pH Directly Regulates Epidermal Permeability Barrier Homeostasis, and Stratum Corneum Integrity/Cohesion

Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased β-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities. Both exposure of stratum corneum to neutral pH buffers and blockade of acidification mechanisms disturb cutaneous permeability barrier homeostasis and stratum corneum integrity/cohesion, but these approaches all introduce potentially confounding variables. To study the consequences of stratum corneum neutralization, independent of hydration, we applied two chemically unrelated superbases, 1,1,3,3-tetramethylguanidine or 1,8-diazabicyclo [5,4,0] undec-7-ene, in propylene glycol:ethanol (7:3) to hairless mouse skin and assessed whether discrete pH changes alone regulate cutaneous permeability barrier function and stratum corneum integrity/cohesion, as well as the responsible mechanisms. Both 1,1,3,3-tetramethylguanidine and 1,8-diazabicyclo [5,4,0] undec-7-ene applications increased skin surface pH in parallel with abnormalities in both barrier homeostasis and stratum corneum integrity/cohesion. The latter was attributable to rapid activation (<20 min) of serine proteases, assessed by in situ zymography, followed by serine-protease-mediated degradation of corneodesmosomes. Western blotting revealed degradation of desmoglein 1, a key corneodesmosome structural protein, in parallel with loss of corneodesmosomes. Coapplication of serine protease inhibitors with the superbase normalized stratum corneum integrity/cohesion. The superbases also delayed permeability barrier recovery, attributable to decreased β-glucocerebrosidase activity, assessed zymographically, resulting in a lipid-processing defect on electron microscopy. These studies demonstrate unequivocally that stratum corneum neutralization alone provokes stratum corneum functional abnormalities, including aberrant permeability barrier homeostasis and decreased stratum corneum integrity/cohesion, as well as the mechanisms responsible for these abnormalities. β-glucocerebrosidase corneodesmosome 1,8-diazabicyclo [5,4,0] undec-7-ene desmoglein 1 Z-[N-morpholino] ethanesulfonic acid sodium-proton exchanger stratum corneum serine protease serine protease inhibitor secretory phospholipase A2 transepidermal water loss 1,1,3,3-tetramethyl-guanidine Although the skin has long been known to display an acid surface (“acid mantle”) (Schade and Marchionini, 1928Schade H. Marchionini A. Der sauremantel der haut.Klin Wochenschr. 1928; 7: 12-14Crossref Scopus (71) Google Scholar), even today little is known about either the origin or the function(s) of this acidic surface. Prior students of the acid mantle ascribed its origin principally to exogenous sources, of microbial, sebaceous gland, and/or eccrine gland origin (Marchionini and Hausknecht, 1938Marchionini A. Hausknecht W. Sauremantel der haut und bakterienabwehr.Sauremantel Haut Bakterienabwehr. 1938; 17: 663-666Google Scholar). Furthermore, based upon a variety of indirect evidence, its function has been assumed to be principally antimicrobial (Aly et al., 1975Aly R. Maibach H.I. Rahman R. Shinefield H.R. Mandel A.D. Correlation of human in vivo and in vitro cutaneous antimicrobial factors.J Infect Dis. 1975; : 579-583Crossref PubMed Scopus (48) Google Scholar;Puhvel et al., 1975Puhvel S.M. Reisner R.M. Sakamoto M. Analysis of lipid composition of isolated human sebaceous gland homogenates after incubation with cutaneous bacteria. Thin-layer chromatography.J Invest Dermatol. 1975; 64: 406-411Crossref PubMed Scopus (45) Google Scholar). Recent studies support alternative views of the origin of the acid mantle, as well as additional functions of stratum corneum (SC) acidity (Ohman and Vahlquist, 1994Ohman H. Vahlquist A. In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis.Acta Dermato-Venereologica. 1994; 74: 375-379PubMed Google Scholar;Ohman and Vahlquist, 1998Ohman H. Vahlquist A. The pH gradient over the stratum corneum differs in X-linked recessive and autosomal dominant ichthyosis: A clue to the molecular origin of the ‘acid skin mantle’?.J Invest Dermatol. 1998; 111: 674-677Crossref PubMed Scopus (142) Google Scholar;Chikakane and Takahashi, 1995Chikakane K. Takahashi H. Measurement of skin pH and its significance in cutaneous diseases.Clin Dermatol. 1995; 13: 299-306Abstract Full Text PDF PubMed Scopus (72) Google Scholar;Denda et al., 2000Denda M. Hosoi J. Asida Y. Visual imaging of ion distribution in human epidermis.Biochem Biophys Res Comms. 2000; 272: 134-137Crossref PubMed Scopus (84) Google Scholar). In addition to exogenous mechanisms, three endogenous pathways have been identified as potential contributors to SC acidity: (1) generation of urocanic acid by histidase-catalyzed deimination of histidine (Krien and Kermici, 2000Krien P.M. Kermici M. Evidence for the existence of a self-regulated enzymatic process within the human stratum corneum - An unexpected role for urocanic acid.J Invest Dermatol. 2000; 115: 414-420Crossref PubMed Scopus (119) Google Scholar); (2) secretory phospholipase A2 (sPLA2) generation of free fatty acids from phospholipids (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar); and (3) a nonenergy-dependent sodium-proton exchanger (NHE1) (Behne et al., 2002Behne M.J. Meyer J.W. Hanson K.M. et al.NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.J Biol Chem. 2002; 277: 47399-47406Crossref PubMed Scopus (165) Google Scholar). Of these three mechanisms, the histidase pathway, though quantitatively capable of acidifying the SC (Krien and Kermici, 2000Krien P.M. Kermici M. Evidence for the existence of a self-regulated enzymatic process within the human stratum corneum - An unexpected role for urocanic acid.J Invest Dermatol. 2000; 115: 414-420Crossref PubMed Scopus (119) Google Scholar), appears least likely to mediate functions in the lower SC, because (1) lack of substrate would make this mechanism nonoperative at the high relative humidities of the stratum compactum (Scott and Harding, 1986Scott I.R. Harding C.R. Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment.Dev Biol. 1986; 115: 84-92Crossref PubMed Scopus (256) Google Scholar), and (2) urocanic acid is a water-soluble metabolite, which may not reach lipophilic membrane domains from the corneocyte cytosol, where it is generated. Yet, it is in membrane sites in the lower SC that the permeability barrier is formed (Elias and Friend, 1975Elias P.M. Friend D.S. The permeability barrier in mammalian epidermis.J Cell Biol. 1975; 65: 180-191Crossref PubMed Scopus (541) Google Scholar), and it is in these sites that SC integrity/cohesion localizes (Chapman and Walsh, 1990Chapman S.J. Walsh A. Desmosomes, corneosomes and desquamation.Arch Dermatol Res. 1990; 282: 304-310Crossref PubMed Scopus (121) Google Scholar;Fartasch et al., 1993Fartasch M. Bassukas I.D. Diepgen T.L. Structural relationship between epidermal lipid lamellae, lamellar bodies, and desmosomes; An ultrastructural study.Br J Dermatol. 1993; 128: 1-9Crossref PubMed Scopus (110) Google Scholar;Elias et al., 2001Elias P.M. Matsuyoshi N. Wu H. Lin C. Wang Z.H. Brown B.E. Stanley J.R. Desmoglein isoform distribution affects stratum corneum structure and function.J Cell Biol. 2001; 153: 243-249Crossref PubMed Scopus (107) Google Scholar). Hence, our laboratory has focused recently on the role and functional importance of the sPLA2 and NHE1 pathways, which both appear to influence membrane acidity in the lower SC (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar;Behne et al., 2002Behne M.J. Meyer J.W. Hanson K.M. et al.NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.J Biol Chem. 2002; 277: 47399-47406Crossref PubMed Scopus (165) Google Scholar). Indeed, inhibition and/or blockade of either sPLA2 or NHE1 result in an elevated SC pH; and more importantly, interference with these mechanisms perturbs permeability barrier homeostasis and/or SC integrity/cohesion (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar;Behne et al., 2002Behne M.J. Meyer J.W. Hanson K.M. et al.NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.J Biol Chem. 2002; 277: 47399-47406Crossref PubMed Scopus (165) Google Scholar). Direct evidence for the importance of pH for permeability barrier homeostasis was first shown by the delay in barrier recovery that occurs when acutely disrupted skin sites are immersed in neutral pH buffers (Mauro et al., 1998Mauro T. Holleran W.M. Grayson S. et al.Barrier recovery is impeded at neutral pH, independent of ionic effects: Implications for extracellular lipid processing.Arch Dermatol Res. 1998; 290: 215-222Crossref PubMed Scopus (221) Google Scholar). Moreover, the barrier abnormality resulting from either sPLA2 or NHE1 blockade could be overridden by coexposure of inhibitor-treated sites to an acidic (normal pH) buffer (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar;Behne et al., 2002Behne M.J. Meyer J.W. Hanson K.M. et al.NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.J Biol Chem. 2002; 277: 47399-47406Crossref PubMed Scopus (165) Google Scholar). An acidic pH is critical for barrier homeostasis, in part because two key lipid-processing enzymes, β-glucocerebrosidase (βGlcCer'ase) and acidic sphingomyelinase, which generate a family of ceramides from glucosylceramide and sphingomyelin precursors, respectively (Uchida et al., 2002Uchida Y. Hara M. Nishio H. et al.Epidermal sphingomyelins are precursors for selected stratum corneum ceramides.J Lipid Res. 2002; 41: 2071-2082Google Scholar), exhibit low pH optima (Vaccaro et al., 1985Vaccaro A.M. Muscillo M. Suzuki K. Characterization of human glucosylsphingosine glucosyl hydrolase and comparison with glucosylceramidase.Eur J Biochem. 1985; 146: 315-321Crossref PubMed Scopus (29) Google Scholar;Holleran et al., 1993Holleran W.M. Takagi Y. Menon G.K. Legler G. Feingold K.R. Elias P.M. Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function.J Clin Invest. 1993; 91: 1656-1664Crossref PubMed Scopus (226) Google Scholar;Jensen et al., 1999Jensen J.M. Schutze S. Forl M. Kronke M. Proksch E. Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier.J Clin Invest. 1999; 104: 1761-1770Crossref PubMed Scopus (153) Google Scholar;Schmuth et al., 2000Schmuth M. Man M.-Q. Weber F. et al.Permeability barrier disorder in Nieman-Pick disease: Sphingomyelin-ceramide processing required for normal barrier homeostasis.J Invest Dermatol. 2000; 115: 459-466Crossref PubMed Scopus (129) Google Scholar). It has also been proposed that an acidic pH directly impacts lipid–lipid interactions in the SC extracellular lamellar bilayers (Bouwstra et al., 1999Bouwstra J.A. Gooris G.S. Dubbelaar F.E. Ponec M. Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range.J Lipid Res. 1999; 40: 2303-2312PubMed Google Scholar). Together, these mechanisms appear to regulate the competence of the extracellular lamellar bilayer system. An acidic SC pH also clearly promotes SC integrity and cohesion. Exposure to either neutral pH buffers or sPLA2 blockade results in an enhanced tendency for the SC to be removed by tape stripping (=integrity), as well as increased amounts of protein removed per stripping (=cohesion) (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar). In the case of sPLA2 blockade, the alterations in SC integrity/cohesion could be further attributed to a decreased density of corneodesmosomes (CD), and to a decline in at least one of its constituent proteins, desmoglein 1 (DSG1) (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar). Furthermore, as both the chymotryptic and tryptic serine proteases (SCCE and SCTE), which mediate degradation leading to desquamation, exhibit neutral pH optima (Ekholm et al., 2000Ekholm I.E. Brattsand M. Egelrud T. Stratum corneum tryptic enzyme in normal epidermi: A missing link in the desquamation process?.J Invest Dermatol. 2000; 114: 56-63Crossref PubMed Scopus (194) Google Scholar), they could become activated as SC pH increases. All prior attempts to manipulate SC pH, and to assess the effects of pH on epidermal function, have utilized either buffers, which could exert effects on function independent of pH (e.g., from hydration or occlusion), or inhibitor/knockout models (e.g., a variety of unrelated changes can occur). To establish definitively the apparent link between SC pH and barrier homeostasis and SC integrity/cohesion, we developed a new model in which SC pH could be modulated directly, utilizing topical applications of low concentrations of two “superbases”, 1,1,3,3-tetramethyl-guanidine (TMG) and 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) (Kaljurand et al., 2000Kaljurand I.I. Rodima T. Leito I.I. Koppel I.A. Schwesinger R. Self-consistent spectrophotometric basicity scale in acetonitrile covering the range between pyridine and DBU.J Org Chem. 2000; 65: 6202-6208Crossref PubMed Scopus (171) Google Scholar;Oyama and Kondo, 2003Oyama K. Kondo T. A novel and convenient chemoselective deprotection method for both silyl and acetyl groups on acidic hydroxyl groups such as phenol and carboxylic acid by using a nitrogen organic base, 1,1,3,3-tetramethylguanidine.Org Lett. 2003; 23: 209-212Crossref Scopus (34) Google Scholar), to raise and sustain pH to specific levels within the SC, without evidence of toxicity at the low concentrations employed. Because superbases and superacids are by definition at least an order of magnitude more basic or more acidic than 1 N NaOH and 1 N H2SO4 (DesMarteau, 2000DesMarteau D.D. Superacids - It's a lot about anions.Science. 2000; 289: 72-73Crossref PubMed Scopus (26) Google Scholar;Oyama and Kondo, 2003Oyama K. Kondo T. A novel and convenient chemoselective deprotection method for both silyl and acetyl groups on acidic hydroxyl groups such as phenol and carboxylic acid by using a nitrogen organic base, 1,1,3,3-tetramethylguanidine.Org Lett. 2003; 23: 209-212Crossref Scopus (34) Google Scholar), they can be used to manipulate pH locally when applied in very low concentrations. In addition, their internal naphthalene structure should favor absorption throughout the SC. Elevations of SC pH utilizing this approach were accompanied by abnormalities in both SC integrity/cohesion and permeability barrier homeostasis, attributable to accelerated serine protease (SP) mediated degradation of CD, and defects in lipid processing, respectively. Male hairless mice (Skh1/Hr), 6–8 wk old, were purchased from Charles River Laboratories (Wilmington, MA) and fed Purina mouse diet and water ad libitum. Propylene glycol, ethanol, and HCl were from Fisher Scientific (Fairlane, NJ), whereas TMG, DBU, phenylmethylsulfonyl fluoride (PMSF), chymostatin, soybean trypsin inhibitor, and aprotinin were from Sigma Chemical (St Louis, MO). Rabbit polyclonal antibody against mouse DSG1 was a gift from Dr John Stanley (University of Pennsylvania). Horseradish peroxidase conjugated with antirabbit IgG was purchased from Vector Laboratories (Burlingame, CA). EnzChek Protease Assay Kit, green fluorescence, and resorufin-D-glucopyranoside were purchased from Molecular Probes (Eugene, OR). 22 mm D-Squame-100 tapes were purchased from CuDerm (Dallas, TX). Bradford protein assay kit and bovine plasma γ globulin were purchased from Bio-Rad (Hercules, CA). Mice were anesthetized with chloral hydrate (Morton Grove Pharmaceuticals, Morton Grove, IL). Normal hairless mice were treated topically with a single application of either TMG or DBU (dose range 1:100–1:1000 vol/vol) in propylene glycol:ethanol (7:3 vol/vol) on 5–6 cm2 areas on both flanks. Controls were treated with HCl-neutralized TMG (nTMG) in the same propylene glycol:ethanol vehicle. The general idea of using superbases is related to their ability to accept protons, thereby acting as “proton sponges”. The chelating function of superbasic TMG is based on its 1,8-diaminonaphthalene skeleton. TMG not only shows a high thermodynamic basicity (pKBH+ value of 25.1), but it also reveals unusually high kinetics for basic reactions, which makes this superbase highly attractive for base-catalyzed applications. Superbases such as TMG and DBU break water to generate a hydroxyl group and chelate a proton to become positively charged. As noted above, the positively charged superbase, with its lipid-soluble core structure, confers on TMG and DBU physicochemical properties that allow penetration through the SC. Surface pH was measured with a flat, glass surface electrode from Mettler-Toledo (Giessen, Germany), attached to a pH meter (PH 900; Courage & Khazaka, Cologne, Germany), immediately before and at 1, 2, 4, 6, 9, 12, 18, and 24 h after TMG, DBU, and nTMG applications. For the experiments with serine protease inhibitors (SPI), animals received coapplications of TMG or DBU with either 10 mM PMSF, chymostatin, aprotinin, or soybean trypsin inhibitor (concentrations specified in the legend to Table I). Normal hairless mice were treated topically with a single application of TMG (1:100 vol/vol) in propylene glycol:ethanol (7:3 vol/vol) on a 5–6 cm2 area on one flank versus nTMG, TMG plus PMSF, or vehicle alone to the contralateral flank, or to the flanks of littermates, immediately after barrier disruption by sequential tape stripping (transepidermal water loss (TEWL) rates ≥4 mg per cm2 per h). Surface pH was measured immediately after disruption and at the same time points as above, followed by assessment of function and mechanistic studies (see below). To assess the kinetics of epidermal permeability barrier recovery, TEWL levels were measured on the flanks of hairless mice using an electrolytic water analyzer (MEECO, Warrington, PA), immediately before and after, as well as 3 h after, acute barrier disruption by repeated D-Squame tape stripping, and after a single application of TMG, nTMG, DBU, or nDBU (see above). SC water content, as the sum of hydration of all SC layers, was determined by capacitance measurements with a corneometer (CM820, Courage & Khazaka, Cologne, Germany) 3 h after application of TMG, nTMG, DBU, or nDBU to intact skin. To study SC integrity (rate of change in TEWL with repeated strippings), sequential tape stripping was performed on the flanks of hairless mice 3 h after prior application of either TMG or nTMG. TEWL levels were measured after each tape stripping, until TEWL rates exceeded 4 mg per cm2 per h (five to six strippings in normal murine SC). Three hours after superbase applications, and immediately before stripping the SC, the skin surface was cleaned with a single ethanol wipe. D-Squame tapes were then placed sequentially onto the test areas for about 3 s each, removed with forceps, and stored in glass scintillation vials at 5°C. SC cohesion is reflected by the amount of protein removed from pooled, sequential D-Squame strippings (whole SC down to the stratum compactum from one site per mouse), extracted in 2 ml of 1 N NaOH, and measured as previously described (Dreher et al., 1998Dreher F. Arens A. Hostynek J.J. Mudumba S. Ademola J. Maibach H.I. Colorimetric method for quantifying human stratum corneum removed by adhesive-tape stripping.Acta Derm Venereol. 1998; 78: 186-189Crossref PubMed Scopus (121) Google Scholar). This microassay system was shown again to be linear for human SC in the range 1–10 μg per ml, using SC from human callus to generate standard curves (calculated slope Rf±SD is 0.0297±0.00062; Spearman coefficient 0.999; p<0.0001). The protein content per stripping was determined with the Bio-Rad Protein Assay Kit, as described recently, using bovine γ globulin as the standard in all assays (Fluhr et al., 2001Fluhr J.W. Kao J. Jain M. Ahn S.K. Feingold K.R. Elias P.M. Generation of free fatty acids from phospholipids regulates stratum corneum acidification and integrity.J Invest Dermatol. 2001; 117: 44-51Crossref PubMed Google Scholar). Briefly, tapes were incubated with 1 ml of 1 N NaOH for 1 h at 37°C in an incubator shaker and neutralized thereafter with 1 ml of 1 N HCl in the scintillation vials. Subsequently, 0.2 ml of this solution was incubated in 0.6 ml distilled water plus 0.2 ml of the Bio-Rad protein dye for 5 min in borosilicate tubes. After incubations, the reagents were transferred to polystyrene cuvettes, and absorption was measured with a Genesys 5 spectrophotometer (Spectronic, Rochester, NY) at 595 nm. Blank D-Squame tapes were processed and assayed as a negative control. The amount of protein removed was then normalized to skin surface area (μg per cm2). The amount of removed protein per D-Squame strip was comparable to previous reports in untreated skin of hairless mice (i.e., range 2.5–4 μg per strip) (Dreher et al., 1998Dreher F. Arens A. Hostynek J.J. Mudumba S. Ademola J. Maibach H.I. Colorimetric method for quantifying human stratum corneum removed by adhesive-tape stripping.Acta Derm Venereol. 1998; 78: 186-189Crossref PubMed Scopus (121) Google Scholar). Biopsies were obtained from hairless mouse flanks after treatment with superbase or neutralized superbase, and the subcutaneous fat was removed by scraping with a #10 Bard-Parker blade. Frozen sections (10 μm thick) were rinsed with a washing solution (2% Tween 20 in deionized water) and incubated at 37°C for 2 h with 250 μl of BODIPY-Fl-casein in deionized water (2 μl per ml). To obtain en face views harvested skin was placed in a chamber slide system with the SC facing toward the beam. Some superbase-treated sections were exposed to the fluorophore substrate in an acidic buffer (10 mM Z-[N-morpholino] ethanesulfonic acid (MES) buffer, pH 5.5). All sections were rinsed with the washing solution, counterstained with propidium iodide, mounted, and visualized directly in a confocal microscope (Leica TCS SP, Heidelberg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Biopsies were obtained from treated sites as above, sectioned (6 μm), rinsed with the washing solution, and incubated with 250 μl of resorufin β-D-glucopyranoside in deionized water (1 mM) at 37°C for 2 h. Acidification of some superbase-treated sections was again performed with 10 mM MES buffer, pH to 5.5, as above. Sections were then visualized in the confocal microscope at an excitation wavelength of 588 nm and an emission wavelength of 644 nm. SC was isolated from hairless mouse flanks, previously treated as above, using D-Squame sequential tape strippings until no further SC could be removed (=whole SC; typically five to six strippings). Hematoxylin and eosin staining was performed on paraffin sections (6 μm) from biopsies of the tape-stripped areas to ensure equivalent SC removal for each experiment group. D-Squame tapes were then incubated overnight at 4°C in 1% Triton X and a protease inhibitor cocktail (Complete Mini, EDTA-Free, Roche, 1 tablet per 10 ml) in deionized water and sonicated for 5 min at room temperature to extract protein from the tapes. The protein content per stripping was then determined, as above. An equal amount of extracted protein from each experimental group was loaded onto 4%–12% Tris-glycine polyacrylamide gels (PAGE Gold Precast Gels, BioWhittaker Molecular Applications, Rockland, ME). After electrophoresis, proteins were transferred from slab gels onto nitrocellulose membranes and immunoblotted with the rabbit antimouse DSG1 antibody, and antibody binding to DSG1 was detected with the Western Lighting chemiluminescence kit (PerkinElmer Life Sciences, Boston, MA). Skin biopsy samples were taken at 1 and 3 h after the various treatments (n=3 from each group) and processed for light and electron microscopy. Samples were minced to less than 0.5 mm3, fixed in modified Karnovsky's fixative overnight, and postfixed in either ruthenium tetroxide (RuO4) or 2% aqueous osmium tetroxide (OsO4), both containing 1.5% potassium ferrocyanide (Hou et al., 1991Hou S.Y. Mitra A.K. White S.H. Menon G.K. Ghadially R. Elias P.M. Membrane structures in normal and essential fatty acid-deficient stratum corneum: Characterization by ruthenium tetroxide staining and x-ray diffraction.J Invest Dermatol. 1991; 96: 215-223Abstract Full Text PDF PubMed Google Scholar). After postfixation, all samples were dehydrated in graded ethanol solutions and embedded in an Epon epoxy mixture. Ultrathin sections were examined, with or without further lead citrate contrasting, in a Zeiss 10A electron microscope (Carl Zeiss, Thornwood, NY), operated at 60 kV. In order to quantify CD density in electron micrographs, 10 or more pictures were taken by an independent observer from three or more blocks from three animals in each experiment at 31,500 magnification; i.e., a total of at least 30 micrographs. The ratio of the total length of intact CD to the total length of the cornified envelopes in the first and second cell layers of the lower SC was determined using a planimeter (Morris, 2000Morris R.E. The use of nonparametric statistics in quantitative electron microscopy.J Electron Microsc. 2000; 49: 545-549Crossref PubMed Scopus (10) Google Scholar). Nonparametric Mann–Whitney statistical analyses were performed to compare percentage ratios between different groups of treatments (Morris, 2000Morris R.E. The use of nonparametric statistics in quantitative electron microscopy.J Electron Microsc. 2000; 49: 545-549Crossref PubMed Scopus (10) Google Scholar). Statistical analyses were performed using Prism 2 (GraphPad Software, San Diego, CA) Before assessing the consequences of increased pH on SC function, we first measured SC pH after applications of two chemically unrelated superbases, TMG and DBU. Surface pH of hairless mouse skin, assessed 3 h after a single TMG or DBU application, increased significantly in comparison to nTMG and nDBU, respectively (Figure 1a,–c). The changes in skin surface pH were concentration dependent at TMG concentrations between 1:100 and 1:1000 (vol/vol) (Figure 1d), and regardless of applied dose, recovery of an acidic surface pH occurred over the subsequent 24 h (Figure 1e). The superbase-induced increase in pH extended throughout the SC, with pH in TMG-treated sites deep within the SC remaining significantly higher than the pH at comparable depths of nTMG-treated sites (Figure 1f). Despite the sustained elevations in surface pH, histologic sections, taken 3 and 24 h following TMG or nTMG treatment, revealed neither histologic abnormalities nor evidence of cytotoxicity or inflammation (Figure 2). Moreover, despite the sustained elevation in SC pH, both basal TEWL and SC hydration levels remained unchanged in superb