The pH of the SC influences at least four key epidermal functions (permeability barrier homeostasis, integrity/cohesion (desquamation), initiation of inflammation, and antimicrobial defense) (Elias, 2005
). Hence, the maintenance of low acidity provides a mechanism that coordinately integrates these key functions (Elias, 2005
). The importance of pH in vivo
was first shown in experiments where permeability barrier function is acutely abrogated, producing a parallel elevation in pH (Mauro et al., 1998
). Under these conditions, it was pH itself, rather than barrier requirements, that upregulated endogenous acidification mechanisms, that is, sodium/hydrogen exchanger 1 (NHE1) antiporter expression— barrier restoration alone did not influence the rate of reacidification (Hachem et al., 2005a
). As inflammatory dermatoses often show parallel alterations in all four of the above key functions, we asked here first whether maintenance of a normal or hyperacidic pH could reverse or prevent either hapten-induced acute allergic contact dermatitis or AD. Although exogenous SC acidification reversed neither hapten-induced acute allergic contact dermatitis nor preexistent Ox-AD, exogenous co-acidification alone restricted development of hapten-induced AD. Indeed, acidification of SC not only normalized epidermal structure and function in the face of ongoing hapten challenges, but also significantly attenuated most components of the inflammatory response, including Th2 markers, epidermal hyperplasia, tissue eosinophil and mast cell densities, and Th2 cell counts within the residual infiltrate. Even serum IgE levels decreased significantly with topical co-acidification. As neither pre- nor co-acidification prevented the development of inflammation during the AACD elicitation phase, despite normalizing epidermal function, it seems safe to assume that our results reflect neither a physical–chemical effect of acidification nor interference with hapten absorption. Instead, our results appear to show that it is pH-induced maintenance of normal epidermal structure and function that most likely accounts for the efficacy of this strategy in Ox-AD.
Normalization of barrier function most likely decreases inflammation by two unrelated mechanisms. First, it most likely restricts antigen access during repeated-challenge phase, but this supposition must still be verified. These studies suggest that an additional anti-inflammatory mechanism is operative, that is, reduced evidence for cytokine activation. Previous studies have shown that barrier disruption stimulates epidermal cytokine generation (Wood et al., 1992
; Nickoloff and Naidu, 1994
); and conversely, normalization of barrier function by occlusion decreases epidermal primary cytokine generation in models of chronic inflammation (Wood et al., 1994a
). The acidification-induced reduction in cytokine generation most likely reflects the observed reduction in SP activity (Nylander-Lundqvist and Egelrud, 1997
). In summary, it is most likely that both mechanisms are operative (that is, decreased hapten ingress and decreased cytokine activation), together accounting for the ability of SC acidification to largely protect against the development of inflammation in this mouse model of AD.
Our study provided additional mechanistic insights about how maintenance of an acidic pH most likely prevents emergence of the barrier abnormality in hapten-induced AD. A characteristic structural feature of human AD is a reduction in extracellular lamellar bilayers (Chamlin et al., 2002
), as also shown here for Ox-AD, most likely rendering the SC more porous to transcutaneous water loss (and simultaneously more susceptible to pro-inflammatory ingress of haptens). We confirmed here that the basis for this abnormality in the Ox-AD model could be a pH-induced increase in SP activity in the outer epidermis (Man et al., 2008
), with several potentially adverse consequences. The increased pH of Ox-AD epidermis downregulates the activities of the key ceramide-generating enzyme, β-glucocerebrosidase, which shows an acidic pH optimum (reviewed by Holleran et al., 2006
). A sustained increase in SP activity not only inactivates lipid-processing enzymes, but also degrades the lipid-processing enzymes that generate the lamellar bilayers that provide for barrier function (Hachem et al., 2005b
). However, whether lipid-processing enzyme protein content decreases in AD or in Ox-AD mice is not known. SP-mediated inhibition of LB secretion also occurs in Ox-AD, which most likely results from increased SP signaling of the PAR2 receptor (Hachem et al., 2006a
). It should be noted that such a failure in both lipid secretion and ceramide generation correlates with the reported reduction in total SC lipids and further decrease in ceramide content that occurs in human AD (Melnik et al., 1988
; Imokawa et al., 1991
; Di Nardo et al., 1998
). Finally, and in parallel, abnormal/heightened SP-PAR2 binding could accelerate terminal differentiation (physiological apoptosis or programmed cell death), a process that traps unsecreted LB within the cytosol of nascent corneocytes (Demerjian et al., 2008
). Taken together, these mechanisms most likely account for the observed abnormalities in lamellar bilayer structure, and in an overall reduction in the quantities of extracellular lamellae in Ox-AD, as well as the abnormal desquamation (poor integrity) of the SC in AD (Cork et al., 2006
). Conversely, we show here that the maintenance of a reduced pH improves permeability barrier function by preventing emergence of many of the above-described pathogenic mechanisms. Reduction in pH would not only allow optimal lipid-processing enzyme activity, but also block SP-mediated degradation of these enzymes (previously shown to be a pH-dependent process) (Hachem et al., 2005b
). In support of these proposed mechanisms, we showed here β-glucocerebrosidase activity increases in acidified, hapten-challenged skin, as well as ultrastructural evidence of accelerated lamellar membrane “maturation” in such acidified sites, consistent with normalization of this enzyme activity in the SC. Thus, the maintenance of an acidic pH could normalize barrier function in murine AD by several distinct, yet interdependent, mechanisms.
This work has implications for the primary or ancillary prevention of AD, as topical PHAs, such as LBA, are “GRAS” ingredients, that is, “Generally Regarded As Safe.” Applications of PHA have been shown to improve barrier function in both neonatal and aged rodent skin (Fluhr et al., 2004
; Choi et al., 2007a
), and even to “super-normalize” barrier function in normal mice (Hachem et al., 2009
), and in humans (Gunathilake, 2009
). Current forms of therapy for AD, regardless of type, could readily exploit these findings simply by ensuring that the vehicle used for topical applications is buffered to achieve a sustained reduction in the pH of diseased SC. Alternatively, SC pH could also be manipulated indirectly by stimulating activity or expression of endogenous epidermal acidifying mechanisms, such as the NHE1 antiporter and/or secretory phospholipase A2
). Both topical peroxisome proliferator activator receptor-α and liver-X receptor activators have already been deployed successfully to normalize SC acidity (and epidermal structure and function) in neonatal murine skin (Fluhr et al., 2005
), and our recent results suggest their use in attenuating AD in a murine model (Hatano et al
., in preparation). Nevertheless, the use of all of these approaches for human AD remains to be assessed.