care and use committee at the San Francisco VA Medical Center.

      Testosterone-replete (adult male vs. adult female) mice

      To assess the impact of physiological levels of testosterone, previously shown to compromise permeability barrier function (
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      ), we compared AMP expression in library samples of adult male versus female mice (aged 8–10 weeks; n=4 each), processed for immunofluorescence, as described below. Serum testosterone levels were >500 pg ml-1 in the male animals and <200 pg ml-1 in the female animals (
      • Kao J.S.
      • Garg A.
      • Mao-Qiang M.
      • et al.
      Testosterone perturbs epidermal permeability barrier homeostasis.
      ).

      Erythemogenic UVB exposure

      Hairless, 8–10-week-old female hairless mice were purchased from Charles River Laboratories (Wilmington, MA), and fed Purina mouse diet (Ralston Purina, St Louis, MO) and water ad libitum. Natural sunlight was excluded and animals were exposed only to low levels of incandescent light before UVB irradiation. UVB irradiation was delivered with Phillips TL20W/12 fluorescent lamps (Eindhoven, The Netherlands), emitting 280–320 nm. The dorsal skin of each mouse was either sham irradiated or irradiated with single-dose equivalents of either 5 or 10 minimal erythemal doses (n=5 each). One minimal erythema dose, determined previously on the same strain of mice, equals approximately 20 mJ cm-2 (60–100 mJ cm-2 hour-1 equals 1 minimal erythema dose in human beings with type II/III pigmentation). In all, 20 animals were treated in each group, and samples were taken before, immediately after, and then 1, 3, and 5 days following UVB exposure, followed by processing for immunofluorescence studies (see below).

      Chronologically aged mouse skin

      Epidermal AMP expression was compared in library samples from aged (15–18 months, equivalent to an age range of 50–60 years in humans) versus young adult (3–4 months) hairless mice (Skh1; Jackson Labs, Bar Harbor, ME; n=4 each) (
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      ). The analogous age of mice and humans was determined from optimal life spans (≈120 years in humans and 24 months in mice). Hairless mice began to display a progressive permeability barrier abnormality after 15 months (
      • Ghadially R.
      • Brown B.E.
      • Sequeira-Martin S.M.
      • et al.
      The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.
      ;
      • Choi E.H.
      • Man M.Q.
      • Xu P.
      • et al.
      Stratum corneum acidification is impaired in moderately aged human and murine skin.
      ).

      Models with enhanced permeability barrier function

      Not only blockade of glucocorticoid production/action (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ), but also suberythemogenic UVB irradiation has already been shown to stimulate mCAMP and mBD3 production (
      • Hong S.P.
      • Kim M.J.
      • Jung M.Y.
      • et al.
      Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
      ;
      • Glaser R.
      • Navid F.
      • Schuller W.
      • et al.
      UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo.
      ) (Table 1). Here, we assessed changes in mCAMP expression after several additional approaches that enhance barrier function. We focused on changes in mCAMP in this subset of studies, because it most closely paralleled changes in barrier status in the previously assessed models with reduced function.

      IMQ and calcipotriol

      Previous studies have shown that both 1,25 (OH)2 vitamin D3 and its analogs (
      • Bikle D.D.
      Vitamin D and the skin.
      ), as well as IMQ (
      • Barland C.O.
      • Zettersten E.
      • Brown B.S.
      • et al.
      Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis.
      ), enhance barrier function in a variety of settings. The dorsal skin of each mouse was treated with topical IMQ (Aldara, Bristol, TN) 5% cream, calcipotriol (Dovonex, Rockaway, NJ) cream 50 μg g-1, or vehicle two times daily for 7 days (n=4 mice in each group). Parallel control groups of hairless mice were treated with the vehicles for the equivalent drug alone at the same time points.
      Library biopsy samples from comparable cohorts of 4–5 normal hairless mice each also were assessed after following approaches that are known to enhance barrier function.

      Chinese herbal mixture and urea

      We recently showed that various Chinese herbal mixtures improve barrier function in normal hairless mice (
      • Man M.
      • Hupe M.
      • Mackenzie D.
      • et al.
      A topical Chinese herbal mixture improves epidermal permeability barrier function in normal murine skin.
      ). Recent studies also have shown that topical urea at concentrations ≥5% improves barrier function in normal human and mouse skin (
      • Grether-Beck S.
      • Felsner I.
      • Brenden H.
      • et al.
      Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression.
      ).

      Tissue processing and immunofluorescence

      Biopsy specimens for immunostaining were obtained at time points when maximal changes in barrier function occurred (see figure and table legends, as well as cited references for further details). Full-thickness skin biopsy specimens, which had either been snap-frozen in liquid nitrogen or library samples embedded in paraffin, were utilized for immunofluorescence studies. Frozen sections (5 μm) were soaked in acetone for 10 minutes, washed in phosphate-buffered saline (PBS), and blocked with 4% BSA and 0.5% cold-water fish gelatin in PBS for 30 minutes. In all, 10 μm paraffin-embedded tissue sections were de-paraffinized, rehydrated, and then rinsed with de-ionized water, followed by three washes in PBS. Sections were incubated for 30 minutes in blocking buffer (4% BSA, 0.5% cold water fish gelatin in PBS), and then incubated overnight at 4 °C with the primary antibodies in blocking buffer. The next morning, sections were washed three times in PBS and incubated for 40 minutes at room temperature with the Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody, diluted 1:2,000 in blocking buffer. Slides were then incubated overnight at 4 °C with primary antibodies (1:500 or 1:1,000) against Cst (from Phoenix Labs, Phoenix, AZ, and Richard Gallo, University of California, San Diego, San Diego, CA), mBD-3 (Alpha Diagnostics, Owings Mills, MD), or mCAMP (from Dr Richard Gallo, UCSD), followed by incubation with FITC-conjugated, goat anti-rabbit secondary antibody (Alpha Diagnostics) for 45 minutes at room temperature, as described (
      • Aberg K.M.
      • Radek K.A.
      • Choi E.H.
      • et al.
      Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
      ;
      • Radek K.A.
      • Lopez-Garcia B.
      • Hupe M.
      • et al.
      The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
      ). Sections were counterstained with propidium iodide and visualized on a Leica TCS-SP laser confocal microscope at excitation and emission wavelengths of 488 and 532 nm, respectively, photographed at original magnification × 40, and the intensity of AMP immunostaining was scored blindly in randomly mixed micrographs (n=20 in each group) as 0 (subnormal), 1 (normal=basal), or 2–5 (increased, with 5=most intense, antigen-positive immunostaining). Sections labeled with only the secondary antibody, and/or sections from mCAMP knockout mice (
      • Nizet V.
      • Ohtake T.
      • Lauth X.
      • et al.
      Innate antimicrobial peptide protects the skin from invasive bacterial infection.
      ;
      • Aberg K.M.
      • Man M.Q.
      • Gallo R.L.
      • et al.
      Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
      ), served as controls.

      ACKNOWLEDGMENTS

      These studies were supported by a grant from the Department of Defense, NIH grants AR019098 and AI059311, and the Medical Research Service, Department of Veterans Affairs. ISDIN S.A. supported post-doctoral fellowships for both Drs Rodríguez-Martín and Martin-Ezquerra. Ms Joan Wakefield provided superb editorial assistance.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

      REFERENCES

        • Aberg K.M.
        • Man M.Q.
        • Gallo R.L.
        • et al.
        Co-regulation and interdependence of the mammalian epidermal permeability and antimicrobial barriers.
        J Invest Dermatol. 2008; 128: 917-925
        • Aberg K.M.
        • Radek K.A.
        • Choi E.H.
        • et al.
        Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice.
        J Clin Invest. 2007; 117: 3339-3349
        • Aly R.
        • Shirley C.
        • Cunico B.
        • et al.
        Effect of prolonged occlusion on the microbial flora, pH, carbon dioxide and transepidermal water loss on human skin.
        J Invest Dermatol. 1978; 71: 378-381
        • Ambach A.
        • Bonnekoh B.
        • Nguyen M.
        • et al.
        Imiquimod, a Toll-like receptor-7 agonist, induces perforin in cytotoxic T lymphocytes in vitro.
        Mol Immunol. 2004; 40: 1307-1314
        • Arikawa J.
        • Ishibashi M.
        • Kawashima M.
        • et al.
        Decreased levels of sphingosine, a natural antimicrobial agent, may be associated with vulnerability of the stratum corneum from patients with atopic dermatitis to colonization by Staphylococcus aureus.
        J Invest Dermatol. 2002; 119: 433-439
        • Barland C.O.
        • Elias P.M.
        • Ghadially R.
        The aged epidermal barrier: basis for functional abnormalities.
        in: Elias P.M. Feingold K.R. Skin Barrier. Marcel Dekker, New York2005: 535-552
        • Barland C.O.
        • Zettersten E.
        • Brown B.S.
        • et al.
        Imiquimod-induced interleukin-1 alpha stimulation improves barrier homeostasis in aged murine epidermis.
        J Invest Dermatol. 2004; 122: 330-336
        • Bibel D.J.
        • Miller S.J.
        • Brown B.E.
        • et al.
        Antimicrobial activity of stratum corneum lipids from normal and essential fatty acid-deficient mice.
        J Invest Dermatol. 1989; 92: 632-638
        • Bikle D.D.
        Vitamin D and the skin.
        J Bone Miner Metab. 2010; 28: 117-130
        • Bikle D.D.
        • Teichert A.
        • Arnold L.A.
        • et al.
        Differential regulation of epidermal function by VDR coactivators.
        J Steroid Biochem Mol Biol. 2010; 121: 308-313
        • Braff M.H.
        • Di Nardo A.
        • Gallo R.L.
        Keratinocytes store the antimicrobial peptide cathelicidin in lamellar bodies.
        J Invest Dermatol. 2005; 124: 394-400
        • Chamlin S.L.
        • Kao J.
        • Frieden I.J.
        • et al.
        Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: changes in barrier function provide a sensitive indicator of disease activity.
        J Am Acad Dermatol. 2002; 47: 198-208
        • Choi E.H.
        • Brown B.E.
        • Crumrine D.
        • et al.
        Mechanisms by which psychologic stress alters cutaneous permeability barrier homeostasis and stratum corneum integrity.
        J Invest Dermatol. 2005; 124: 587-595
        • Choi E.H.
        • Demerjian M.
        • Crumrine D.
        • et al.
        Glucocorticoid blockade reverses psychological stress-induced abnormalities in epidermal structure and function.
        Am J Physiol Regul Integr Comp Physiol. 2006; 291: R1657-R1662
        • Choi E.H.
        • Man M.Q.
        • Xu P.
        • et al.
        Stratum corneum acidification is impaired in moderately aged human and murine skin.
        J Invest Dermatol. 2007; 127: 2847-2856
        • Cork M.J.
        • Robinson D.A.
        • Vasilopoulos Y.
        • et al.
        New perspectives on epidermal barrier dysfunction in atopic dermatitis: gene–environment interactions.
        J Allergy Clin Immunol. 2006; 118 (quiz 22–3): 3-21
        • Corsini E.
        • Racchi M.
        • Lucchi L.
        • et al.
        Skin immunosenescence: decreased receptor for activated C kinase-1 expression correlates with defective tumour necrosis factor-alpha production in epidermal cells.
        Br J Dermatol. 2009; 160: 16-25
        • de Jongh G.J.
        • Zeeuwen P.L.
        • Kucharekova M.
        • et al.
        High expression levels of keratinocyte antimicrobial proteins in psoriasis compared with atopic dermatitis.
        J Invest Dermatol. 2005; 125: 1163-1173
        • Denda M.
        • Tsuchiya T.
        • Elias P.M.
        • et al.
        Stress alters cutaneous permeability barrier homeostasis.
        Am J Physiol Regul Integr Comp Physiol. 2000; 278: R367-R372
        • Denda M.
        • Tsuchiya T.
        • Hosoi J.
        • et al.
        Immobilization-induced and crowded environment-induced stress delay barrier recovery in murine skin.
        Br J Dermatol. 1998; 138: 780-785
        • Drake D.R.
        • Brogden K.A.
        • Dawson D.V.
        • et al.
        Thematic review series: skin lipids. Antimicrobial lipids at the skin surface.
        J Lipid Res. 2008; 49: 4-11
        • Elias P.M.
        Stratum corneum defensive functions: an integrated view.
        J Invest Dermatol. 2005; 125: 183-200
        • Elias P.M.
        The skin barrier as an innate immune element.
        Sem Immunopathol. 2007; 29: 3-14
        • Elias P.M.
        • Choi E.H.
        Interactions among stratum corneum defensive functions.
        Exp Dermatol. 2005; 14: 719-726
        • Elias P.M.
        • Schmuth M.
        Abnormal skin barrier in the etiopathogenesis of atopic dermatitis.
        Curr Opin Allergy Clin Immunol. 2009; 9: 437-446
        • Elias P.M.
        • Steinhoff M.
        “Outside-to-inside” (and now back to “outside”) pathogenic mechanisms in atopic dermatitis.
        J Invest Dermatol. 2008; 128: 1067-1070
        • Eyerich K.
        • Pennino D.
        • Scarponi C.
        • et al.
        IL-17 in atopic eczema: linking allergen-specific adaptive and microbial-triggered innate immune response.
        J Allergy Clin Immunol. 2009; 123 (e4): 59-66
        • Georgel P.
        • Crozat K.
        • Lauth X.
        • et al.
        A toll-like receptor 2-responsive lipid effector pathway protects mammals against skin infections with Gram-positive bacteria.
        Infect Immun. 2005; 73: 4512-4521
        • Ghadially R.
        • Brown B.E.
        • Sequeira-Martin S.M.
        • et al.
        The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.
        J Clin Invest. 1995; 95: 2281-2290
        • Glaser R.
        • Meyer-Hoffert U.
        • Harder J.
        • et al.
        The antimicrobial protein psoriasin (S100A7) is upregulated in atopic dermatitis and after experimental skin barrier disruption.
        J Invest Dermatol. 2009; 129: 641-649
        • Glaser R.
        • Navid F.
        • Schuller W.
        • et al.
        UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo.
        J Allergy Clin Immunol. 2009; 123: 1117-1123
        • Grayson S.
        • Johnson-Winegar A.G.
        • Wintroub B.U.
        • et al.
        Lamellar body-enriched fractions from neonatal mice: preparative techniques and partial characterization.
        J Invest Dermatol. 1985; 85: 289-294
        • Grether-Beck S.
        • Felsner I.
        • Brenden H.
        • et al.
        Urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression.
        J Invest Dermatol. 2011 (in press)
        • Haratake A.
        • Uchida Y.
        • Schmuth M.
        • et al.
        UVB-induced alterations in permeability barrier function: roles for epidermal hyperproliferation and thymocyte-mediated response.
        J Invest Dermatol. 1997; 108: 769-775
        • Hatano Y.
        • Man M.Q.
        • Uchida Y.
        • et al.
        Maintenance of an acidic stratum corneum prevents emergence of murine atopic dermatitis.
        J Invest Dermatol. 2009; 129: 1824-1835
        • Holick M.F.
        Photosynthesis of vitamin D in the skin: effect of environmental and life-style variables.
        Fed Proc. 1987; 46: 1876-1882
        • Holleran W.M.
        • Uchida Y.
        • Halkier-Sorensen L.
        • et al.
        Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function.
        Photodermatol Photoimmunol Photomed. 1997; 13: 117-128
        • Hong S.P.
        • Kim M.J.
        • Jung M.Y.
        • et al.
        Biopositive effects of low-dose UVB on epidermis: coordinate upregulation of antimicrobial peptides and permeability barrier reinforcement.
        J Invest Dermatol. 2008; 128: 2880-2887
        • Howell M.D.
        The role of human beta defensins and cathelicidins in atopic dermatitis.
        Curr Opin Allergy Clin Immunol. 2007; 7: 413-417
        • Kao J.S.
        • Garg A.
        • Mao-Qiang M.
        • et al.
        Testosterone perturbs epidermal permeability barrier homeostasis.
        J Invest Dermatol. 2001; 116: 443-451
        • Kobayashi M.
        • Yoshiki R.
        • Sakabe J.
        • et al.
        Expression of toll-like receptor 2, NOD2 and dectin-1 and stimulatory effects of their ligands and histamine in normal human keratinocytes.
        Br J Dermatol. 2009; 160: 297-304
        • Komuves L.G.
        • Schmuth M.
        • Fowler A.J.
        • et al.
        Oxysterol stimulation of epidermal differentiation is mediated by liver X receptor-beta in murine epidermis.
        J Invest Dermatol. 2002; 118: 25-34
        • Korting H.C.
        • Hubner K.
        • Greiner K.
        • et al.
        Differences in the skin surface pH and bacterial microflora due to the long-term application of synthetic detergent preparations of pH 5.5 and pH 7.0. Results of a crossover trial in healthy volunteers.
        Acta Derm Venereol. 1990; 70: 429-431
        • Lai Y.
        • Gallo R.L.
        Toll-like receptors in skin infections and inflammatory diseases.
        Infect Disord Drug Targets. 2008; 8: 144-155
        • Man M.
        • Hupe M.
        • Mackenzie D.
        • et al.
        A topical Chinese herbal mixture improves epidermal permeability barrier function in normal murine skin.
        Exp Dermatol. 2011; 20: 285-288
        • Man M.Q.
        • Fowler A.J.
        • Schmuth M.
        • et al.
        Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.
        J Invest Dermatol. 2004; 123: 305-312
        • McInturff J.E.
        • Modlin R.L.
        • Kim J.
        The role of toll-like receptors in the pathogenesis and treatment of dermatological disease.
        J Invest Dermatol. 2005; 125: 1-8
        • Miller S.J.
        • Aly R.
        • Shinefeld H.R.
        • et al.
        In vitro and in vivo antistaphylococcal activity of human stratum corneum lipids.
        Arch Dermatol. 1988; 124: 209-215
        • Morizane S.
        • Yamasaki K.
        • Kabigting F.D.
        • et al.
        Kallikrein expression and cathelicidin processing are independently controlled in keratinocytes by calcium, vitamin D(3), and retinoic acid.
        J Invest Dermatol. 2010; 130: 1297-1306
        • Nizet V.
        • Ohtake T.
        • Lauth X.
        • et al.
        Innate antimicrobial peptide protects the skin from invasive bacterial infection.
        Nature. 2001; 414: 454-457
        • Nomura I.
        • Goleva E.
        • Howell M.D.
        • et al.
        Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.
        J Immunol. 2003; 171: 3262-3269
        • Oren A.
        • Ganz T.
        • Liu L.
        • et al.
        In human epidermis, beta-defensin 2 is packaged in lamellar bodies.
        Exp Mol Pathol. 2003; 74: 180-182
        • Peric M.
        • Koglin S.
        • Kim S.M.
        • et al.
        IL-17A enhances vitamin D3-induced expression of cathelicidin antimicrobial peptide in human keratinocytes.
        J Immunol. 2008; 181: 8504-8512
        • Proksch E.
        • Brandner J.M.
        • Jensen J.M.
        The skin: an indispensable barrier.
        Exp Dermatol. 2008; 17: 1063-1072
        • Proksch E.
        • Jensen J.M.
        • Elias P.M.
        Skin lipids and epidermal differentiation in atopic dermatitis.
        Clin Dermatol. 2003; 21: 134-144
        • Radek K.
        • Gallo R.
        Antimicrobial peptides: natural effectors of the innate immune system.
        Semin Immunopathol. 2007; 29: 27-43
        • Radek K.A.
        • Elias P.M.
        • Taupenot L.
        • et al.
        Neuroendocrine nicotinic receptor activation increases susceptibility to bacterial infections by suppressing antimicrobial peptide production.
        Cell Host Microbe. 2010; 7: 277-289
        • Radek K.A.
        • Lopez-Garcia B.
        • Hupe M.
        • et al.
        The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.
        J Invest Dermatol. 2008; 128: 1525-1534
        • Reed J.T.
        • Elias P.M.
        • Ghadially R.
        Integrity and permeability barrier function of photoaged human epidermis.
        Arch Dermatol. 1997; 133: 395-396
        • Sauder D.N.
        The role of epidermal cytokines in inflammatory skin diseases.
        J Invest Dermatol. 1990; 95: 27S-28S
        • Sauder D.N.
        Mechanism of action and emerging role of immune response modifier therapy in dermatologic conditions.
        J Cutan Med Surg. 2004; 8: 3-12
        • Schauber J.
        • Gallo R.L.
        The vitamin D pathway: a new target for control of the skin's immune response?.
        Exp Dermatol. 2008; 17: 633-639
        • Schroder J.M.
        • Harder J.
        Antimicrobial skin peptides and proteins.
        Cell Mol Life Sci. 2006; 63: 469-486
        • Takeuchi O.
        • Akira S.
        Innate immunity to virus infection.
        Immunol Rev. 2009; 227: 75-86
        • Yano S.
        • Banno T.
        • Walsh R.
        • et al.
        Transcriptional responses of human epidermal keratinocytes to cytokine interleukin-1.
        J Cell Physiol. 2008; 214: 1-13
        • Ye J.
        • Calhoun C.
        • Feingold K.
        • et al.
        Age-related changes in the IL-1 gene family and their receptors before and after barrier abrogation.
        J Invest Dermatol. 1999; 112: 543
        • Zasloff M.
        Sunlight, vitamin D, and the innate immune defenses of the human skin.
        J Invest Dermatol. 2005; 125: xvi-xvii