Methods to assess barrier function
Barrier function can be assessed in a variety of ways.10,11 The integrity of the skin barrier can be assessed objectively in a noninvasive way by measuring transepidermal water loss (TEWL).12,13 Skin barrier function as assessed by TEWL is intrinsically compromised in children with AD but not in children with other allergic conditions.14 The magnitude of increase in TEWL correlates with AD severity.14,15 The TEWL is higher in AD patients16–19 than in healthy control subjects, even in clinically unaffected areas.20 In humans, TEWL varies between anatomical sites,21–23 and it is affected by a variety of factors, such as the age of the patient,22,24,25 but it is not linked to particular genetic mutations.26 Measurement of TEWL, moreover, allows assessment of otherwise unnoticed damage to the epidermal water barrier in nonlesional skin. Interestingly, TEWL has been reported to be higher in patients with extrinsic AD than in patients with the intrinsic form (patients with AD in whom allergen-specific IgE is not detectable).27 Patients with the intrinsic form have been documented to retain a normal barrier function and sensory reactivity to external pruritic stimuli.27 The advantages and disadvantages of various TEWL-measuring devices (i.e. evaporimeters) have been investigated.28 In open-chamber devices, the probe consists of a cylindrical open chamber with two sensors mounted in the diffusion chamber to measure the humidity and the temperature. The open-chamber devices are based on the diffusion principle, in which the water vapour pressure gradient is indirectly measured by two pairs of combined thermistors and hygrosensors, present at two different heights inside a hollow cylinder. The water pressure measured in the chamber is used to calculate the evaporated water over a constant skin area. The probe head is placed horizontally on the skin at a constant pressure, and its small size minimizes the influence of air turbulence inside the probe. The measuring usually takes between 30 and 40 s. Criticisms of this traditional open system are related to effects of ambient and body-induced airflows near the probe, probe size, the limitation of measurement sites and application/probe angles. Other important factors to consider during TEWL measurements with an open-chamber method are air convection, room temperature and ambient humidity. Another consideration is that volatile agents, other than water, might affect the readings (e.g. when measurements are made immediately after application of moisturizers).
Closed-chamber devices use a closed, unventilated chamber system, which is not affected by ambient or body-induced airflows. Closed-chamber conditions are created upon skin contact with a surface area of 1 cm diameter, and the measuring time is between 8 and 10 s. It has been proposed that closed-chamber devices allowed measurements at any angle, had short reading times and were insensitive to external air currents. A recent study reported that both open- and closed-chamber devices are able to estimate water loss rates accurately when held in a vertical position.28 Even though closed-chamber evaporimeters might be easier to use than open-chamber devices, their tendency to become saturated in conditions of high water loss are a disadvantage when assessing TEWL dynamically.28 Open- and closed-chamber TEWL readings, furthermore, correlate well.29 The TEWL values of healthy volunteers were measured simultaneously with three closed- and open-chamber devices in the same room according to the guidelines of the standardization group of the European Society of Contact Dermatitis, and there was no statistically significant difference between the mean forearm TEWL values measured by all three instruments. The authors concluded that the TEWL values measured by all instruments were constant, with small standard deviations.30
As TEWL measurements appear to be variable, it has been suggested that continuous assessment of the epidermal water barrier by multiple TEWL measurements over longer periods of time would decrease the risk of mistakes and increase accuracy.31
Skin capacitance is another parameter used to assess the skin barrier function. Electrical skin capacitance measures the hydration of the stratum corneum and provides an estimate of cutaneous capacity to retain moisture. Capacitance has been shown to affect the balance of epidermal cell proliferation and differentiation. Higher skin hydration values indicate greater cutaneous water capacitance.32
Chemical and enzymatic abnormalities in the atopic epidermis
The stratum corneum, composed of flattened keratinocytes and lipids, is crucial for the protective functions of the skin. Lipids in this layer are composed primarily of free fatty acids, cholesterol and ceramides. Ceramides, in particular, are the dominant lipids, constituting approximately 50% of the human stratum corneum lipids,33 and play a crucial role in skin barrier function.34,35 Ceramides are also the the most heterogeneous of the epidermal lipids and include 11 different molecules.36 Ceramides are generated from a sphingoid base and a fatty acid. Sphingoid bases include, but are not limited to, sphingosin, dihydro-sphingosin and phyto-sphingosin. Over the last few years, critical enzymes in ceramide biosynthesis, including ceramide synthases and ceramide hydroxylase/desaturase, have been identified.37 Ceramide synthesis is upregulated in the basal cell layer. Ceramides are then rapidly converted into glucosyl-ceramides and sphingomyelins that are then packed into the lamellar bodies. Once the content of these organelles is released at the interface between the stratum granulosum and stratum corneum, ceramides are regenerated by hydrolysis by β-glucocerebrosidase and sphingomyelinase Thus, ceramide levels in the stratum corneum are regulated by a balance between synthetic enzymes (e.g. β-glucocerebrosidase and sphingomyelinase) and ceramidases (which are responsible for their degradation). Ceramidases may be endogenous or exogenous (e.g. from bacteria).4
Impaired skin barrier function in human AD has been linked to a variety of epidermal abnormalities, although it is unclear whether they are primary, secondary or both. The stratum corneum of atopic skin is characterized biochemically by a reduction in the amounts of ceramides, especially ceramide-1, sebum lipids and water-soluble amino acids,38 although one study reported normal levels of ceramides in uninvolved atopic skin.39 In atopic individuals, decreased epidermal sphingomyelinase activity is found in both nonlesional and, more significantly, lesional skin, correlating with reduced stratum corneum ceramide content and disturbed barrier function.40 This is associated with impaired expression of cornified envelope proteins and keratins important for skin barrier function. Another mechanism for decreased ceramides in atopic skin is the decreased biosynthesis of free glucosylceramides and ceramides.38
Some studies have proposed that ceramide deficiency is linked to a novel sphingolipid-metabolizing enzyme, termed sphingomyelin glucosylceramide deacylase,41–43 which hydrolyses sphingomyelin or glucosylceramide, decreasing their availability for synthesis of ceramides. The enzymatic characteristics of the sphingomyelin glucosylceramide deacylase are completely distinct from ceramidase as well as the other known deacylases.41,42 Sphingomyelin glucosylceramide deacylase activity is enhanced more than fivefold in lesional stratum corneum, more than threefold in uninvolved stratum corneum and approximately threefold in the involved epidermis from patients with AD compared with healthy control subjects.41,42 This increased enzymatic activity appears to be specific to AD and is not detected in patients with contact dermatitis, who have the same enzymatic activity as healthy control subjects.41 It is interesting to note that, in peripheral blood lymphocytes of AD patients, there is no increase in activity compared with healthy control subjects, indicating that the high expression of sphingomyelin deacylase is highly associated with the skin of AD patients.44 In one study, a lack of phosphatidylcholine–sphingomyelin transacylase activity was also proposed as an underlying defect in human AD, although the number of samples evaluated in that study was too small to draw final conclusions.45
In summary, lipid deficiency, especially of ceramides, in the skin of atopic patients is considered an important component of the disease. Furthermore, treatments aimed at correcting such deficiencies, by topical application of lipid preparations, have been reported to ameliorate clinical signs.46,47
Proteins important in the epidermal differentiation/cornification process have also been reported to be decreased in patients with AD. Filaggrin is undoubtedly the most discussed protein at the moment. An early study reported decreased expression of filaggrin in the skin of atopic patients in both lesional and clinically unaffected skin.48 The more recent report of loss-of-function mutations in the gene encoding for filaggrin (FLG)49 has triggered large-scale investigation of the genetic mutations in FLG in a variety of populations.50–52 Of all the genes previously investigated as candidates for the development of AD, FLG is currently considered the most important, and FLG mutations are thought to be a major risk factor for AD.53–55 It is accepted that FLG mutations predispose to allergic sensitizations and to the development of early-onset, extrinsic disease that persists into adulthood.56–59 It is, however, important to note that decreased filaggrin expression does not necessarily imply a genetic mutation, because filaggrin can be reduced by Th2 cytokines.60 Decreased expression in the skin of some patients may therefore be a secondary phenomenon or may result from epigenetic modifications. Nevertheless, decreased filaggrin expression may still be clinically relevant in atopic individuals lacking the FLG mutations. It is estimated that only 18–48% of all eczema patients have FLG mutations,61 and that 40% of individuals with the FLG null alleles have never had signs of eczema.62 These considerations highlight the fact that that the link between FLG mutations and AD is not a simple and direct one and that AD is the result of a complex interaction between genetic and environmental factors.63
Interestingly, flaky tail (Flgft) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin in AD. In specific pathogen-free conditions, the majority of Flgft mice develop clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction, suggesting that the Flgft mouse genotype has potential as an animal model of AD corresponding to FLG mutation in human AD.64
Epidermal proteases and protease inhibitors
Skin barrier function in AD is impaired by a variety of mechanisms. Besides mutations in the genes encoding for proteins, there is also evidence of mutations in the genes encoding for proteases and protease inhibitors. This could lead to increased desquamation and an impaired skin barrier. Desquamation is determined by a cocktail of proteases (e.g. serine, cysteine and aspartic proteases) regulated by protease inhibitors.65
Human tissue kallikreins (KLKs) are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1–KLK15) important for desquamation.66 Many KLKs have been identified in normal stratum corneum and sweat, including KLK7 (also called stratum corneum chymotryptic enzyme, SCCE) and KLK5 (also called stratum corneum tryptic enzyme, SCTE). A mutation in the gene encoding for KLK7/SCCE has been described in some human patients with AD. This mutation leads to a change in activity and increased desquamation, particularly at alkaline pH.67 This genetic associationwas not confirmed in other studies, however, and there are no additive effects of mutations on phenotype.26 In the stratum corneum of AD patients, all kallikreins, except KLK11, have been reported to be significantly elevated.68 Alterations of the levels of KLKs in the stratum corneum of atopic patients are more pronounced than those in the serum.66
In a recent study investigating stratum corneum thickness and proteases, it was found that lesional atopic skin had a significantly thinner stratum corneum and that serine protease activity [stratum corneum tryptase-like enzyme (45-fold), plasmin (30-fold), urokinase (7.1-fold), trypsin-like KLKs (5.8-fold) and chymotrypsin-like KLKs (3.9-fold)] were increased in atopic skin when compared with healthy control subjects.69 The increased serine protease activities were found in acute eczematous AD, especially in deeper layers of the stratum corneum.67 These elevations in protease activities were associated with impaired barrier function, irritation and reduced skin capacitance (a measurement of the stratum corneum hydration). Elevations of some serine proteases (e.g. plasmin and urokinase) are, however, not specific for AD, but can be found in other circumstances in which the skin barrier is disrupted.70 Protease activity was also found to correlate with clinical staging of AD (i.e. clinical scores) and tended to normalize as the disease regressed.71 Whether increased proteases are the cause or simply a marker of disease activity remains unknown. Cells within the inflammatory infiltrate (e.g. mast cells) can produce proteases (e.g. chymase, a chymotrypsin-like serine proteinase),72 which can further contribute to the disruption of the skin barrier. This makes it difficult to separate cause from effect.
Proteases are regulated by protease inhibitors. A particularly important, pH-dependent protease inhibitor is lymphoepithelial Kazal-type 5 serine protease inhibitor (LEKTI), which is encoded by the serine protease inhibitor Kazal-type 5 (SPINK5) gene.73 The most convincing evidence for a role of excessive serine protease activity in AD comes from Netherton syndrome, a disease associated with loss-of-function mutations in SPINK5 and decreased LEKTI. Patients with this condition have severe AD and high IgE. Their stratum corneum is extremely thin, resulting in severe skin barrier defects. A significant association with atopy, AD and Netherton syndrome has been reported.74 The pathogenic role of serine protease inhibitor LEKTI in AD has been recently questioned owing to contradictory results from the analyses of an association between genetic polymorphisms of SPINK5 and AD. The association of AD with SPINK5 polymorphisms and AD has been reported by some studies75,76 but not confirmed by other authors.26 A recent study reported that expression of LEKTI was significantly decreased in atopic patients compared with healthy volunteers.77 Due to reduced protease inhibition, trypsin-like hydrolytic activity in AD was slightly increased, although not significantly.74 The authors of the study concluded that functional analyses in addition to genetic investigations are necessary to gain further and more detailed insights into the role of LEKTI in AD.
It has long been known that water permeability of the stratum corneum is regulated primarily by the lamellar arrangement of lipid bilayers between the corneocytes.78 The process of postsecretory extracellular development of the lamellar body has been investigated in detail in healthy skin in electron microscopy studies.79 These investigations found that there are differences between inner and outer parts of the stratum corneum, in terms of both lipid lamellae and corneodesmosomes.77 Differences have been reported in the relative volume of lamellar bodies in the upper layers of the stratum corneum between atopic patients and healthy control subjects.80 Lamellar bodies remain undelivered within the cells of the uppermost stratum granulosum cell layer of humans with AD (26% in atopics versus 8% in control subjects, P < 0.01). The authors concluded that a pathological failure to extrude lamellar bodies in AD may be at least partly responsible for the lipid abnormalities and skin barrier impairments observed in affected patients. Additionally, lipid lamellae appear misshapen and decreased in number when compared with healthy control subjects.78 No additional studies have specifically evaluated this proposed defect of extrusion.