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Hydrogen Antiporter-1 Acidify Neonatal Rat Stratum Corneum
Stratum Corneum Acidification in Neonatal Skin: Secretory Phospholipase A2 and the Sodium/Hydrogen Antiporter-1 Acidify Neonatal Rat Stratum Corneum Joachim W. Fluhr,w Martin J. Behne, Barbara E. Brown, David G. Moskowitz, Clare Selden,z Man Mao-Qiang, Theodora M. Mauro, Peter M. Elias, and Kenneth R. Feingold
Dermatology and Medical Service, Veterans Affairs Medical Center, San Francisco, and Departments of Dermatology and Medicine, University of California, San Francisco, California, USA; zCenter for Hepatology, Department of Medicine, Royal Free and University College Medical School, Hampstead, London, UK; wDepartment of Dermatology and Allergology, Friedrich-Schiller-University, Jena, Germany
At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, b-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum–SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth.
Key words: neonatal rat/stratum corneum pH/barrier function. J Invest Dermatol 122:320 – 329, 2004
Stratum corneum (SC) maturation late in fetal development leads to the generation of a competent cutaneous permeability barrier, crucial both for postnatal survival in a terrestrial environment and for defense against exogenous environmental insults. In rodent skin (e.g., rat), the final steps of fetal epidermal development occur between days 20 and 22 (term) in a patterned fashion (dorsal–ventral) (Aszterbaum et al, 1992; Hardman et al, 1998, 1999). Both a fully developed cronified envelope (Akiyama et al, 2000; Lee et al, 1999) and abundant extracellular lamellar bilayers are present late in gestation (Hanley et al, 1997a). Moreover, basal permeability barrier function is competent at birth
(Aszterbaum et al, 1992; Aszterbaum et al, 1993; Williams et al, 1993). Yet, skin surface pH is neutral at birth both in humans and in various animal models (Behrendt and Green, 1971; Hardman et al, 1998; Visscher et al, 2000; Yosipovitch et al, 2000), and the absence of an acidic SC at birth has been associated with an increased risk of bacterial and yeast infections in neonates (Leyden and Kligman, 1978). In contrast, the acidic pH both of older neonates and adults is associated with decreased colonization by pathogenic bacteria (Aly et al, 1975; Puhvel et al, 1975) and favors adhesion of nonpathogenic bacteria to the SC (Bibel et al, 1987). Thus, the acidic pH of the SC has been postulated to mediate at least one important SC function, that is, its antibacterial properties, and thus the absence of an acidic pH at birth may have clinical consequences for the neonate. In addition to its putative antimicrobial activity, development of an acidic pH could be important for other SC
Abbreviations: BPB, bromophenacylbromide; cUCA, cis-urocanic acid; FFA, free fatty acids; HAL, histidase ammonia-lyase; NHE1, sodium/hydrogen antiporter-1; SC, stratum corneum; sPLA2, secretory phospholipase A2; TEWL, transepidermal water loss.
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
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functions. Prior studies have shown that permeability barrier recovery after acute insults is impeded at a neutral versus acidic pH (Mauro et al, 1998), explicable by the acidic pH optimum of certain critical lipid-processing enzymes in the SC, that is, b-glucocerebrosidase (Takagi et al, 1999) and acidic sphingomyelinase (Jensen et al, 2000) (Schmuth et al, 2000), which generate a family of ceramides required for permeability barrier homeostasis (Holleran et al, 1993) (Jensen et al, 2000). Although basal barrier function is normal in neonates, we have shown recently that barrier homeostasis is impaired owing to the neutral pH of neonatal SC.1 Finally, the integrity and cohesion of adult SC are pHdependent; that is, an acidic pH favors a SC that is more resistant to mechanical disruption (Fluhr et al, 2001). Thus, the neutral pH of neonatal SC could explain other clinical abnormalities in neonatal skin, including an increased propensity to develop irritant contact (diaper) dermatitis and increased skin fragility. Although the SC acid mantle was first recognized decades ago (Schade, 1928; Blank, 1939; Draize, 1942; Beare et al, 1959; Jolly et al, 1961; Baden and Pathak, 1967), its origin is incompletely understood. Traditionally, exogenous (originating outside the epidermis) mechanisms have been invoked to explain the formation of the acid mantle. Such exogenous mechanisms include the generation of (1) microbial metabolites (e.g., it is widely believed that bacterial colonization, by generating acidic metabolites, is important for the postnatal development of an acidic surface pH); (2) free fatty acids (FFA) of pilosebaceous origin (Puhvel et al, 1975); and (3) eccrine-gland-derived products, such as lactic acid (Ament et al, 1997). Alternatively, endogenous processes have been implicated recently in acidification of adult SC including: (1) generation of cisurocanic acid (cUCA) from histidine (Krien and Kermici, 2000); (2) FFA generation from phospholipid hydrolysis (Fluhr et al, 2001); and (3) a sodium/proton antiporter, sodium/hydrogen antiporter-1 (NHE1) (Behne et al, 2002). Because neonatal SC is incompletely acidified, we hypothesized that one or more of these mechanisms might not be functional at birth. To begin to assess the mechanisms involved in the progressive, postnatal development of an acidic SC, we assessed acidification in newborn rats over the first week after birth. We report here that neonatal rats generate adult levels of SC acidification by 5 to 6 d after birth, compressing processes that require weeks to months in human neonates into less than 1 wk. In assessing the origins of postnatal SC acidification in this rodent model, and in available mutant murine models, we excluded roles for exogenous mechanisms, including bacterial colonization and sebaceous gland activity. Our results further exclude one endogenous mechanism, namely, the histidase pathway, as a major contributor to the development of an acid mantle during the immediate postnatal period. Instead, we provide here evidence that both secretory phospholipase A2 (sPLA2) and NHE1 activity contribute to, but do not completely account for, postnatal acidification. 1 Fluhr J, Fowler AJ, Ichachem J, Crumine D, Elias PM, Feingold KR: Consequences for the neonate of a neutral stratum corneum pH: Permeability barrier homeostasis. J Invest Dermatol 119: 249 2002 (abstr).
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Results Formation of an acidic surface pH occurs during the first week after birth We first assessed changes in surface pH and in the pH gradient in neonatal rats between birth and day 7. Surface pH decreased progressively from initial, elevated values of 6.63 0.02 on days 0 to 1 to values of 5.35 0.02 on days 7 to 8 after birth. The amniotic fluid on day 19 of gestation exhibited a pH of 7.14 0.02, whereas the surface pH of fetal skin at the same gestational age was 6.80 0.02 (p ¼ o0.0001; Fig 1A). The surface pH in adult rats was 5.90 0.08. Neonatal pH levels were significantly higher not only at the surface of the SC (days 0–1 pH 6.65 0.10 vs. day 4 pH 5.91 0.08; ANOVA, po0.0001), but also in the deeper layers of the SC, both in young and in older neonates (Fig 1B). The pH-gradient of neonates was comparable to that observed in adult rats. The data shown in Fig 1B suggest that acidity starts in lower layers, consistent with recent reports (Behne et al, 2002). This recent publication showed a good correlation between FLIM measurements and the surface pH electrode suggesting that both techniques provide an accurate reflection of SC pH. These results show that the attainment of adult levels of acidification occurs much more quickly in the neonatal rat (i.e., within 7–8 d) than in human newborn SC, where it takes several weeks to months (Behrendt and Green, 1958; Fox et al, 1998; Visscher et al, 2000; Yosipovitch et al, 2000; Giusti et al, 2001).
b-Glucocerebrosidase activity increases in parallel with development of an acidic SC pH Prior studies have shown that lipid processing enzymes are expressed in epidermis late in fetal development (Hanley et al, 1997a, b). We also have shown that the activities of one of the key lipid-processing enzymes, b-glucocerebrosidase, is highly pH-dependent (Holleran et al, 1992), becoming inactive when SC is neutralized (Takagi et al, 1999). Therefore, we next assessed changes in the in situ activity of b-glucocerebrosidase as a surrogate marker of changes in SC acidification during the postnatal period. Zymographic analysis of b-glucocerebrosidase activity demonstrated that most enzyme activity localized within the SC extracellular spaces (Fig 2, note pronounced red signal), which is in accordance with previous publications of our group (Takagi et al, 1999). Sequential confocal sections revealed that the highest b-glucocerebrosidase activity on days 0 to 1 was detected at the stratum granulosum/SC level, whereas on days 5 to 6 the highest activity was located in both the lower and the upper SC (data from sequential sections not shown). These results further demonstrate the progressive postnatal, apical shift in SC acidification, using a surrogate marker, that is, changes in the activity of a pH-sensitive lipid-processing enzyme, b-glucocerebrosidase. The sequential confocal microscopy showed an activity pattern that suggests that the b-glucocerebrosidase activity is located intercellular. Nevertheless, the topical applied substrate does not exclude the presence of intracellular activity. Topical application was employed to closely mimic in vivo conditions.
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Although histomorphology and differentiation do not change between days 0 to 1 and 5 to 6, neonatal epidermis is hyperproliferative To further characterize the neonatal rat model, we first compared epidermal differentiation and proliferation by light microscopy, in day 0 to 1 versus day 5 to 6 neonatal rats. Hematoxylin and eosin staining showed no morphologic differences at the two time points (data not shown). Moreover, the expression of two differentiation-specific proteins, loricrin and involucrin, appeared unchanged by immunohistochemistry in day 0 to 1 versus day 5 to 6 animals (data not shown). Yet, the proportion of proliferating cell nuclear antigen-positive cells was significantly higher in basal cells of days 0 to 1 than in day 5 to 6 epidermis (60.2 2.7 positive cells per field on day 1 vs. 28.0 2.4 on day 5; mean SEM, n ¼ 6, po0.0001). These results show that neonatal epidermis, although morphologically similar to day 5 to 6 epidermis, is more hyperproliferative at birth compared to day 5 to 6 epidermis.
Figure 1 Formation of an acidic surface pH develops during the first postnatal week. (A) Surface pH decreases from an initial value of 6.63 0.02 on days 0 to 1 and 7.14 0.02 of the amniotic fluid (white bar) and on intrauterine fetuses 6.80 0.02 on day 19 of gestation to values of 5.35 0.02 on days 7 to 8 after birth. The pH value of adult, shaved rats was 5.90 0.08. (B) Neonates’ pH levels are significantly higher not only at the surface of the SC, but also in deeper SC layers than in older neonates (anova, po0.0001). Deeper layers at both ages are more acidic than superficial layers, suggesting that acidification starts in the lower SC. The pH gradient of adult rats is not different from those of day 4 newborns. No. of D-Squames, the number of attached and removed cellophane tapes. Each D-Squame removes approximately one cell layer.
We next assessed zymographically whether the reduced b-glucocerebrosidase activity on days 0 to 1 reflected pH differences versus decreased enzyme mass. The fluorophore in an acidic pH buffer (MES) was directly applied to sections of day 0 to 1 skin. As seen in Fig 2C, enzyme activity increased under these conditions to levels comparable to day 4 to 5 skin in the acidic buffer. Fig 2C serves as a positive control and demonstrates that the enzyme is present at birth but is inactive owing to the high pH on days 0 to 1. Together, these results show that the neutral pH of neonatal skin results in reduced activity of one key lipidprocessing enzyme, b-glucocerebrosidase.
Despite an elevated pH, neonatal rats display almost normal basal barrier function and SC hydration Although the morphology of the epidermis in hematoxylin and eosin-stained paraffin sections is normal at birth, and does not change substantially as acidification occurs over the first week after birth, we next assessed whether basal barrier function, quantified as TEWL, changes during postnatal development. Despite evidence of reduced b-glucocerebrosidase activity (see above), basal barrier function in neonatal rats was competent at birth (see also Aszterbaum et al, 1992), and it did not change throughout the newborn period (Fig 3). In adult rats TEWL is lower than in newborns suggesting that permeability barrier function is partially compromised in neonatal rats. Nevertheless, it must be recognized that the absolute TEWL level in the neonatal rat is still very low and is not indicative of a dysfunctional permeability barrier (for example, we often see similar levels in normal adult mice). Likewise, SC hydration, measured with a capacitance-based Corneometer, is both normal at birth and remains unchanged over the first postnatal week (data not shown). These results demonstrate that, despite a postnatal delay in acidification, basal barrier function and SC hydration are not abnormal in neonatal rat skin. Colonization with normal flora does not correlate with SC acidification Whereas the studies described above delineate certain changes that occur in neonatal rat epidermis during the first week of life, we next assessed a variety of exogenous and endogenous mechanisms as the basis for development of the acid mantle. We first assessed whether the progressive decrease in pH correlates with colonization by normal bacterial flora. Neither a positive nor a negative correlation could be detected for colonization by either aerobic (i.e., Micrococcaceae) or microaerophilic (Propionibacteriae) bacteria. For Propionibacteriae, bacterial counts tend to decline between days 1 to 2 and days 5 to 6, in parallel with the decrease in surface pH, and Micrococcaceae counts remain relatively constant over the entire postnatal first week (Fig 4A,B). Thus, the pattern of surface colonization by normal flora does not correlate with the
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Figure 2 Zymographic activity of b-glucocerebrosidase in neonatal rat SC. Zymographic analysis of b-glucocerebrosidase activity and localization showed low levels of activity in the intercellular spaces at birth (days 0–1, A), increasing markedly by days 5 to 6 (B). Enzyme activity can be induced on day 1 by acidifying the tissue (C). (C) Positive control to show that the enzyme is present at birth but inactive owing to the high pH. Lowering the pH results in an increase of b-glucocerebrosidase activity. The dotted line represents the dermal/epidermal junction. Negative controls both without substrate (vehicle alone) as well with an inhibitor (CBE) showed the absence of any activity (black figures not shown). Bar, 20 mm.
are not likely to account for the postnatal attainment of adult levels of pH.
Figure 3 Despite an elevated SC pH, basal barrier function of neonatal skin is competent. The TEWL, assessed by a closed-loop system (MEECO), showed similar values under basal conditions throughout the entire postnatal period (n ¼ 10, differences not significant between different newborn ages). Nevertheless, in adult rats, 24 h after shaving, lower TEWL are observed (n ¼ 3).
development of an acid mantle, and therefore it does not appear to account for the progressive, postnatal decline in SC surface pH.
Sebaceous gland products are not required for acidification of rodent SC Although rodent epidermis generally elaborates no eccrine glands, pilosebaceous structures appear during the first week after birth. Thus, nascent sebaceous gland products could, in theory, account for postnatal acidification. To assess the role of sebaceous gland products in SC acidification in general, we next compared SC pH and the pH gradient across the SC in adult asebia-J1 homozygote (profound hypoplasia of sebaceous glands) versus heterozygote and wild-type mouse littermates. The surface pH of asebia-J1 mice was not elevated in comparison to controls; instead, pH appeared to be slightly lower in asebia-J (6.20 0.29, n ¼ 6) than in wild-type mice (6.44 0.13, n ¼ 6), but the differences did not achieve statistical significance. These results show that sebaceous gland products are not required for acidification of adult rodent SC and therefore
Histidase activity does not account for sequential changes in postnatal acidification Whereas the studies described above appear to exclude certain exogenous mechanisms as the basis for postnatal SC acidification, one or more endogenous mechanisms could be required. In fact, it has been suggested that histidase-mediated generation of UCA accounts for the bulk of SC acidification (Krien and Kermici, 2000). Yet, we found no changes in histidase activity between day 0 to 1 and day 5 to 6 normal neonatal rats (Fig 5A). Moreover, his/his (Peruvian) mice, with histidase mutations that result in less than 10% of normal enzyme levels (Selden et al, 1995) display a normal (acidic) SC pH: HIS–/– 5.62 0.06, HIS þ /– 5.63 0.04, and wild-type mice 5.58 0.08 (Fig 5B). These results show, first, that changes in histidase activity do not correlate with the progressive postnatal acidification of neonatal SC and, second, that histidase activity is not required to generate the acidic pH of adult rodent SC. Epidermal NHE1 contributes to postnatal acidification Prior studies have shown that the non-energy-dependent, epidermal antiporter NHE1 contributes to bulk acidification of adult rodent SC, beginning in microdomains at the SC/ stratum granulosum interface; that is, NHE1 knockout mice exhibit one-third unit higher SC pH than do their wild-type littermates (Behne et al, 2002). To assess directly the role of NHE1 in postnatal acidification, we applied a specific inhibitor daily from day 0. Blockade of NHE1 antiporter activity by topical HOE694 treatment of neonatal rat skin delayed postnatal acidification (Fig 6). These results show that epidermal NHE1 activity contributes to postnatal acidification. Nevertheless, the levels of NHE1 in the epidermis, measured by immunohistochemistry or western blotting, did not change during neonatal development (M.J. Behne et al, manuscript in preparation). sPLA2 increases after birth and contributes to postnatal acidification We next assessed the role of sPLA2 activity, which has been implicated recently in the acidification of adult SC (Fluhr et al, 2001), in postnatal acidification. Total SC sPLA2, assessed by a fluorometric assay, increased by
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Figure 4 Colonization with normal flora does not correlate with postnatal SC acidification. (A) Colony-forming units per square centimeter of Micrococcaceae collected from the surface of the newborn rats at different time points after birth with a modified detergent scrub method. The bacteria were grown on sheep blood agar and counted after 48 h of aerobic incubation at 371C. No correlation could be detected between the formation of an acidic surface pH and surface colonization with Micrococcaceae. (B) Colony-forming units per square centimeter of Propionibacteriae collected from newborn rats as above. Bacteria were grown on reinforced clostridium medium agar supplemented with 60 mg per L furoxine and 1 mL per L Tween 80 and cultivated for 72 h under microareobic conditions at 371C. No correlation could be detected between the formation of an acidic surface pH and surface colonization with Propionibacteriae.
66.4% between day 0 and day 4 after birth (p ¼ 0.0018) (Fig 7A). Moreover, analysis of individual, sequentially obtained D-Squame strips showed that sPLA2 activity was limited to deeper layers of the SC on days 0 to 1, followed by progressive extension of activity to all SC layers by day 5 (data not shown). To further assess the role of sPLA2 in postnatal acidification, we next applied a sPLA2 inhibitor daily from days 0 to 1 to days 3 to 4 to neonatal rat skin. Single daily applications of bromophenacylbromide (BPB), an inhibitor of most mammalian sPLA2, delayed, but did not completely prevent, the emergence of an acidic SC (Fig 7B). A chemically unrelated inhibitor, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, showed similar results (data not shown). We tested the inhibitory capacity of both BPB and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phos-
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Figure 5 Changes in histidase activity do not correlate with postnatal acidification. (A) No significant changes in SC histidase activity occurred between day 0 to 1 and day 4 to 5 neonatal rat SC (values expressed as means SEM, ns; days 0–1, n ¼ 6; days 4–5, n ¼ 5). (B) No significant changes could be detected in the surface pH of HIS–/– (n ¼ 4) 5.62 0.06, HIS þ /– (n ¼ 7) 5.63 0.04, and wild-type mice (n ¼ 2) 5.58 0.08.
phomethanol on sPLA2 activity in vitro. Both inhibitors showed a concentration-dependent inhibition of PLA2 activity (data not shown). Further, to determine whether the inhibitor-induced delay in acidification results from lack of generation of pathway end products (i.e., FFA), we next coapplied a FFA product of sPLA2-mediated hydrolysis of epidermal phospholipids, palmitic acid, along with BPB. As seen in Fig 7C, coapplications of palmitic acid with BPB normalized the time table of postnatal acidification in neonatal rat skin. Finally, coapplications of BPB and the NHE1 inhibitor HOE694 together also blocked postnatal acidification of neonatal rat skin, but not more than did either inhibitor alone (not shown). Together, these studies implicate both pathways, sPLA2-generated FFA and NHE1, as contributors to the postnatal acidification of neonatal rat SC.
Discussion Late in gestation, fetal skin develops a permeability barrier, sufficient for life in a terrestrial environment, which is competent under basal conditions at birth (see also Aszterbaum et al, 1992). In fetal rat skin, the key steps leading to development of a competent barrier occur between days 20 and 21 (day 22 is term). Both normal cornified envelopes and mature lamellar bilayers are
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Figure 6 NHE1 antiporter contributes to postnatal acidification. Blockade of the NHE1 antiporter by topical HOE694 to for 3 d leads to a significant delay in neonatal acidification in comparison to vehicle-treated animals (n ¼ 8 and 11, inhibitor and vehicle, respectively).
present by 21 d, accounting for barrier competence (Aszterbaum et al, 1993; Williams et al, 1993). Yet, certain developmental changes continue after birth; for example, whereas adult SC is acidified, skin surface pH is neutral at birth in both humans and various animal models (Behrendt and Green, 1971; Hardman et al, 1998; Visscher et al, 2000; Yosipovitch et al, 2000). An acidic surface pH, the so-called ‘‘acid mantle,’’ develops within the first weeks to months of life in humans (Visscher et al, 2000). Whereas human SC does not achieve adult levels of acidification for up to 6 mo, the mechanisms responsible for the delayed acidification of the SC remain unknown. In this article, we describe a neonatal rat model in which adult levels of acidification are achieved in less than 1 wk. Using this model, we could assess a variety of mechanisms that have been either proposed or shown to contribute to the acidic pH of adult SC. Our results exclude a variety of exogenous and endogenous mechanisms previously implicated in SC acidification, pointing instead to contributory roles for two endogenous mechanisms, the sPLA2 pathway and NHE1, in the generation of the postnatal acid mantle. It has been assumed that microbial colonization, with its metabolic products, is a key acidification mechanism and, conversely, that an acidic pH, once established, favors colonization by normal flora over pathogens. The SC becomes colonized with normal flora during birth, a process that could be favored by a neutral pH as seen in some clinical conditions like diaper dermatitis. Subsequent microbial growth could then generate products that form the acid mantle, thereby limiting subsequent colonization by pathogens, such as Staphylococcus aureus and Streptococceae. Yet, we could not show here a convincing relationship between the establishment of neonatal normal flora and SC acidification. In fact, colonization by the major representatives of normal flora either leveled off immediately after birth (for Micrococcaceae) or actually declined (for Propionibacteriae). Moreover, if this flora were responsible for the progressive acidification of neonatal SC, we would expect that the surface of the SC would become acidic before the deeper levels. Instead, the deeper layers of the SC appeared to become acidic first, as shown both by sequential tape stripping and by zymography of the lipid-
Figure 7 sPLA2 activity increases after birth and contributes to postnatal acidification. (A) sPLA2 activity, assessed as total sPLA2 activity by a fluorometric assay on pooled tape strips, increased by 66.4% between days 0 to 1 and days 4 to 5 after birth (p ¼ 0.0018). (B) Moreover, a single daily applications of the global sPLA2 inhibitor, BPB, delayed, but did not completely prevent, emergence of an acidic SC. (C) Coapplications of palmitic acid, the product of phospholipid hydrolysis by sPLA2, along with BPB, normalized the rate of postnatal acidification.
processing enzyme b-glucocerebrosidase. In Fig 1B surface pH is higher than the pH seen after a single tape stripping, indicating that surface factors such as bacterial growth are not likely to be the major factor inducing the decrease in pH. Together, these results suggest that microbial colonization is not the mechanism for postnatal SC acidification. To definitively demonstrate that microbial colonization does not contribute to SC pH one would have to maintain animals in a sterile environment, which is extremely difficult to achieve. Thus, although our studies suggest that microbial colonization is not a major contributor, they do not completely rule out the potential impact of an early
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microbial colonization on the initiation of the development of an acidic SC pH. In addition to, or instead of, microbes, eccrine and/or sebaceous gland products (i.e., lactic acid and FFA, respectively) are commonly implicated, exogenous mechanisms for SC acidification. Although rodent skin contains no eccrine glands, sebaceous glands appear in parallel with hair follicles by 2 d after birth. Yet, our studies demonstrate a normal acidic SC in asebia mice, who exhibit a profound hypoplasia (498% loss) of sebaceous glands. These results appear to exclude a requirement for sebaceous gland products in SC acidification. Having excluded commonly implicated exogenous mechanisms, we focused attention on three potential endogenous acidification mechanisms. cUCA generation from histidine by the enzyme, HAL (histidase), was proposed recently as the principal source of SC acidification (Krien and Kermici, 2000). Histidase is a neutral pH-optimum enzyme, which localizes to the cytosol of corneocytes. Activation of histidase occurs only after prior hydrolysis of filaggrin to histidine, above the stratum compactum, and only at relative humidities of less than 80%. Moreover, because cUCA is a highly polar metabolite, it may remain restricted to the cytosol and not influence events in membrane domains. Further, to what extent this mechanism would be operative under conditions of high humidity, when less histidine would become available as a precursor for cUCA generation (Scott et al, 1982; Scott and Harding, 1986), is unknown. Thus, this mechanism may not be relevant for hydrolytic processes that occur deep in the SC and, more specifically, within the hydrophobic, extracellular compartments of the lower SC. Yet, it is in these domains that the processing events that lead to barrier formation (Elias and Menon, 1991) and desquamation (Williams and Elias, 1987) largely occur. Our results also tend to further exclude histidase in acidification of neonatal SC, because (1) histidase activity in neonatal SC does not increase between days 0 to 1 and days 5 to 6; and (2) adult his/his (Peruvian) mice, with loss-of-function mutations in histidase, display a normal (acidic) SC pH. It is of course possible that in the his/his (Peruvian) mice that the small amount of remaining histidase activity (o10%) might contribute to SC acidification. Nevertheless, our studies suggest that this pathway does not contribute significantly to bulk SC acidification. Exclusion of histidase as a potential endogenous acidifying mechanism led us to consider two alternate endogenous pathways, that is, the NHE1 antiporter and sPLA2 generation of FFA. NHE1 knockout mice exhibit a one-third pH unit increase in surface pH that contributes to bulk acidification of adult SC, but this exchanger more specifically acidifies the stratum granulosum–SC interface (Behne et al, 2002). In this study inhibition of NHE1 activity delayed postnatal acidification. These results, coupled with previous studies, suggest that the NHE1 antiporter contributes to the progressive acidification of neonatal SC. Finally, we found that the sPLA2 pathway, previously shown to account for at about two-third to one pH unit of the bulk acidification of adult SC (Fluhr et al, 2001), is also critical for postnatal acidification. Not only does sPLA2 activity in SC progressively increase after birth, but most
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importantly, sPLA2 blockade also delayed postnatal acidification. Moreover, we identified the sPLA2 product, FFA, as required for neonatal acidification, as previously demonstrated in adult SC (Fluhr et al, 2001). Yet, it should be noted that both NHE1 and sPLA2 blockade alone delayed, but did not completely prevent, postnatal acidification and that the combined application of both inhibitors did not increase neonatal SC pH above levels achieved with either inhibitor alone (data not shown). These results suggest that both mechanisms may influence the same or overlapping domains in the SC and that other, as yet unidentified acidification mechanisms also contribute to the progressive, postnatal acidification of the SC. In summary, this study demonstrated that acidification of the SC pH in neonatal rats occurs during the first 5 to 7 days and is mediated, in part, by increased activity and/or expression of NHE1 and sPLA2 in the SC. Materials and Methods Animals and materials Timed, pregnant Sprague-Dawley rats were obtained from Simonson Laboratories (Gilroy, CA) and fed Purina mouse diet and water ad libitum. Functional measurements were performed between postnatal days 0 and 6. Measurements on adult rats were performed 24 h after careful shaving. Propylene glycol, ethanol, NaOH, and HCl were from Fisher Scientific (Fairlane, NJ), whereas palmitic acid, bromphenacyl bromide (BPB), and all other chemicals were from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Sebaceous-gland-derived lipids as a source of surface pH were studied in asebia-J mice and their wild-type littermates (Jackson Laboratory Inc., Bar Harbor, ME; gift from Dr J. Sundberg). Asebia-J mice display profound sebaceous gland hypoplasia (Sundberg et al, 2000), and thus the importance of sebaceous gland products for SC pH can be studied by comparing affected homozygote mice to heterozygote and wild-type littermates. The pH of the SC was also compared in histidinemic (his/his; Peruvian) and wild-type mice, backcrossed to C57BL6 mice (Taylor et al, 1993; Selden et al, 1995), to assess whether endogenous urocanic acid, derived from histidase, contributes to an acidic SC pH. Both asebia and his/his mice were carefully shaved 24 h before the measurements. D-Squame tapes (22 mm), which were used for sequential tape stripping and the quantification of sPLA2 activity, were purchased from CuDerm (Dallas, TX). D-Squame cellophane tapes were attached and removed in a standardized fashion (Fluhr et al, 2001). Each D-Squame removes approximately one cell layer (validation study, data not shown). All experiments were performed with the approval of the Institutional Animals Studies Committee. Experimental procedures: surface pH, SC hydration, and transepidermal water loss measurements Permeability barrier function was determined as changes in levels of transepidermal water loss (TEWL), measured with an electrolytic water analyzer (MEECO, Warrington, PA). Surface pH was measured with a flat, glass surface electrode from Mettler-Toledo (Giessen, Germany), attached to a pH meter (skin pH meter Model PH 900, Courage & Khazaka, Cologne, Germany). The pH gradient within the SC was determined by measuring surface pH after each of four consecutive strippings with the 22-mm D-Squame disks, as described previously (Ohman and Vahlquist, 1994, 1998), and in some experiments pooled SC strippings were utilized for quantification of total sPLA2 activity (see below). SC hydration was measured with the capacitance-based Corneometer CM 825 (Courage & Khazaka), with values reported in arbitrary units (Fluhr et al, 1999a, b). To assess the importance of sPLA2, neonatal animals were treated topically twice daily for three d with BPB (4 mg/mL) in propylene glycol: ethanol (7:3, vol/vol) vehicle, the vehicle alone
122 : 2 FEBRUARY 2004 applied to the backs and flanks of the newborn rats ( 10 mL/ cm2), or a chemically unrelated sPLA2 inhibitor, 1-hexadecyl-3trifluoroethylglycero-sn-2-phosphomethanol (4 mg/mL), in propylene glycol:ethanol (7:3, vol/vol) vehicle, as described previously for hairless mice (Mao-Qiang et al, 1996; Fluhr et al, 2001). To prevent the maternal rats from licking applied substances off the newborns, we placed the pups in a plastic container in a 37 to 381C incubator for about 3 h after each application. The inhibitor doses that we employed have been shown previously to inhibit secretory PLA2 activity selectively in different tissues and cell types (Gelb et al, 1994; Jain et al, 1991) and to be nontoxic to murine skin, without evidence of inhibition of other synthetic activities (Mao-Qiang et al, 1995, 1996). For override experiments, animals received coapplications of palmitic acid (10 mg/mL) at the same time points that these sites were treated with sPLA2 inhibitors (Fluhr et al, 2001). The NHE1 antiporter was blocked with the specific inhibitor HOE 694 (gift from Aventis Pharma, Frankfurt, Germany). HOE 694 has shown to be a specific NHE1 inhibitor (Loh et al, 1996). Surface bacterial colonization Surface colonization with bacteria was assessed within the first 12 h after birth by a modified detergent scrub method (Williamson and Kligman, 1965). Briefly, 1 mL of phosphate buffer was pipetted into an open stainless-steel cylinder applied to the skin, and the surface was gently scrubbed with a Teflon rod for 60 s. The wash fluid was removed into a sterile tube, followed by a second, repeat scrubbing again with an additional 1 mL of phosphate buffer. Aliquots of the wash samples were pipetted onto blood agar plates, incubated at 371C for 48 h, and quantitated as log colony-forming units per cm2 for Micrococacceae. To assess the growth of Propionibacteria sp., we incubated an aliquot on propionibacteria-selective, reinforced clostridium medium, supplemented with 1 mL per L Tween 80 agar plates, grown under microareobic conditions for 72 h at 371C, and quantitated as log colony-forming units per cm2. No further identification of the bacterial species in the retrieved normal flora was performed. Propionibacteria and Micrococacceae are both mixtures of bacteria species representing the majority of the resident flora (Hoffler et al, 1980; Korting et al, 1988; Leyden, 2001). Microscopy and imaging Epidermal structure was assessed in 5-mm hematoxylin and eosin-stained sections (Zeiss AxioMat 2 microscope; Zeiss, Heidelberg, Germany) using a 20 objective. Digital images were captured by an AxioVision digital camera (Zeiss, Heidelberg, Germany), and pictures were assembled using Adobe Illustrator 9.0 and Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA). Immunohistochemistry Immunohistochemistry for differentiation markers was performed according to previously published protocols (Komuves et al, 2002). Briefly, the primary, affinity-purified rabbit antibodies, specific for involucrin, loricrin, and proliferating cell nuclear antigen were from BabCo (Richmond, CA). The secondary antibody was affinity-purified, biotinylated, goat antirabbit IgG from Vector Laboratories (Burlingame, CA). Either omission of the first antibody or incubation with the substrate solution alone resulted in negative immunostaining. Sections were examined without further counterstaining.
In situ zymographic assay of a-glucocerebrosidase activity Enzyme activity was measured according to a modification of a previously described method (Takagi et al, 1999). Briefly, 20 mL of 10 mM resorufin b-D-glucopyranoside dissolved in dimethyl sulfoxide (Molecular Probes, Eugene, OR) was applied to the backs of 0- to 5-d-old rats. The resorufin dye becomes fluorescent once the substrate is enzymatically cleaved. Following topical application, neonatal pups were placed for 2 to 3 h in a plastic container at 37 to 381C, as above. At the end of these in vivo incubations, 4-mm punchout biopsies were taken from treated and control (dimethyl sulfoxide alone) sites. The biopsies were placed on a plastic coverslip with a punched out center, inverted onto a microscopic slide, and covered with a second, nonpunched
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coverslip. The cleaved compound was visualized with a confocal microscope (as above), at an excitation wavelength of 568 nm and an emission wavelength of 580 nm. Resorufin alone served as an additional negative control and as a further check for specificity. a-Glucocerebrosidase enzyme activity was blocked when the fluorogenic substrate was coapplied with 10 mM conduritol B epoxide (Toronto Research Chemicals, Toronto, Ontario, Canada) (Takagi et al, 1999). Finally, to further assess whether postnatal changes in a-Glucocerebrosidase activity reflected pH effects alone, or developmental changes in enzyme expression, we also assessed whether an acidic buffer (MES at pH 5.5), added to day 0 to 1 sections, would increase in situ enzyme activity. Quantitation of sPLA2 activity sPLA2 activity was assessed in pooled, sequential SC tape strips (D-Squame) with a fluorometric assay, as described by Mazereeuw-Hautier et al (2000) and Radvanyi et al (1989). Briefly, sequential tape strips were collected down to the glistening layer, that is, until the surface of the newborn rats was shiny. In newborn rats on days 0 to 1, this level was achieved with approximately three strippings, whereas on days 4 to 5, approximately four to five strippings were necessary. To ensure uniform removal, the D-Squame tapes were placed on the skin surface with forceps, and the tape surface was gently rubbed three times over its entire surface. The tapes were stored individually in glass scintillation vials at 41C until assayed. D-Squame strips were each incubated at 41C for 1 h with 500 mL of 0.36 M H2SO4 and 500 mL of distilled water. The incubation solutions then were removed from the vials and centrifuged for 20 min at 400g at room temperature. Total sPLA2 activity in SC was quantified as the release of pyrenyldecanoic acid from 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phophocholine (Molecular Probes). The assay buffer consisted of 100 mM TrisHCl (pH 7.5), 200 mM NaCl, 2 mM ethylenediaminetetraacetate, and 2 mM ethylene glycol bis(b-aminoethyl ether) N,N0 -tetraacetic acid, to ensure that cytosolic (Ca2 þ -dependent) PLA2 activity is not measured. Ten microliters of bovine serum albumin (10%) and 20 mL of the substrate 2 mM 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phophocholine were added. Reactions were initiated by addition of 50 mL of the enzyme extract and 200 mL of 30 mM CaCl2, and fluorescence was recorded at time 0 and after 45 min of incubation at 371C. A standard curve (0–1.9 mU/mL final concentration) was generated using Naja naja sPLA2 (Sigma). Additional experiments also showed a comparable activity for sPLA2 from bee venom (Apis mellifera, Sigma). The PLA2 substrate and dilutions were freshly prepared before each set of experiments. Fluorescence was monitored with a spectrofluorometer at excitation and emission wavelengths of 341 and 376 nm, respectively. Quantitation of histidase activity SC was removed by sequential stripping with D-Squame 22-mm tapes down to the glistening layer as above, and histidase was extracted, as described by Jin et al (1994). Briefly, each D-Squame tape was incubated immediately after removal in 2 mL of extraction buffer:10.56 mM phosphate buffer, adjusted to pH 7.4, containing 1 mM ethylenediaminetetraacetate, 1 mM ethylene glycol bis(b-aminoethyl ether) N,N0 tetraacetic acid, 0.5 mM phenylmethylsulfonyl fluoride, 11.6 mM sodium cholate at 41C for 30 min, followed by disruption on ice with 30-s bursts in a sonicator for a total of 3 min. After an overnight incubation at 41C, the extraction liquid was concentrated by spinning the pooled D-Squame extraction liquid at 41C in Amicon Centriprep YM-10 (Millipore, Bedford, MA) columns. The concentrated extraction liquid was stored at 201C until histidase ammonia-lyase (HAL) activity could be quantified on all samples from the same series of experiments. Enzyme activity was measured according to a recently published modification (Rother et al, 2001) of a standard method (Tabor and Mehler, 1955). Briefly, HAL activity was assessed by preincubating 50 mL of the HAL extraction in 900 mL of aqueous incubation buffer containing 0.1 M sodium pyrophosphate, pH 9.3, 10 mM ZnCl2, and 2 mM
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glutathione at 251C for 5 min. The assays were performed in 1-cm quartz cuvettes after adding 50 mL of 0.5 M L-histidine in distilled H2O to initiate the reaction. The rate of change was measured at 277 mm at 30-s intervals over a total period of 5 min. A standard curve was generated using HAL from Sigma, dissolved in 0.1 M glutathione.
Statistical analyses and data presentation Statistical analyses were performed using Prism 3 software (Graph Pad Software Inc., San Diego, CA). Normal distributions were tested before calculating comparisons with an unpaired t test. When three or more experimental groups were compared, an ANOVA was performed, followed by a pairwise, post hoc Bonferroni test. Values are given as means SEM.
We thank Ja´nos Re´tey and Damar Ro¨ther for helpful discussions regarding the histidine ammonia-lyase assay. Sandra Chang assisted with the histologic and immunohistochemical preparation of samples. This study was supported by NIH grants AR 19098, AR 39448 (PP), RR00173, and HD29706; the VA Merit Review (MAU 3) ‘‘Creation and Maintenance of the Epidermal pH Gradient’’ (TM); and the Medical Research Service, Department of Veterans Affairs. DOI: 10.1046/j.0022-202X.2003.22204.x Manuscript received December 5, 2002; revised March 7, 2003; accepted for publication May 20, 2003 Address correspondence to: Joachim W. Fluhr, MD, Dermatology Service (190), Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. Email: [email protected]
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122 : 2 FEBRUARY 2004 Ohman H, Vahlquist A: In vivo studies concerning a pH gradient in human stratum corneum and upper epidermis. Acta Derm Venereol 74:375–379, 1994 Ohman H, Vahlquist A: The pH gradient over the stratum corneum differs in Xlinked recessive and autosomal dominant ichthyosis: A clue to the molecular origin of the ‘‘acid skin mantle’’? J Invest Dermatol 111:674– 677, 1998 Puhvel SM, Reisner RM, Sakamoto M: Analysis of lipid composition of isolated human sebaceous gland homogenates after incubation with cutaneous bacteria: Thin-layer chromatography. J Invest Dermatol 64:406–411, 1975 Radvanyi F, Jordan L, Russo-Marie F, Bon C: A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin. Anal Biochem 177:103–109, 1989 Rother D, Poppe L, Viergutz S, Langer B, Retey J: Characterization of the active site of histidine ammonia-lyase from Pseudomonas putida. Eur J Biochem 268:6011–6019, 2001 Schade HM: Zur physikalischen chemie der hautoberfla¨che. Arch Dermatol Syphil 154:690–716, 1928 Schmuth M, Man MQ, Weber F, et al: Permeability barrier disorder in NiemannPick disease: Sphingomyelin-ceramide processing required for normal barrier homeostasis. J Invest Dermatol 115:459–466, 2000 Scott IR, Harding CR: Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment. Dev Biol 115:84–92, 1986 Scott IR, Harding CR, Barrett JG: Histidine-rich protein of the keratohyalin granules: Source of the free amino acids, urocanic acid and pyrrolidone carboxylic acid in the stratum corneum. Biochim Biophys Acta 719:110– 117, 1982
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Selden C, Calnan D, Morgan N, Wilcox H, Carr E, Hodgson HJ: Histidinemia in mice: A metabolic defect treated using a novel approach to hepatocellular transplantation. Hepatology 21:1405–1412, 1995 Sundberg JP, Boggess D, Sundberg BA, Eilertsen K, Parimoo S, Filippi M, Stenn K: Asebia-2J (Scd1 (ab2J). A new allele and a model for scarring alopecia. Am J Pathol 156:2067–2075, 2000 Tabor H, Mehler AH: Histidase and urocanase. Methods Enzymol 2:228–233, 1955 Takagi Y, Kriehuber E, Imokawa G, Elias PM, Holleran WM: Beta-glucocerebrosidase activity in mammalian stratum corneum. J Lipid Res 40:861–869, 1999 Taylor RG, Grieco D, Clarke GA, McInnes RR, Taylor BA: Identification of the mutation in murine histidinemia (his) and genetic mapping of the murine histidase locus (Hal) on chromosome 10. Genomics 16:231–240, 1993 Visscher MO, Chatterjee R, Munson KA, Pickens WL, Hoath SB: Changes in diapered and nondiapered infant skin over the first month of life. Pediatr Dermatol 17:45–51, 2000 Williams ML, Aszterbaum M, Menon GK, Moser AH, Feingold KR, Hoath SB: Preservation of permeability barrier ontogenesis in the intrauterine growth-retarded fetal rat. Pediatr Res 33:418–424, 1993 Williams ML, Elias PM: The extracellular matrix of stratum corneum: Role of lipids in normal and pathological function. Crit Rev Ther Drug Carrier Syst 3:95– 122, 1987 Williamson P, Kligman AM: A new method for the quantitative investigation of cutaneous bacteria. J Invest Dermatol 45:498–503, 1965 Yosipovitch G, Maayan-Metzger A, Merlob P, Sirota L: Skin barrier properties in different body areas in neonates. Pediatrics 106:105–108, 2000
Dermatology and Medical Service, Veterans Affairs Medical Center, San Francisco, and Departments of Dermatology and Medicine, University of California, San Francisco, California, USA; zCenter for Hepatology, Department of Medicine, Royal Free and University College Medical School, Hampstead, London, UK; wDepartment of Dermatology and Allergology, Friedrich-Schiller-University, Jena, Germany
At birth, human stratum corneum (SC) displays a near-neutral surface pH, which declines over several days to weeks to months to an acidic pH, comparable to that of adults. Recent studies suggest that an acidic pH is required for normal permeability barrier homeostasis and SC integrity/cohesion. We assessed here the basis for postnatal acidification in the neonatal rat, where SC pH, as measured with a flat surface electrode, declines progressively from near-neutral levels (pH 6.63) on postnatal days 0 to 1 to adult levels (pH 5.9) or even below over the subsequent 7 to 8 d. The postnatal decline in SC pH was paralleled by a progressive activation of a pH-dependent hydrolytic enzyme, b-glucocerebrosidase. Because SC acidification could not be linked to commonly implicated exogenous factors, such as bacterial colonization, or the deposition of sebaceous gland products. We next assessed whether changes in one or more of three endogenous mechanisms demonstrate postnatal activity changes that contribute to the progressive development of an acidic SC pH. Although the histidine-to-urocanic acid pathway has been implicated in acidification of the adult SC, surface pH is completely normal in histidase-deficient (his/his, Peruvian) mice, ruling out a requirement for this mechanism. In contrast, when sodium/hydrogen antiporter-1 (NHE1), which predominantly acidifies membrane domains at the stratum granulosum–SC interface, is inhibited, postnatal acidification of the SC is partially blocked. Likewise, SC secretory phospholipase A2 (sPLA2) activity, measured with a fluorometric assay, is low at birth, but increases progressively (by 66%) over the first 5 d after birth, and inhibition of sPLA2 between days 0 to 1 and days 5 to 6 delays postnatal SC acidification. Together, these results describe a neonatal model, in which the development of an acidic surface pH can be ascribed, in part, to progressive SC acidification by two endogenous mechanisms, namely, sPLA2 and NHE1, which are known to be important for acidification of adult rodent SC. Conversely, the impaired acidification of neonatal SC, which has important functional and clinical consequences, can be explained by the relatively low activities of one or both of these mechanisms at birth.
Key words: neonatal rat/stratum corneum pH/barrier function. J Invest Dermatol 122:320 – 329, 2004
Stratum corneum (SC) maturation late in fetal development leads to the generation of a competent cutaneous permeability barrier, crucial both for postnatal survival in a terrestrial environment and for defense against exogenous environmental insults. In rodent skin (e.g., rat), the final steps of fetal epidermal development occur between days 20 and 22 (term) in a patterned fashion (dorsal–ventral) (Aszterbaum et al, 1992; Hardman et al, 1998, 1999). Both a fully developed cronified envelope (Akiyama et al, 2000; Lee et al, 1999) and abundant extracellular lamellar bilayers are present late in gestation (Hanley et al, 1997a). Moreover, basal permeability barrier function is competent at birth
(Aszterbaum et al, 1992; Aszterbaum et al, 1993; Williams et al, 1993). Yet, skin surface pH is neutral at birth both in humans and in various animal models (Behrendt and Green, 1971; Hardman et al, 1998; Visscher et al, 2000; Yosipovitch et al, 2000), and the absence of an acidic SC at birth has been associated with an increased risk of bacterial and yeast infections in neonates (Leyden and Kligman, 1978). In contrast, the acidic pH both of older neonates and adults is associated with decreased colonization by pathogenic bacteria (Aly et al, 1975; Puhvel et al, 1975) and favors adhesion of nonpathogenic bacteria to the SC (Bibel et al, 1987). Thus, the acidic pH of the SC has been postulated to mediate at least one important SC function, that is, its antibacterial properties, and thus the absence of an acidic pH at birth may have clinical consequences for the neonate. In addition to its putative antimicrobial activity, development of an acidic pH could be important for other SC
Abbreviations: BPB, bromophenacylbromide; cUCA, cis-urocanic acid; FFA, free fatty acids; HAL, histidase ammonia-lyase; NHE1, sodium/hydrogen antiporter-1; SC, stratum corneum; sPLA2, secretory phospholipase A2; TEWL, transepidermal water loss.
Copyright r 2004 by The Society for Investigative Dermatology, Inc.
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functions. Prior studies have shown that permeability barrier recovery after acute insults is impeded at a neutral versus acidic pH (Mauro et al, 1998), explicable by the acidic pH optimum of certain critical lipid-processing enzymes in the SC, that is, b-glucocerebrosidase (Takagi et al, 1999) and acidic sphingomyelinase (Jensen et al, 2000) (Schmuth et al, 2000), which generate a family of ceramides required for permeability barrier homeostasis (Holleran et al, 1993) (Jensen et al, 2000). Although basal barrier function is normal in neonates, we have shown recently that barrier homeostasis is impaired owing to the neutral pH of neonatal SC.1 Finally, the integrity and cohesion of adult SC are pHdependent; that is, an acidic pH favors a SC that is more resistant to mechanical disruption (Fluhr et al, 2001). Thus, the neutral pH of neonatal SC could explain other clinical abnormalities in neonatal skin, including an increased propensity to develop irritant contact (diaper) dermatitis and increased skin fragility. Although the SC acid mantle was first recognized decades ago (Schade, 1928; Blank, 1939; Draize, 1942; Beare et al, 1959; Jolly et al, 1961; Baden and Pathak, 1967), its origin is incompletely understood. Traditionally, exogenous (originating outside the epidermis) mechanisms have been invoked to explain the formation of the acid mantle. Such exogenous mechanisms include the generation of (1) microbial metabolites (e.g., it is widely believed that bacterial colonization, by generating acidic metabolites, is important for the postnatal development of an acidic surface pH); (2) free fatty acids (FFA) of pilosebaceous origin (Puhvel et al, 1975); and (3) eccrine-gland-derived products, such as lactic acid (Ament et al, 1997). Alternatively, endogenous processes have been implicated recently in acidification of adult SC including: (1) generation of cisurocanic acid (cUCA) from histidine (Krien and Kermici, 2000); (2) FFA generation from phospholipid hydrolysis (Fluhr et al, 2001); and (3) a sodium/proton antiporter, sodium/hydrogen antiporter-1 (NHE1) (Behne et al, 2002). Because neonatal SC is incompletely acidified, we hypothesized that one or more of these mechanisms might not be functional at birth. To begin to assess the mechanisms involved in the progressive, postnatal development of an acidic SC, we assessed acidification in newborn rats over the first week after birth. We report here that neonatal rats generate adult levels of SC acidification by 5 to 6 d after birth, compressing processes that require weeks to months in human neonates into less than 1 wk. In assessing the origins of postnatal SC acidification in this rodent model, and in available mutant murine models, we excluded roles for exogenous mechanisms, including bacterial colonization and sebaceous gland activity. Our results further exclude one endogenous mechanism, namely, the histidase pathway, as a major contributor to the development of an acid mantle during the immediate postnatal period. Instead, we provide here evidence that both secretory phospholipase A2 (sPLA2) and NHE1 activity contribute to, but do not completely account for, postnatal acidification. 1 Fluhr J, Fowler AJ, Ichachem J, Crumine D, Elias PM, Feingold KR: Consequences for the neonate of a neutral stratum corneum pH: Permeability barrier homeostasis. J Invest Dermatol 119: 249 2002 (abstr).
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Results Formation of an acidic surface pH occurs during the first week after birth We first assessed changes in surface pH and in the pH gradient in neonatal rats between birth and day 7. Surface pH decreased progressively from initial, elevated values of 6.63 0.02 on days 0 to 1 to values of 5.35 0.02 on days 7 to 8 after birth. The amniotic fluid on day 19 of gestation exhibited a pH of 7.14 0.02, whereas the surface pH of fetal skin at the same gestational age was 6.80 0.02 (p ¼ o0.0001; Fig 1A). The surface pH in adult rats was 5.90 0.08. Neonatal pH levels were significantly higher not only at the surface of the SC (days 0–1 pH 6.65 0.10 vs. day 4 pH 5.91 0.08; ANOVA, po0.0001), but also in the deeper layers of the SC, both in young and in older neonates (Fig 1B). The pH-gradient of neonates was comparable to that observed in adult rats. The data shown in Fig 1B suggest that acidity starts in lower layers, consistent with recent reports (Behne et al, 2002). This recent publication showed a good correlation between FLIM measurements and the surface pH electrode suggesting that both techniques provide an accurate reflection of SC pH. These results show that the attainment of adult levels of acidification occurs much more quickly in the neonatal rat (i.e., within 7–8 d) than in human newborn SC, where it takes several weeks to months (Behrendt and Green, 1958; Fox et al, 1998; Visscher et al, 2000; Yosipovitch et al, 2000; Giusti et al, 2001).
b-Glucocerebrosidase activity increases in parallel with development of an acidic SC pH Prior studies have shown that lipid processing enzymes are expressed in epidermis late in fetal development (Hanley et al, 1997a, b). We also have shown that the activities of one of the key lipid-processing enzymes, b-glucocerebrosidase, is highly pH-dependent (Holleran et al, 1992), becoming inactive when SC is neutralized (Takagi et al, 1999). Therefore, we next assessed changes in the in situ activity of b-glucocerebrosidase as a surrogate marker of changes in SC acidification during the postnatal period. Zymographic analysis of b-glucocerebrosidase activity demonstrated that most enzyme activity localized within the SC extracellular spaces (Fig 2, note pronounced red signal), which is in accordance with previous publications of our group (Takagi et al, 1999). Sequential confocal sections revealed that the highest b-glucocerebrosidase activity on days 0 to 1 was detected at the stratum granulosum/SC level, whereas on days 5 to 6 the highest activity was located in both the lower and the upper SC (data from sequential sections not shown). These results further demonstrate the progressive postnatal, apical shift in SC acidification, using a surrogate marker, that is, changes in the activity of a pH-sensitive lipid-processing enzyme, b-glucocerebrosidase. The sequential confocal microscopy showed an activity pattern that suggests that the b-glucocerebrosidase activity is located intercellular. Nevertheless, the topical applied substrate does not exclude the presence of intracellular activity. Topical application was employed to closely mimic in vivo conditions.
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Although histomorphology and differentiation do not change between days 0 to 1 and 5 to 6, neonatal epidermis is hyperproliferative To further characterize the neonatal rat model, we first compared epidermal differentiation and proliferation by light microscopy, in day 0 to 1 versus day 5 to 6 neonatal rats. Hematoxylin and eosin staining showed no morphologic differences at the two time points (data not shown). Moreover, the expression of two differentiation-specific proteins, loricrin and involucrin, appeared unchanged by immunohistochemistry in day 0 to 1 versus day 5 to 6 animals (data not shown). Yet, the proportion of proliferating cell nuclear antigen-positive cells was significantly higher in basal cells of days 0 to 1 than in day 5 to 6 epidermis (60.2 2.7 positive cells per field on day 1 vs. 28.0 2.4 on day 5; mean SEM, n ¼ 6, po0.0001). These results show that neonatal epidermis, although morphologically similar to day 5 to 6 epidermis, is more hyperproliferative at birth compared to day 5 to 6 epidermis.
Figure 1 Formation of an acidic surface pH develops during the first postnatal week. (A) Surface pH decreases from an initial value of 6.63 0.02 on days 0 to 1 and 7.14 0.02 of the amniotic fluid (white bar) and on intrauterine fetuses 6.80 0.02 on day 19 of gestation to values of 5.35 0.02 on days 7 to 8 after birth. The pH value of adult, shaved rats was 5.90 0.08. (B) Neonates’ pH levels are significantly higher not only at the surface of the SC, but also in deeper SC layers than in older neonates (anova, po0.0001). Deeper layers at both ages are more acidic than superficial layers, suggesting that acidification starts in the lower SC. The pH gradient of adult rats is not different from those of day 4 newborns. No. of D-Squames, the number of attached and removed cellophane tapes. Each D-Squame removes approximately one cell layer.
We next assessed zymographically whether the reduced b-glucocerebrosidase activity on days 0 to 1 reflected pH differences versus decreased enzyme mass. The fluorophore in an acidic pH buffer (MES) was directly applied to sections of day 0 to 1 skin. As seen in Fig 2C, enzyme activity increased under these conditions to levels comparable to day 4 to 5 skin in the acidic buffer. Fig 2C serves as a positive control and demonstrates that the enzyme is present at birth but is inactive owing to the high pH on days 0 to 1. Together, these results show that the neutral pH of neonatal skin results in reduced activity of one key lipidprocessing enzyme, b-glucocerebrosidase.
Despite an elevated pH, neonatal rats display almost normal basal barrier function and SC hydration Although the morphology of the epidermis in hematoxylin and eosin-stained paraffin sections is normal at birth, and does not change substantially as acidification occurs over the first week after birth, we next assessed whether basal barrier function, quantified as TEWL, changes during postnatal development. Despite evidence of reduced b-glucocerebrosidase activity (see above), basal barrier function in neonatal rats was competent at birth (see also Aszterbaum et al, 1992), and it did not change throughout the newborn period (Fig 3). In adult rats TEWL is lower than in newborns suggesting that permeability barrier function is partially compromised in neonatal rats. Nevertheless, it must be recognized that the absolute TEWL level in the neonatal rat is still very low and is not indicative of a dysfunctional permeability barrier (for example, we often see similar levels in normal adult mice). Likewise, SC hydration, measured with a capacitance-based Corneometer, is both normal at birth and remains unchanged over the first postnatal week (data not shown). These results demonstrate that, despite a postnatal delay in acidification, basal barrier function and SC hydration are not abnormal in neonatal rat skin. Colonization with normal flora does not correlate with SC acidification Whereas the studies described above delineate certain changes that occur in neonatal rat epidermis during the first week of life, we next assessed a variety of exogenous and endogenous mechanisms as the basis for development of the acid mantle. We first assessed whether the progressive decrease in pH correlates with colonization by normal bacterial flora. Neither a positive nor a negative correlation could be detected for colonization by either aerobic (i.e., Micrococcaceae) or microaerophilic (Propionibacteriae) bacteria. For Propionibacteriae, bacterial counts tend to decline between days 1 to 2 and days 5 to 6, in parallel with the decrease in surface pH, and Micrococcaceae counts remain relatively constant over the entire postnatal first week (Fig 4A,B). Thus, the pattern of surface colonization by normal flora does not correlate with the
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Figure 2 Zymographic activity of b-glucocerebrosidase in neonatal rat SC. Zymographic analysis of b-glucocerebrosidase activity and localization showed low levels of activity in the intercellular spaces at birth (days 0–1, A), increasing markedly by days 5 to 6 (B). Enzyme activity can be induced on day 1 by acidifying the tissue (C). (C) Positive control to show that the enzyme is present at birth but inactive owing to the high pH. Lowering the pH results in an increase of b-glucocerebrosidase activity. The dotted line represents the dermal/epidermal junction. Negative controls both without substrate (vehicle alone) as well with an inhibitor (CBE) showed the absence of any activity (black figures not shown). Bar, 20 mm.
are not likely to account for the postnatal attainment of adult levels of pH.
Figure 3 Despite an elevated SC pH, basal barrier function of neonatal skin is competent. The TEWL, assessed by a closed-loop system (MEECO), showed similar values under basal conditions throughout the entire postnatal period (n ¼ 10, differences not significant between different newborn ages). Nevertheless, in adult rats, 24 h after shaving, lower TEWL are observed (n ¼ 3).
development of an acid mantle, and therefore it does not appear to account for the progressive, postnatal decline in SC surface pH.
Sebaceous gland products are not required for acidification of rodent SC Although rodent epidermis generally elaborates no eccrine glands, pilosebaceous structures appear during the first week after birth. Thus, nascent sebaceous gland products could, in theory, account for postnatal acidification. To assess the role of sebaceous gland products in SC acidification in general, we next compared SC pH and the pH gradient across the SC in adult asebia-J1 homozygote (profound hypoplasia of sebaceous glands) versus heterozygote and wild-type mouse littermates. The surface pH of asebia-J1 mice was not elevated in comparison to controls; instead, pH appeared to be slightly lower in asebia-J (6.20 0.29, n ¼ 6) than in wild-type mice (6.44 0.13, n ¼ 6), but the differences did not achieve statistical significance. These results show that sebaceous gland products are not required for acidification of adult rodent SC and therefore
Histidase activity does not account for sequential changes in postnatal acidification Whereas the studies described above appear to exclude certain exogenous mechanisms as the basis for postnatal SC acidification, one or more endogenous mechanisms could be required. In fact, it has been suggested that histidase-mediated generation of UCA accounts for the bulk of SC acidification (Krien and Kermici, 2000). Yet, we found no changes in histidase activity between day 0 to 1 and day 5 to 6 normal neonatal rats (Fig 5A). Moreover, his/his (Peruvian) mice, with histidase mutations that result in less than 10% of normal enzyme levels (Selden et al, 1995) display a normal (acidic) SC pH: HIS–/– 5.62 0.06, HIS þ /– 5.63 0.04, and wild-type mice 5.58 0.08 (Fig 5B). These results show, first, that changes in histidase activity do not correlate with the progressive postnatal acidification of neonatal SC and, second, that histidase activity is not required to generate the acidic pH of adult rodent SC. Epidermal NHE1 contributes to postnatal acidification Prior studies have shown that the non-energy-dependent, epidermal antiporter NHE1 contributes to bulk acidification of adult rodent SC, beginning in microdomains at the SC/ stratum granulosum interface; that is, NHE1 knockout mice exhibit one-third unit higher SC pH than do their wild-type littermates (Behne et al, 2002). To assess directly the role of NHE1 in postnatal acidification, we applied a specific inhibitor daily from day 0. Blockade of NHE1 antiporter activity by topical HOE694 treatment of neonatal rat skin delayed postnatal acidification (Fig 6). These results show that epidermal NHE1 activity contributes to postnatal acidification. Nevertheless, the levels of NHE1 in the epidermis, measured by immunohistochemistry or western blotting, did not change during neonatal development (M.J. Behne et al, manuscript in preparation). sPLA2 increases after birth and contributes to postnatal acidification We next assessed the role of sPLA2 activity, which has been implicated recently in the acidification of adult SC (Fluhr et al, 2001), in postnatal acidification. Total SC sPLA2, assessed by a fluorometric assay, increased by
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Figure 4 Colonization with normal flora does not correlate with postnatal SC acidification. (A) Colony-forming units per square centimeter of Micrococcaceae collected from the surface of the newborn rats at different time points after birth with a modified detergent scrub method. The bacteria were grown on sheep blood agar and counted after 48 h of aerobic incubation at 371C. No correlation could be detected between the formation of an acidic surface pH and surface colonization with Micrococcaceae. (B) Colony-forming units per square centimeter of Propionibacteriae collected from newborn rats as above. Bacteria were grown on reinforced clostridium medium agar supplemented with 60 mg per L furoxine and 1 mL per L Tween 80 and cultivated for 72 h under microareobic conditions at 371C. No correlation could be detected between the formation of an acidic surface pH and surface colonization with Propionibacteriae.
66.4% between day 0 and day 4 after birth (p ¼ 0.0018) (Fig 7A). Moreover, analysis of individual, sequentially obtained D-Squame strips showed that sPLA2 activity was limited to deeper layers of the SC on days 0 to 1, followed by progressive extension of activity to all SC layers by day 5 (data not shown). To further assess the role of sPLA2 in postnatal acidification, we next applied a sPLA2 inhibitor daily from days 0 to 1 to days 3 to 4 to neonatal rat skin. Single daily applications of bromophenacylbromide (BPB), an inhibitor of most mammalian sPLA2, delayed, but did not completely prevent, the emergence of an acidic SC (Fig 7B). A chemically unrelated inhibitor, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, showed similar results (data not shown). We tested the inhibitory capacity of both BPB and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phos-
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Figure 5 Changes in histidase activity do not correlate with postnatal acidification. (A) No significant changes in SC histidase activity occurred between day 0 to 1 and day 4 to 5 neonatal rat SC (values expressed as means SEM, ns; days 0–1, n ¼ 6; days 4–5, n ¼ 5). (B) No significant changes could be detected in the surface pH of HIS–/– (n ¼ 4) 5.62 0.06, HIS þ /– (n ¼ 7) 5.63 0.04, and wild-type mice (n ¼ 2) 5.58 0.08.
phomethanol on sPLA2 activity in vitro. Both inhibitors showed a concentration-dependent inhibition of PLA2 activity (data not shown). Further, to determine whether the inhibitor-induced delay in acidification results from lack of generation of pathway end products (i.e., FFA), we next coapplied a FFA product of sPLA2-mediated hydrolysis of epidermal phospholipids, palmitic acid, along with BPB. As seen in Fig 7C, coapplications of palmitic acid with BPB normalized the time table of postnatal acidification in neonatal rat skin. Finally, coapplications of BPB and the NHE1 inhibitor HOE694 together also blocked postnatal acidification of neonatal rat skin, but not more than did either inhibitor alone (not shown). Together, these studies implicate both pathways, sPLA2-generated FFA and NHE1, as contributors to the postnatal acidification of neonatal rat SC.
Discussion Late in gestation, fetal skin develops a permeability barrier, sufficient for life in a terrestrial environment, which is competent under basal conditions at birth (see also Aszterbaum et al, 1992). In fetal rat skin, the key steps leading to development of a competent barrier occur between days 20 and 21 (day 22 is term). Both normal cornified envelopes and mature lamellar bilayers are
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Figure 6 NHE1 antiporter contributes to postnatal acidification. Blockade of the NHE1 antiporter by topical HOE694 to for 3 d leads to a significant delay in neonatal acidification in comparison to vehicle-treated animals (n ¼ 8 and 11, inhibitor and vehicle, respectively).
present by 21 d, accounting for barrier competence (Aszterbaum et al, 1993; Williams et al, 1993). Yet, certain developmental changes continue after birth; for example, whereas adult SC is acidified, skin surface pH is neutral at birth in both humans and various animal models (Behrendt and Green, 1971; Hardman et al, 1998; Visscher et al, 2000; Yosipovitch et al, 2000). An acidic surface pH, the so-called ‘‘acid mantle,’’ develops within the first weeks to months of life in humans (Visscher et al, 2000). Whereas human SC does not achieve adult levels of acidification for up to 6 mo, the mechanisms responsible for the delayed acidification of the SC remain unknown. In this article, we describe a neonatal rat model in which adult levels of acidification are achieved in less than 1 wk. Using this model, we could assess a variety of mechanisms that have been either proposed or shown to contribute to the acidic pH of adult SC. Our results exclude a variety of exogenous and endogenous mechanisms previously implicated in SC acidification, pointing instead to contributory roles for two endogenous mechanisms, the sPLA2 pathway and NHE1, in the generation of the postnatal acid mantle. It has been assumed that microbial colonization, with its metabolic products, is a key acidification mechanism and, conversely, that an acidic pH, once established, favors colonization by normal flora over pathogens. The SC becomes colonized with normal flora during birth, a process that could be favored by a neutral pH as seen in some clinical conditions like diaper dermatitis. Subsequent microbial growth could then generate products that form the acid mantle, thereby limiting subsequent colonization by pathogens, such as Staphylococcus aureus and Streptococceae. Yet, we could not show here a convincing relationship between the establishment of neonatal normal flora and SC acidification. In fact, colonization by the major representatives of normal flora either leveled off immediately after birth (for Micrococcaceae) or actually declined (for Propionibacteriae). Moreover, if this flora were responsible for the progressive acidification of neonatal SC, we would expect that the surface of the SC would become acidic before the deeper levels. Instead, the deeper layers of the SC appeared to become acidic first, as shown both by sequential tape stripping and by zymography of the lipid-
Figure 7 sPLA2 activity increases after birth and contributes to postnatal acidification. (A) sPLA2 activity, assessed as total sPLA2 activity by a fluorometric assay on pooled tape strips, increased by 66.4% between days 0 to 1 and days 4 to 5 after birth (p ¼ 0.0018). (B) Moreover, a single daily applications of the global sPLA2 inhibitor, BPB, delayed, but did not completely prevent, emergence of an acidic SC. (C) Coapplications of palmitic acid, the product of phospholipid hydrolysis by sPLA2, along with BPB, normalized the rate of postnatal acidification.
processing enzyme b-glucocerebrosidase. In Fig 1B surface pH is higher than the pH seen after a single tape stripping, indicating that surface factors such as bacterial growth are not likely to be the major factor inducing the decrease in pH. Together, these results suggest that microbial colonization is not the mechanism for postnatal SC acidification. To definitively demonstrate that microbial colonization does not contribute to SC pH one would have to maintain animals in a sterile environment, which is extremely difficult to achieve. Thus, although our studies suggest that microbial colonization is not a major contributor, they do not completely rule out the potential impact of an early
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microbial colonization on the initiation of the development of an acidic SC pH. In addition to, or instead of, microbes, eccrine and/or sebaceous gland products (i.e., lactic acid and FFA, respectively) are commonly implicated, exogenous mechanisms for SC acidification. Although rodent skin contains no eccrine glands, sebaceous glands appear in parallel with hair follicles by 2 d after birth. Yet, our studies demonstrate a normal acidic SC in asebia mice, who exhibit a profound hypoplasia (498% loss) of sebaceous glands. These results appear to exclude a requirement for sebaceous gland products in SC acidification. Having excluded commonly implicated exogenous mechanisms, we focused attention on three potential endogenous acidification mechanisms. cUCA generation from histidine by the enzyme, HAL (histidase), was proposed recently as the principal source of SC acidification (Krien and Kermici, 2000). Histidase is a neutral pH-optimum enzyme, which localizes to the cytosol of corneocytes. Activation of histidase occurs only after prior hydrolysis of filaggrin to histidine, above the stratum compactum, and only at relative humidities of less than 80%. Moreover, because cUCA is a highly polar metabolite, it may remain restricted to the cytosol and not influence events in membrane domains. Further, to what extent this mechanism would be operative under conditions of high humidity, when less histidine would become available as a precursor for cUCA generation (Scott et al, 1982; Scott and Harding, 1986), is unknown. Thus, this mechanism may not be relevant for hydrolytic processes that occur deep in the SC and, more specifically, within the hydrophobic, extracellular compartments of the lower SC. Yet, it is in these domains that the processing events that lead to barrier formation (Elias and Menon, 1991) and desquamation (Williams and Elias, 1987) largely occur. Our results also tend to further exclude histidase in acidification of neonatal SC, because (1) histidase activity in neonatal SC does not increase between days 0 to 1 and days 5 to 6; and (2) adult his/his (Peruvian) mice, with loss-of-function mutations in histidase, display a normal (acidic) SC pH. It is of course possible that in the his/his (Peruvian) mice that the small amount of remaining histidase activity (o10%) might contribute to SC acidification. Nevertheless, our studies suggest that this pathway does not contribute significantly to bulk SC acidification. Exclusion of histidase as a potential endogenous acidifying mechanism led us to consider two alternate endogenous pathways, that is, the NHE1 antiporter and sPLA2 generation of FFA. NHE1 knockout mice exhibit a one-third pH unit increase in surface pH that contributes to bulk acidification of adult SC, but this exchanger more specifically acidifies the stratum granulosum–SC interface (Behne et al, 2002). In this study inhibition of NHE1 activity delayed postnatal acidification. These results, coupled with previous studies, suggest that the NHE1 antiporter contributes to the progressive acidification of neonatal SC. Finally, we found that the sPLA2 pathway, previously shown to account for at about two-third to one pH unit of the bulk acidification of adult SC (Fluhr et al, 2001), is also critical for postnatal acidification. Not only does sPLA2 activity in SC progressively increase after birth, but most
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importantly, sPLA2 blockade also delayed postnatal acidification. Moreover, we identified the sPLA2 product, FFA, as required for neonatal acidification, as previously demonstrated in adult SC (Fluhr et al, 2001). Yet, it should be noted that both NHE1 and sPLA2 blockade alone delayed, but did not completely prevent, postnatal acidification and that the combined application of both inhibitors did not increase neonatal SC pH above levels achieved with either inhibitor alone (data not shown). These results suggest that both mechanisms may influence the same or overlapping domains in the SC and that other, as yet unidentified acidification mechanisms also contribute to the progressive, postnatal acidification of the SC. In summary, this study demonstrated that acidification of the SC pH in neonatal rats occurs during the first 5 to 7 days and is mediated, in part, by increased activity and/or expression of NHE1 and sPLA2 in the SC. Materials and Methods Animals and materials Timed, pregnant Sprague-Dawley rats were obtained from Simonson Laboratories (Gilroy, CA) and fed Purina mouse diet and water ad libitum. Functional measurements were performed between postnatal days 0 and 6. Measurements on adult rats were performed 24 h after careful shaving. Propylene glycol, ethanol, NaOH, and HCl were from Fisher Scientific (Fairlane, NJ), whereas palmitic acid, bromphenacyl bromide (BPB), and all other chemicals were from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Sebaceous-gland-derived lipids as a source of surface pH were studied in asebia-J mice and their wild-type littermates (Jackson Laboratory Inc., Bar Harbor, ME; gift from Dr J. Sundberg). Asebia-J mice display profound sebaceous gland hypoplasia (Sundberg et al, 2000), and thus the importance of sebaceous gland products for SC pH can be studied by comparing affected homozygote mice to heterozygote and wild-type littermates. The pH of the SC was also compared in histidinemic (his/his; Peruvian) and wild-type mice, backcrossed to C57BL6 mice (Taylor et al, 1993; Selden et al, 1995), to assess whether endogenous urocanic acid, derived from histidase, contributes to an acidic SC pH. Both asebia and his/his mice were carefully shaved 24 h before the measurements. D-Squame tapes (22 mm), which were used for sequential tape stripping and the quantification of sPLA2 activity, were purchased from CuDerm (Dallas, TX). D-Squame cellophane tapes were attached and removed in a standardized fashion (Fluhr et al, 2001). Each D-Squame removes approximately one cell layer (validation study, data not shown). All experiments were performed with the approval of the Institutional Animals Studies Committee. Experimental procedures: surface pH, SC hydration, and transepidermal water loss measurements Permeability barrier function was determined as changes in levels of transepidermal water loss (TEWL), measured with an electrolytic water analyzer (MEECO, Warrington, PA). Surface pH was measured with a flat, glass surface electrode from Mettler-Toledo (Giessen, Germany), attached to a pH meter (skin pH meter Model PH 900, Courage & Khazaka, Cologne, Germany). The pH gradient within the SC was determined by measuring surface pH after each of four consecutive strippings with the 22-mm D-Squame disks, as described previously (Ohman and Vahlquist, 1994, 1998), and in some experiments pooled SC strippings were utilized for quantification of total sPLA2 activity (see below). SC hydration was measured with the capacitance-based Corneometer CM 825 (Courage & Khazaka), with values reported in arbitrary units (Fluhr et al, 1999a, b). To assess the importance of sPLA2, neonatal animals were treated topically twice daily for three d with BPB (4 mg/mL) in propylene glycol: ethanol (7:3, vol/vol) vehicle, the vehicle alone
122 : 2 FEBRUARY 2004 applied to the backs and flanks of the newborn rats ( 10 mL/ cm2), or a chemically unrelated sPLA2 inhibitor, 1-hexadecyl-3trifluoroethylglycero-sn-2-phosphomethanol (4 mg/mL), in propylene glycol:ethanol (7:3, vol/vol) vehicle, as described previously for hairless mice (Mao-Qiang et al, 1996; Fluhr et al, 2001). To prevent the maternal rats from licking applied substances off the newborns, we placed the pups in a plastic container in a 37 to 381C incubator for about 3 h after each application. The inhibitor doses that we employed have been shown previously to inhibit secretory PLA2 activity selectively in different tissues and cell types (Gelb et al, 1994; Jain et al, 1991) and to be nontoxic to murine skin, without evidence of inhibition of other synthetic activities (Mao-Qiang et al, 1995, 1996). For override experiments, animals received coapplications of palmitic acid (10 mg/mL) at the same time points that these sites were treated with sPLA2 inhibitors (Fluhr et al, 2001). The NHE1 antiporter was blocked with the specific inhibitor HOE 694 (gift from Aventis Pharma, Frankfurt, Germany). HOE 694 has shown to be a specific NHE1 inhibitor (Loh et al, 1996). Surface bacterial colonization Surface colonization with bacteria was assessed within the first 12 h after birth by a modified detergent scrub method (Williamson and Kligman, 1965). Briefly, 1 mL of phosphate buffer was pipetted into an open stainless-steel cylinder applied to the skin, and the surface was gently scrubbed with a Teflon rod for 60 s. The wash fluid was removed into a sterile tube, followed by a second, repeat scrubbing again with an additional 1 mL of phosphate buffer. Aliquots of the wash samples were pipetted onto blood agar plates, incubated at 371C for 48 h, and quantitated as log colony-forming units per cm2 for Micrococacceae. To assess the growth of Propionibacteria sp., we incubated an aliquot on propionibacteria-selective, reinforced clostridium medium, supplemented with 1 mL per L Tween 80 agar plates, grown under microareobic conditions for 72 h at 371C, and quantitated as log colony-forming units per cm2. No further identification of the bacterial species in the retrieved normal flora was performed. Propionibacteria and Micrococacceae are both mixtures of bacteria species representing the majority of the resident flora (Hoffler et al, 1980; Korting et al, 1988; Leyden, 2001). Microscopy and imaging Epidermal structure was assessed in 5-mm hematoxylin and eosin-stained sections (Zeiss AxioMat 2 microscope; Zeiss, Heidelberg, Germany) using a 20 objective. Digital images were captured by an AxioVision digital camera (Zeiss, Heidelberg, Germany), and pictures were assembled using Adobe Illustrator 9.0 and Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA). Immunohistochemistry Immunohistochemistry for differentiation markers was performed according to previously published protocols (Komuves et al, 2002). Briefly, the primary, affinity-purified rabbit antibodies, specific for involucrin, loricrin, and proliferating cell nuclear antigen were from BabCo (Richmond, CA). The secondary antibody was affinity-purified, biotinylated, goat antirabbit IgG from Vector Laboratories (Burlingame, CA). Either omission of the first antibody or incubation with the substrate solution alone resulted in negative immunostaining. Sections were examined without further counterstaining.
In situ zymographic assay of a-glucocerebrosidase activity Enzyme activity was measured according to a modification of a previously described method (Takagi et al, 1999). Briefly, 20 mL of 10 mM resorufin b-D-glucopyranoside dissolved in dimethyl sulfoxide (Molecular Probes, Eugene, OR) was applied to the backs of 0- to 5-d-old rats. The resorufin dye becomes fluorescent once the substrate is enzymatically cleaved. Following topical application, neonatal pups were placed for 2 to 3 h in a plastic container at 37 to 381C, as above. At the end of these in vivo incubations, 4-mm punchout biopsies were taken from treated and control (dimethyl sulfoxide alone) sites. The biopsies were placed on a plastic coverslip with a punched out center, inverted onto a microscopic slide, and covered with a second, nonpunched
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coverslip. The cleaved compound was visualized with a confocal microscope (as above), at an excitation wavelength of 568 nm and an emission wavelength of 580 nm. Resorufin alone served as an additional negative control and as a further check for specificity. a-Glucocerebrosidase enzyme activity was blocked when the fluorogenic substrate was coapplied with 10 mM conduritol B epoxide (Toronto Research Chemicals, Toronto, Ontario, Canada) (Takagi et al, 1999). Finally, to further assess whether postnatal changes in a-Glucocerebrosidase activity reflected pH effects alone, or developmental changes in enzyme expression, we also assessed whether an acidic buffer (MES at pH 5.5), added to day 0 to 1 sections, would increase in situ enzyme activity. Quantitation of sPLA2 activity sPLA2 activity was assessed in pooled, sequential SC tape strips (D-Squame) with a fluorometric assay, as described by Mazereeuw-Hautier et al (2000) and Radvanyi et al (1989). Briefly, sequential tape strips were collected down to the glistening layer, that is, until the surface of the newborn rats was shiny. In newborn rats on days 0 to 1, this level was achieved with approximately three strippings, whereas on days 4 to 5, approximately four to five strippings were necessary. To ensure uniform removal, the D-Squame tapes were placed on the skin surface with forceps, and the tape surface was gently rubbed three times over its entire surface. The tapes were stored individually in glass scintillation vials at 41C until assayed. D-Squame strips were each incubated at 41C for 1 h with 500 mL of 0.36 M H2SO4 and 500 mL of distilled water. The incubation solutions then were removed from the vials and centrifuged for 20 min at 400g at room temperature. Total sPLA2 activity in SC was quantified as the release of pyrenyldecanoic acid from 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phophocholine (Molecular Probes). The assay buffer consisted of 100 mM TrisHCl (pH 7.5), 200 mM NaCl, 2 mM ethylenediaminetetraacetate, and 2 mM ethylene glycol bis(b-aminoethyl ether) N,N0 -tetraacetic acid, to ensure that cytosolic (Ca2 þ -dependent) PLA2 activity is not measured. Ten microliters of bovine serum albumin (10%) and 20 mL of the substrate 2 mM 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phophocholine were added. Reactions were initiated by addition of 50 mL of the enzyme extract and 200 mL of 30 mM CaCl2, and fluorescence was recorded at time 0 and after 45 min of incubation at 371C. A standard curve (0–1.9 mU/mL final concentration) was generated using Naja naja sPLA2 (Sigma). Additional experiments also showed a comparable activity for sPLA2 from bee venom (Apis mellifera, Sigma). The PLA2 substrate and dilutions were freshly prepared before each set of experiments. Fluorescence was monitored with a spectrofluorometer at excitation and emission wavelengths of 341 and 376 nm, respectively. Quantitation of histidase activity SC was removed by sequential stripping with D-Squame 22-mm tapes down to the glistening layer as above, and histidase was extracted, as described by Jin et al (1994). Briefly, each D-Squame tape was incubated immediately after removal in 2 mL of extraction buffer:10.56 mM phosphate buffer, adjusted to pH 7.4, containing 1 mM ethylenediaminetetraacetate, 1 mM ethylene glycol bis(b-aminoethyl ether) N,N0 tetraacetic acid, 0.5 mM phenylmethylsulfonyl fluoride, 11.6 mM sodium cholate at 41C for 30 min, followed by disruption on ice with 30-s bursts in a sonicator for a total of 3 min. After an overnight incubation at 41C, the extraction liquid was concentrated by spinning the pooled D-Squame extraction liquid at 41C in Amicon Centriprep YM-10 (Millipore, Bedford, MA) columns. The concentrated extraction liquid was stored at 201C until histidase ammonia-lyase (HAL) activity could be quantified on all samples from the same series of experiments. Enzyme activity was measured according to a recently published modification (Rother et al, 2001) of a standard method (Tabor and Mehler, 1955). Briefly, HAL activity was assessed by preincubating 50 mL of the HAL extraction in 900 mL of aqueous incubation buffer containing 0.1 M sodium pyrophosphate, pH 9.3, 10 mM ZnCl2, and 2 mM
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glutathione at 251C for 5 min. The assays were performed in 1-cm quartz cuvettes after adding 50 mL of 0.5 M L-histidine in distilled H2O to initiate the reaction. The rate of change was measured at 277 mm at 30-s intervals over a total period of 5 min. A standard curve was generated using HAL from Sigma, dissolved in 0.1 M glutathione.
Statistical analyses and data presentation Statistical analyses were performed using Prism 3 software (Graph Pad Software Inc., San Diego, CA). Normal distributions were tested before calculating comparisons with an unpaired t test. When three or more experimental groups were compared, an ANOVA was performed, followed by a pairwise, post hoc Bonferroni test. Values are given as means SEM.
We thank Ja´nos Re´tey and Damar Ro¨ther for helpful discussions regarding the histidine ammonia-lyase assay. Sandra Chang assisted with the histologic and immunohistochemical preparation of samples. This study was supported by NIH grants AR 19098, AR 39448 (PP), RR00173, and HD29706; the VA Merit Review (MAU 3) ‘‘Creation and Maintenance of the Epidermal pH Gradient’’ (TM); and the Medical Research Service, Department of Veterans Affairs. DOI: 10.1046/j.0022-202X.2003.22204.x Manuscript received December 5, 2002; revised March 7, 2003; accepted for publication May 20, 2003 Address correspondence to: Joachim W. Fluhr, MD, Dermatology Service (190), Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. Email: [email protected]
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