Download Skin barrier function and SC hydration - Pre-congress

Didier Pin
Dermatology and dermatopathology Unit
LONG-TERM PARTNERS PRE-CONGRESS SYMPOSIUM / ESVD - ECVD 2011
ICF session
Skin barrier function and stratum corneum hydration
A major function of the skin is to provide a protective barrier at the interface between the
hostile external environment, the “outside”, and the organism, the “inside” (Elias et al., 2003;
Proksch et al., 2008).
This protective barrier is mainly represented by the epidermis which comprises the physical,
the chemical or biochemical (antimicrobial, innate immunity) and the adaptive immunological
barriers. Table 1 shows the multiple protective functions of mammalian stratum corneum (SC)
and their principal compartment, structural and chemical bases, and known regulatory signals.
Here, we are focusing on the permeability barrier, since the formation of a permeability
barrier that impedes transcutaneous loss of water is of major importance, because it is
required for life in a desiccating, terrestrial environment. While each protective function of
the skin can be considered as a discrete process, these individual functions are often kinked
and even co-regulated.
Under normal circumstances, epidermis must be as impermeable as possible except for a
small amount of water loss to hydrate the outer layers of the SC. Although all living cells
require water to function, some “dead” cells also require water to be metabolically active. As
it is the case of corneocytes, epidermis must retain sufficient water to (a) hydrate the outer
layers of the SC to maintain its flexibility and deformability and (b) to provide enough water
to allow enzyme reactions that facilitate SC maturation events, together with
corneodesmolysis and ultimately desquamation (Rawlings, 2006).
Where is the barrier and how does it form?
Although the deep nucleated epidermis play a role, particularly in the generation of the SC,
most of the critical protective functions against external insults localize to the outermost,
anucleated layers of the epidermis, the SC and are mediated by either the corneocytes or the
extracellular matrix (Elias, 2007).
The corneocytes are keratinocyte-derived anucleated cells providing structural support for the
SC. They lack intracellular organelles that are degraded during the final differentiation of
keratinocytes. During the final stages of normal differentiation, keratins are aligned into
highly ordered and condensed arrays through interactions with filaggrin, which is the
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Didier Pin
Dermatology and dermatopathology Unit
proteolytic product of profilaggrin, synthesized in the keratinocytes of the stratum
granulosum (SG) and contained in the keratohyalin granules. Filaggrin functions as an
intermediate filament-associated protein (IFAP) to aggregate keratin filaments into
macrofibrils which compose the matrix filling the cytosol of the corneocytes. The corneocytes
are 0.2 to 0.3 µm thick and their diameter is 30 to 50 µm (Pouillot et al., 2008). They are
surrounded by a cell envelope composed of an inner cornified cell envelope and an outer
corneocyte-bound lipid envelope, which is a plasma membrane-like structure and replaces the
plasma membrane on the external aspect of mammalian corneocytes. The cornified envelope
which is 0.015 to 0.02 µm thick, results of cross-linking of specialized cornified envelope
structural proteins, including loricrin, involucrin, trichohyalin and the class of small prolinrich proteins, by both disulphide bonds and N-epsilon-(gamma-glutamyl)lysine isopeptide
bonds formed by transglutaminases. The corneocyte lipid envelope is a 0.05-µm-thick
structure of ω-hydroxyceramides with very long chain N-acyl fatty acids covalently attached
by ester linkage to the proteins of the cornified envelope (Proksch et al., 2008). These lipids
also interdigitate with the intercellular lipid lamellae. Corneocytes are interconnected by
corneodesmosomes and tight junctions which both play a role in the barrier function and
physiological desquamation process (Haftek et al., 2011).
The intercellular lipid matrix, located between corneocytes, constitutes about 10 to 15% of the
dry weight of SC. The lipid species of the SC are composed of 50% ceramides, 10% fatty
acids, 25% cholesterol, and 15% cholesterol and glucosylceramide derivatives. The majority
of lipids of the SC are synthesized by the keratinocytes in the upper layer of the stratum
spinosum (SS) and the SG. At the SS-SG interface, fatty acids, sphingolipids, and other lipid
precursors are extruded to reside in the intercellular lipid matrix where they form
extracellular, nonpolar, lipid-enriched lamellar membranes that are impermeable to water
(Elias et al., 2003). In fact, there are two distinct states of the intercellular lipid matrix: the
non permeable (gel) and the permeable (liquid crystalline) matrix, the latter being permeable
to water and electrons. In contrast, the cornified envelope, the corneocyte lipid envelope and
the intercellular lipid matrix create a dense and impermeable network (Pouillot et al., 2008).
The lamellar bodies play a central role in the barrier edification. The epidermal lamellar body
(LB, often called Odland body) is an ovoid, 0.25 to 0.3 µm membrane bilayer-encircled,
secretory organelle that is unique to mammalian epidermis and some other keratinizing
epithelia. They contain not only pro-barrier lipids and their respective lipid processing
enzymes, but also additional structure proteins (e.g. corneodesmosin), antimicrobial peptides
(e.g. hBD2 and LL-37), proteases (stratum corneum chymotryptic enzyme, SCCE, stratum
corneum tryptic enzyme, SCTE, and probably aspartate or cysteine proteases) and their
inhibitors (elafin or skin-derived antileukoprotease, SKALP, secretory leucocyte protease
inhibitor, SLPI, lymphoepithelial kazal-type protease inhibitor, LEKTI, and the cysteine
protease inhibitor, cystatin C/K) that participate in cohesion, desquamation, and antimicrobial
function (Elias, 2003; Elias, 2007).
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Didier Pin
Dermatology and dermatopathology Unit
Sources of stratum corneum hydration
The state of SC hydration depends on:
-
the rate at which water reaches the SC from the tissue below,
-
the rate at which water leaves the skin surface by evaporation,
-
the ability of the SC to retain water.
The SC uses three main mechanisms to hold onto water:
- the intercellular lamellar lipids, whose physical conformation, predominantly an
orthorhombic laterally-packed gel and 13 nm long periodicity lamellar phase, mixed with the
corneocyte-bound lipid envelope, provide a tight and semi-permeable barrier to the passage of
water through the tissue;
- the presence of fully matured corneodesmosome-bound interdigitating corneocytes which
influence the tortuosity of the SC and thereby the diffusion path length of water;
- the presence of both intracellular and extracellular hygroscopic materials called ‘natural
moisturizing factors” (NMF). Much of the NMF is represented by amino acids (glutamine,
histidine, and arginine) and their deiminated derivatives (pyrrolidone carboxylic acid,
urocanic acid, and ornithine/citrulline/aspartic acid, respectively) derived from the breakdown
of filaggrin. As NMF compounds are present in high concentrations within corneocytes and
represent up to 20% to 30% of the dry weight of the SC, they can trap water within the
corneocyte cytosol. Other components found within but also external to the corneocytes
include lactates, urea, and electrolytes (Table 2). Other natural hygroscopic agents are
important for SC hydration. Although glycerol is a well-known cosmetic ingredient, its role as
natural endogenous humectant has been elucidated recently (Fluhr et al., 2003; Hara et al.,
2002; Choi et al., 2005). The endogenous glycerol is derived from the sebaceous gland
(sebum triglycerides) and also from the circulation, transported to the epidermis by aquaporin
3, a member of the aquaglyceroporin family, through water/glycerol channels (Hara et al.,
2002). Hyaluronic acid is a major component of and provides hydration and structural
integrity to the dermis but this hygroscopic polymer of sugar molecule is also naturally
present in the epidermis (Sakai et al., 2000), binds to the extracellular space via CD44, and
plays a role in epidermal barrier function (regulating both epidermal differentiation and lipid
synthesis and secretion), and probably in SC hydration (Bourguignon et al., 2006).
Then, the water-retaining capacity of the SC is highly dependent upon the thickness of the
SC, the precise phenotype of the corneocytes, their volume, and their organization, the precise
composition and physical packing state of barrier lipids, and finally the presence of highly
hygroscopic compounds largely founds within the corneocytes (Rawlings, 2006). For
examples, first, for the same volume of SC, the diffusion path length of water diffusing
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Didier Pin
Dermatology and dermatopathology Unit
through the SC lipids will be less if the corneocytes are smaller, second, the tight layering of
the corneocytes provides them the ability to retain water while their membrane is permeable
to water but impermeable to proteins.
Naturally, as water is being constantly lost from the skin surface, water gradient are
established within the different layers of the SC. It was established that the natural hydration
levels in the SC are between 15% and 40%-45% (in comparison, the whole body contains a
mean of 65% water), from 15% at the skin surface to 40% at the innermost layer compared
with 70%-80% within the granular layer. These data suggest that a barrier to water loss begins
prior to the formation of the SC and is present at the SC-SG interface and that a selective
retention of water in the different SC cellular layers is required to explain the apparent
discontinuity in hydration between different corneocyte layers (Rawlings, 2006). This
function of the SC is believed to be largely dependent on the presence of the so-called NMF.
All of the NMF components generated from filaggrin decrease in concentration toward the
surface of the SC (Rawlings, 2006).
A fraction of the water is tightly bound to hygroscopic molecules (the NMF) and lipids in the
skin (Verdier-Sévrain and Bonté, 2007). This fraction of water content is proportional to
external relative humidity. Under normal circumstances only a very small amount of water
must be present in the intercellular lipid lamellae (Rawlings, 2006). The remaining fraction of
water is bound within the intracellular keratin and usually does not change in nonpathological
conditions (Verdier-Sévrain and Bonté, 2007).
Method for assessing skin hydration
A lot of techniques have been developed for measuring water in skin such as water flux
analysis, electrical measurements (resistance, capacitance, and impedance), heat conductivity,
photoacoustic spectroscopy viscoelastic properties, microwave propagation, dye fluorescence,
topography, infrared spectroscopy, and electron probe analysis (Warner et al., 1988), all of
these were ex vivo methods. Recently, the ex vivo findings have been proven noninvasively in
vivo using the in vivo confocal Raman microscopy (Caspers et al., 2001). Other innovative
methods have been developed to measure skin hydration such as silicon image sensor
technology that provides sensitive imaging of the skin capacitance, near infrared (NIR)
multispectral imaging method that measures the absorption of NIR light by water in living
tissue from its reflectance spectrum, and optical coherence tomography, nuclear magnetic
resonance spectroscopy, and transient thermal transfer (Verdier-Sévrain and Bouté, 2007). An
easier in vivo method is the measurement of TEWL. In fact, the TEWL is a marker of the
inside-outside barrier only. The outside-inside barrier often correlates with the inside-outside
barrier, but not always. Nevertheless, an inverse relationship between TEWL and SC
hydration is well known. High TEWL values, as a marker of disturbed skin barrier function,
are frequently correlated with low hydration of the SC as shown in experimental settings after
skin cleansing with soaps and detergents or in diseased skin (Proksch et al., 2008).
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Didier Pin
Dermatology and dermatopathology Unit
A model to better understand the barrier function
Disruption of the permeability barrier by a variety of insults, including mechanical trauma
such as tape stripping or contact with solvent such as acetone, stimulates a vigorous
homeostatic repair response in the underlying viable epidermis that leads to the rapid
restoration of permeability barrier function (Proksch et al., 1993).
Function
Permeability*
Hydration*
Principal
compartment
Extracellular
matrix
Structural basis
Chemical basis
Lamellar bilayers
Corneocyte
Cytosol
Ceramides, chol, non
essential FA in proper
ratio
Filaggrin proteolytic
products, glycerol
(AQP3), HA, xylose
Intercellular DSG1/
pH, Ca++ (TRPV)
DSC1 et CDSN, claudin1
and 4, occludin, JAM-1
AMPs, FFA, Sph
1,25(OH)2D3, IL-1α
Cohesion (integrity) Extracellular
and desquamation* matrix
CD, tight junctions
Antimicrobial*
Lamellar bilayers
Mechanical*
Antioxidant*
Extracellular
matrix
Corneocyte
Extracellular
matrix
Extracellular
matrix
Corneocyte
Chemical (Ag
exclusion)
Initiation of
inflammation* (1st
cytokine activation)
Psychosensory
Extracellular
interface*
matrix
UV light
Corneocyte
Cornified envelope,
keratin filaments
Lamellar bilayers
Regulatory signals
(receptors)
IL-1α, Ca++, pH,
liposensors, SP through
PAR2, TRPV1 and 4
Relative humidity
(TRPV4)
γ-Glutamyl isopeptide
bonds
Chol, FFAs, secreted
vitE, redox grandient
Hydrophilic products of
CD
Proteolytic activation of
pro-IL-1 α/β
Ca++, CholSO4,
liposensors
?
Lamellar bilayers
Barrier lipids
GCs, heat (TRPV3)
Cytosol
Trans-urocanic acid
(histidase activity)
Extracellular lacunae
Cytosol
Same as for
permeability function
pH, serine proteases
activation
* Abnormal in atopic dermatitis, SP serine protease, AMPs antimicrobial peptides, Sph sphingosine, DSG1/
DSC1 desmogleïn/desmocollin, CDSN corneodesmosin, TRPV transient receptor potential vanilloid, HA
hyaluronic acid, CD corneodesmosome, AQP3 aquaporin-3, PAR2 proteinase-associated receptor 2, FFA free
fatty acid, JAM-1 junctional adhesion molecule
Table1 Multiple protective functions of mammalian SC (Elias, 2007; Elias et al., 2008;
Verdier-Sévrain and Bonté, 2006)
Chemical
Composition (%)
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Didier Pin
Free amino acids
Pyrrolidone carboxylic acid
Lactate
Sugars
Urea
Chloride
Sodium
Potassium
Ammonia, uric acid, glucosamine, creatine
Calcium
Magnesium
Phosphate
Citrate and formate
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12
12
8.5
7
6
5
4
1.5
1.5
1.5
0.5
0.5
Table 2 Chemical composition of NMF (Verdier-Sévrain and Bonté, 2007)
Microphotograph Epidermis of canine skin: 1 stratum basale, 2 stratum spinosum, 3 stratum
granulosum, 4 stratum corneum compactum, 5 stratum corneum disjunctum, rectangular area
indicates the location of the skin barrier, the hydration gradient through the epidermis is
noted.
References
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PM, Feingold KR. Hyaluronan-CD44 interaction stimulates kératinocytes differentiation,
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Didier Pin
Dermatology and dermatopathology Unit
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Caspers PJ, Lucassen GW, Carter EA, Bruining HA, Puppels GJ. In vivo confocal Raman
microspectroscopy of the skin: noninvasive determination of molecular concentration profiles.
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