Chapter 47. Skin as an Organ of Protection

The skin's most important function is to form an effective barrier between the “inside” and the “outside” of the organism. Life on dry land requires the presence of a barrier to regulate water loss and prevent desiccation, commonly referred to as the “inside–outside” barrier. Additionally, skin provides an “outside–inside” barrier to protect against mechanical, chemical, and microbial assaults from the external environment (Fig. 47-1).1 To perform these functions the epidermis undergoes keratinization, a process in which epidermal cells progressively mature from basal cells with proliferative potential to the lifeless, flattened squames of the stratum corneum (SC) (Fig. 47-2). Both the SC and the deeper skin layers protect the skin from mechanical forces, ultraviolet (UV) radiation, cold and hot temperatures, and invasion of chemicals and microbes. To effectively perform this multiplicity of functions, the skin contains different types of barriers. The physical barrier consists mainly of the SC, but the nucleated epidermis, in particular the tight junctions, provides another important barrier component. The chemical/biochemical (antimicrobial) barrier consists of lipids, acids, lysozymes, and antimicrobial peptides (discussed in Chapter 10). The humoral and cellular immune system provides a barrier to infectious disease, but immune hyperactivity may lead to allergy (Table 47-1).

Although the skin is of central importance for preventing water loss in a dry environment, aquatic animals also require a skin barrier to protect them from the high salinity of their surrounding environment. Terrestrial mammals with dense fur have much thinner skin than animals without this protective coat, demonstrating that fur itself is a considerable barrier. The relatively hairless skin of pigs shows much similarity to human skin and is therefore a good model for skin research.

In addition to the SC the entire skin, as a whole, serves a protective function. The innermost region of human skin, the subcutaneous fat layer, offers mechanical shock protection, insulates the body against external heat and cold, and is active in general energy metabolism and storage. The dermis is composed of collagen bundles and elastic fibers and is very important for the mechanical strength of the skin. The epidermis, the skin's outer layer, consisting primarily of stratified nucleated keratinocytes and the SC, is most important for skin protection—the focus of this chapter. Sweat glands and blood vessels regulate body temperature. Sebaceous glands secrete specialized lipids to protect the hair from the environmental stress (see Chapter 79). In animals, these lipids serve as a water repellent for the fur, aiding in buoyancy and temperature regulation, and also preventing desiccation of the body and UV damage. The role of sebaceous lipids for SC barrier function and for dry skin is under active investigation.2,3 Sebaceous glands also transport glycerol to the skin surface, which is important for SC hydration.4 Nerve fibers are chemosensitive and act as a warning system against external trauma (see Chapter 102).

(Table 47-2)

Research from the 1940s and 1950s established that the SC is the specific location of the physical barrier.5,6 The SC barrier structure visualized by electron microscopy and ruthenium tetroxide fixation (Fig. 47-3) is a better indicator of its barrier function than the typical “basket-weave” appearance of the SC in routine formalin-embedded tissue sections. Regulation of water permeation is not absolute. The normal movement of water from the SC into the atmosphere is known as transepidermal water loss (TEWL), previously called insensible water loss. The SC serves as the principal barrier against the percutaneous penetration of chemicals and microbes1 as well.

The 10–20 μm thick SC forms a continuous sheet of protein-enriched cells, embedded in an intercellular matrix, enriched in nonpolar lipids, and organized as lamellar lipid layers. The viable epidermis is a stratified squamous epithelium, consisting of basal, spinous, and granular cell layers. Upon leaving the basal layer, keratinocytes begin to differentiate and undergo a number of changes in both structure and composition during the apical migration into the stratum spinosum and stratum granulosum (see Chapter 46). Keratinocytes synthesize and express numerous structural proteins and lipids during their maturation. The final steps in keratinocyte differentiation are associated with profound changes in their structure, resulting in their transformation into flat and anucleated squamous cells of the SC, consisting mainly of keratin filaments and surrounded by a cell envelope composed of cross-linked proteins (cornified envelope proteins) as well as a covalently bound lipid envelope (Fig. 47-2). Extracellular nonpolar lipids surround the corneocytes to form a hydrophobic matrix. The cornified envelope proteins as well as the covalently bound lipid envelope are thought to be important for the chemical resistance of the corneocytes (Fig. 47-4). Desmosomes, which interconnect adjacent keratinocytes, are important for SC cohesion and are shed during the desquamation process in the SC. In the upper spinous and granular layers characteristic lamellar vesicles appear, which are called epidermal lamellar bodies (Figs. 47-3 and 47-5). These are enriched in polar lipids, glycosphingolipids, free sterols, phospholipids, and hydrolytic enzymes that deliver the lipids required for the SC's extracellular layers. The lamellar bodies may also contain proteins such as human β-defensin 2.7 In response to certain signals, for example, an increase in calcium concentration during the transition from the granular layers to the SC, the lamellar bodies move to the apex of the uppermost granular cells, fuse with the plasma membrane, and secrete their content into the intercellular spaces through exocytosis. The lipids derived from the lamellar bodies are subsequently modified and rearranged into intercellular lamellae positioned approximately parallel to the cell surface. The covalently bound lipid envelope acts as a scaffold for this process. After the extrusion of the lamellar bodies into the stratum granulosum–SC interface, the polar lipids are enzymatically converted into nonpolar products. Hydrolysis of glycosphingolipids generates ceramides while phospholipids are converted into free fatty acids. These changes in lipid composition and cell structure result in the formation of a very dense structure packed into the interstices of the SC (Table 47-2).8

Lipid Composition and Role of Lipids in the Stratum Corneum Permeability Barrier

Confocal laser scanning microscopy and X-ray microanalysis studies have shown that the major route of penetration results in a tortuous pathway between the corneocytes, confirming that intercellular lipids play an irreplaceable role in regulating skin permeability barrier function.8 The major lipid classes in the SC are cholesterol, free fatty acids, and ceramides (see eFig. 47-5.1).9–11

Cholesterol

Cholesterol is probably the most abundant lipid in the entire body and part of the plasma membrane, but also part of the intercellular lipid lamellae in the SC. Although basal cells are capable of resorbing cholesterol from circulation, most cholesterol in the epidermis is synthesized in situ from acetate.12 The epidermal keratinocyte, the main cell type in the epidermis, is highly active in the synthesis of several lipids, including cholesterol and free fatty acids. The rate-limiting step in cholesterol biosynthesis is catalyzed by hydroxymethylglutaryl CoA (HMG CoA) reductase (Fig. 47-6). Epidermal cholesterol synthesis is regulated by these enzymes and increases during permeability barrier repair.13

Free Fatty Acids

The skin contains free fatty acids as well as fatty acids bound in triglycerides, phospholipids, glycosylceramides, and ceramides. The chain length of free fatty acids in the epidermis ranges from C12 to C24. The rate-limiting enzymes acetyl-CoA carboxylase and fatty acid synthase in the epidermis are largely autonomous (Figs. 47-6 and 47-7).14 Saturated and monounsaturated fatty acids are synthesized in the epidermis, in contrast to di- and polyunsaturated acids. The nomenclature of the fatty acids is determined from the position of the first double bond in the molecule, starting from the terminal methyl group. In particular, the essential ω-6-unsaturated acids are obtained from food and reach the epidermis by the circulation but can also be obtained by topical treatment. The nonessential monounsaturated fatty acid, the oleic acid, is an ω-9-fatty acid. The most important double unsaturated fatty acid, linoleic acid, is an ω-6 fatty acid. Also of importance is α-linoleic acid (ω-3). No skin changes due to ω-3-fatty acid deficiency are currently known; however, it has been proposed that these fatty acids are important for the resolution of inflammation. ω-3-fatty acids are obtained from fish, whereas ω-6-fatty acids are obtained from plant oils.15,16 Essential fatty acid deficiency (EFAD) caused by unusual diets or malabsorption in humans or experimentally induced in rats and mice leads to the EFAD syndrome, characterized by profound changes in epithelia including the epidermis.17 In this condition, the epidermis is rough, scaly, and red and shows a severely disturbed permeability barrier function. In addition, severe bacterial infection, impaired wound healing, and alopecia may occur. Linoleic acid is part of phospholipids, glucosylceramides, ceramide 1, ceramide 4, and ceramide 9.18 It has been proposed that the linoleic acid metabolite γ-linoleic acid is of special importance for atopic eczema.

Ceramides

Ceramide is an amide-linked fatty acid containing a long-chain amino alcohol called sphingoid base. The carbon chain lengths of amide-linked fatty acids and sphingoid bases in most mammalian tissues are 16 to 26 and 18 to 20, respectively (see eFig. 47-5.1). Although sphingolipids, including glycosphingolipids and phosphosphingolipids, are ubiquitously distributed in mammalian tissues, tissue-specific molecular distribution has been described. Glucosylceramide is enriched in the epidermis and spleen while galactosylceramide is enriched in the brain, but is not detected in keratinocytes. Whereas ceramide is a minor lipid component, comprising less than 10% of cholesterol or phospholipids in other mammalian tissues, ceramide is a major lipid component in the SC, accounting for 30%–40% of lipids by weight. Moreover, such a high content of ceramides in the SC is not observed in the epidermal stratum granulosum, stratum spinosum, or stratum basale. This also suggests that terminal differentiation is a key factor in accumulating ceramide. The SC contains at least nine different ceramides.18 In addition, there are two protein-bound ceramides: (1) ceramide A and (2) ceramide B (see eFig. 47-5.1).8 These ceramides are covalently bound to cornified envelope proteins, most importantly to involucrin.

Ceramides are synthesized by serine-palmitoyltransferase as rate-limiting enzyme and by hydrolysis of both glucosylceramide (by β-glucocerebrosidase)19 and sphingomyelin (by acid sphingomyelinase) (Figs. 47-7 and 47-8).20 Whereas all kinds of ceramides are derived by synthesis from serine-palmitoyltransferase and from β-glucocerebrosidase, only ceramide 2 and ceramide 5 are obtained from sphingomyelinase because sphingomyelinase contains nonhydroxy acids.21

Lipid Transport

Keratinocytes require abundant cholesterol for cutaneous permeability barrier function. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a pivotal role in regulating cellular cholesterol levels. It was demonstrated that ABCA1 is expressed in cultured human keratinocytes and murine epidermis. Liver X receptor (LXR) activation and activation of peroxisome proliferator-activated receptor (PPAR)-α, PPAR-ss/δ, and retinoid X receptor (RXR) increased ABCA1 expression in keratinocyte cultures. Thus, cholesterol levels for permeability barrier function are regulated by ABCA1, LXR, and PPARs.22 The cellular fatty acid transport and metabolism is regulated by fatty acid-binding proteins (FABPs).23,24

Additional protective functions of the epidermis are not discussed in this chapter, but are listed in Table 47-3.

To provide the physical barrier of the SC, not only intercellular lipids, but also corneocytes are of crucial importance.25,26 The epidermis undergoes keratinization in which epidermal cells progressively mature from basal cells with proliferative potential to lifeless flattened squames of the SC (Fig. 47-2). Keratinocytes arise from stem cells in the basal layers and transient amplification cells and move to a series of differentiation events until they are finally brought to desquamation.27 Thus, in the normal epidermis, there is a balance between the processes of proliferation and desquamation that results in a complete renewal approximately every 28 days. In some forms of ichthyosis, the rate of desquamation may be decreased, leading to epidermal cell retention (retention hyperkeratosis).28 (See Chapter 49.) In inflammatory skin diseases like psoriasis there is an increase in proliferation resulting in a disturbance in differentiation and parakeratotic squames (hyperproliferative hyperkeratosis).29

Keratins are major structural proteins synthesized in keratinocytes (see Chapter 46). They assemble into a web-like pattern of intermediate filaments that emanate from a perinuclear ring, extend throughout the cytoplasm, and terminate at junctional desmosomes and hemidesmosomes. During the final stages of normal differentiation, keratins are aligned into highly ordered and condensed arrays through interactions with filaggrin, a matrix protein. In keratin disorders, the filament networks collapse around the nucleus, preventing attachment with the filament-matrix complex and the inner surface of squames, and alter interaction between neighboring cells, thereby affecting desquamation. Filaggrin aggregates the keratin filaments into tight bundles. This promotes the collapse of the cell into a flattened shape, which is characteristic of corneocytes in the cornified layer. Together, keratins and filaggrin constitute 80%–90% of the protein mass of mammalian epidermis.25,26

The structural proteins involucrin, loricrin, trichohyalin, and the class of small proline-rich proteins (SPRRs) are synthesized and subsequently cross-linked by transglutaminases to reinforce the cornified envelope just beneath the plasma membrane. The proteins of the cornified envelope constitute about 7%–10% of the mass of the epidermis. These corneocytes provide the bulwark of mechanical and chemical protection, and together with their intercellular lipid surroundings, confer water-impermeability. The cornified cell envelope is a tough protein/lipid polymer structure formed just below the cytoplasmic membrane and subsequently resides on the exterior of the corneocytes (Fig. 47-4). It is resistant to 10% KOH and is the rigid structure seen on KOH skin scrapings. It consists of two parts: (1) a protein envelope and (2) a lipid envelope. The protein envelope contributes to the biomechanical properties of the CE as a result of cross-linking of specialized cornified envelope structural proteins by both disulfide bonds and N(ϵ)-(γ-glutamyl)lysine isopeptide bonds formed by transglutaminases.26,30 The isopeptide bonds are resistant to most common proteolytic enzymes. The corneocyte-bound lipid envelope is plasma membrane-like structure, which replaces the plasma membrane on the external aspect of mammalian corneocytes.31 Involucrin, envoplakin, and periplakin serve as substrates for the covalent attachment of ω-hydroxyceramides with very long-chained N-acyl fatty acids by ester linkage.32 These not only provide a coating to the cell, but also interdigitate with the intercellular lipid lamellae (Table 47-2).26

Experimental barrier disruption leads to changes in epidermal differentiation, epidermal lipid keratin, and cornified envelope protein expression and, vice versa, overexpression and deficiency of these lipids and proteins in mice result in barrier defects. A number of diseases displaying defective epidermal barrier function are also the result of genetic defects in the synthesis and metabolism of either lipids, keratins, cornified envelope proteins, or the transglutaminase 1 cross-linking enzyme.

Inhibition of HMG CoA reductase by topical application of the lipid-lowering drug lovastatin in mice resulted in a disturbed barrier function and in epidermal hyperproliferation. Therefore, the specific relationship between barrier function and epidermal DNA synthesis was examined. After acute skin barrier disruption (local acetone treatment or by tape-stripping) (Fig. 47-9) and in a model of chronic barrier disruption (EFAD diet), an increase in DNA synthesis leading to epidermal hyperplasia was noticed.33 The increase in DNA, and in lipid synthesis, was partially prevented by occlusion.15,33,34

Also, the described acute and chronic barrier disruption leads to specific changes in epidermal keratin and cornified envelope protein expression. Increased expression of the basal keratins K5 and K14 and a reduction of the differentiation-related keratins K1 and K10 were noted. In addition, there was expression of the proliferation-associated keratins K6 and K16 as well as the inflammation-associated keratin K17 (see eFig. 47-9.1).35 The importance of keratins for skin barrier function was supported by studies in K10-deficient mice. Heterozygotes and homozygotes showed a mild or severe permeability barrier disruption, respectively. Importantly, homozygous neonatal K10-deficient mice exhibited an extremely delicate epidermis and died a few hours after birth. Heterozygous littermates showed a normal skin at birth but developed increasing hyperkeratosis as they grew up.36 Barrier repair in heterozygous K10-deficient mice was delayed and skin hydration was impaired.37 Changes in ceramide composition, a reduced amount of glucosylceramide and sphingomyelin, and reduced acid sphingomyelinase activity, as well as increased involucrin content, were also noted.38 This shows that genetically determined changes in structural proteins lead to an impaired skin barrier function and changes in differentiation and lipid composition.

The importance of keratins for skin barrier function is further supported by studies in diseases that are caused by monogenetic defects of these structural proteins. Epidermolysis bullosa simplex (EBS) shows mutation in the basal layer keratins K5 or K14 (details in Chapter 62). Genetic defects in the suprabasal keratins results in hyperkeratosis and a mild barrier defect (details in Chapters 49 and 59). Epidermolytic hyperkeratosis (EHK) has spinous layer K1 or K10 defects, epidermolytic palmoplantar keratoderma (EPPK) has granular layer K9 defects (because this keratin is expressed only in palmar and plantar skin, the disease is restricted to that area), and ichthyosis bullosa of Siemens (IBS) has granular layer defects K2 (formerly K2e) defects.25

Experimental permeability barrier disruption leads to a premature expression of involucrin, but not loricrin.35 Overexpression of filaggrin in mice in the suprabasal epidermis resulted in a delay of barrier repair.39 Loss of normal profilaggrin and filaggrin is the cause for the flaky tail in an autosomal recessive mutation in mice that results in a dry, flaky skin, and annular tail and paw constrictions in the neonatal period. Targeted ablation of the murine involucrin gene did not show changes in skin barrier function under basal conditions40 but resulted in a reduced barrier repair. In addition, knockdown of filaggrin was shown to increase UV-sensitivity in a human skin model.41 Loricrin deficient mice do not show a disturbed barrier function, but a greater susceptibility to mechanical stress which may alter skin barrier function secondarily.42,43

Changes in epidermal proliferation and differentiation are also seen in inflammatory skin diseases with a disturbed skin barrier function (see eFig. 47-9.1). Increased proliferation is one of the main characteristics of psoriasis, but in atopic dermatitis lesional skin also there is a considerable increase in epidermal proliferation. Also, changes in keratins and cornified envelope proteins occur in inflammatory skin diseases.44 Overall, this shows that there is undoubtedly a connection between epidermal proliferation, differentiation, and skin barrier function.

Although the SC is recognized as the most important physical barrier, the lower epidermal layers are also significant in barrier function. A low-to-moderate increase in TEWL occurs after removal of the SC by tape stripping, whereas loss of the entire epidermis through suction blisters leads to a severe disturbance in barrier function. Loss of the SC and parts of the granular layers in staphylococcal scalded skin syndrome (SSSS) are not usually life-threatening.45 In contrast, the suprabasal and subepidermal blistering diseases pemphigus vulgaris, toxic epidermal necrolysis and severe burns, respectively, are life-threatening when large areas of the body are involved. Patients die because of extensive water loss or sepsis induced by external bacteria infection—outcomes directly resulting from perturbed barrier function. Survival rates can be greatly improved with application of an artificial barrier in the form of a foil or a grease ointment, often containing active antimicrobial substances. These clinical observations confirm the importance of the nucleated epidermal layers in skin barrier function in both directions, both in preventing excessive water loss and the entry of harmful substances into the skin.46

Tight Junctions: A Second-Line Epidermal Barrier

Tight junctions are cell-junctions connecting neighboring cells sealing the intracellular space and controlling the paracellular movements of molecules (Fig. 47-2). The most important tight junction proteins in the human epidermis are occludin, claudins, and zonal occluding proteins (ZOs). Localization of occludin is restricted to the stratum granulosum, ZO-1 and claudin 4 are found in suprabasal layers, and claudins 1 and 7 are found in all epidermal layers. In various diseases with perturbed SC barrier function, for example, psoriasis vulgaris, lichen planus, acute and chronic eczema, and ichthyosis vulgaris, tight junction proteins that were formerly restricted to the stratum granulosum and upper stratum spinosum, were also found in deeper layers of the epidermis. Claudin 1-deficient mice die within 1 day of birth due to tremendous water loss.47 Altered barrier function of the skin has also been demonstrated in mice overexpressing claudin 6 in the epidermis.48,49

Desmosomal Proteins: Structural Cell–Cell Interfaces

A perturbation in SC barrier function has also been found after the alteration of desmosomal proteins. Desmogleins are desmosomal cadherins that play a major role in stabilizing cell–cell adhesion in the living layers of the epidermis (Fig. 47-2, see Chapter 53). Autoantibodies against these transmembrane glycoproteins cause blisters in pemphigus vulgaris due to loss of keratinocyte adhesion. In acute eczema, which shows disturbed skin barrier function a reduction in keratinocyte membrane E-cadherin in areas of spongiosis has been found.50,51 In transgenic mice in which the distribution of desmoglein 3 in epidermis was similar to that in mucous membrane, a highly increased TEWL resulted in lethality during the first week of life due to dehydration.52 Mice with conditionally inactivated E-cadherin in the epidermis died perinatally due to the inability to retain a functional epidermal water barrier. Absence of E-cadherin leads to improper localization of key-tight junctional proteins and impermeable tight junctions and thus altered epidermal barrier function.53,54

Connexins: Intercellular Gatekeepers

Connexins are transmembrane proteins that homo- or heteromerize on the plasma membrane to form a connexon. Connexons on adjoining cells that associate to form gap junctions and allow the passage of ions and small molecules between cells. Connexin 26 is one of the most highly upregulated genes in psoriatic plaques. Missense mutations in connexin 26 result in five distinct ichthyosis-like skin disorders. In mice overexpressing connexin 26, a hyperproliferative state, infiltration of immunocells, and a delayed epidermal barrier recovery were noted.55

Proteases

Proteases are important for epidermal differentiation. The characteristic resistance of the cornified envelope is based on the formation of very stable isopeptide bonds that are catalyzed by transglutaminase 1, 3, and 5. Transglutaminase 1-deficient mice showed a defective SC and early neonatal death.56 Mutations in transglutaminases 1 have been found to be the defect in lamellar ichthyosis.57 Cathepsin D is involved in the processing of transglutaminase 1. Cathepsin D-deficient mice expressed a defect in barrier function and hyperproliferation.58 Evidence suggests that cystatin M/E and cathepsin L may be upstream proteases of the cathepsin D–transglutaminase pathway and must exist in a tightly regulated balance in order to ensure tissue integrity in the epidermis, hair follicles, and corneal epithelium.59 There is further evidence that disruption of the cystatin M/E–cathepsin pathway contributes to the underlying skin barrier dysregulation characteristic of inflammatory dermatoses, such as psoriasis and atopic dermatitis.60

Netherton syndrome, a severe autosomal recessive genodermatosis (see Chapter 49) is caused by mutations in SPINK5, encoding the serine protease inhibitor LEKTI. In Netherton syndrome, there is often an atopic eczema-like skin disease with a disrupted permeability barrier. SPINK5−/− mice replicate key features of Netherton syndrome, including altered desquamation, impaired keratinization, hair malformation, and a skin barrier defect. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1 due to SC chymotryptic enzyme (SCCE)-like hyperactivity. This leads to defective SC adhesion and results in loss of skin barrier function.61,62

Cytokine Signaling: Regulation of Epidermal Homeostasis and Repair

Cytokines are very important for the regulation of wound healing in which reepithelization and differentiation to form a competent barrier are the last steps63 (see Chapter 248). Besides the immune cells, keratinocytes are able to produce a large variety and amounts of cytokines (Fig. 47-10). Of special importance are the so-called primary cytokines tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6. IL-1, TNF, and IL-6 are potent mitogens and stimulators of lipid synthesis in cutaneous and extracutaneous tissues. After acute permeability barrier disruption, an increase in the expression of TNF, IL-1, and IL-6 on the mRNA and the protein level occurs.20,64,65,66 In mice deficient in TNF receptor 1 or IL-1 receptor 1/TNF receptor 1-double knockout mice and in IL-6-deficient mice, a delay in permeability barrier occurs.20,66 Moreover, topical application of TNF enhances permeability barrier repair, and topical application of IL-6 in IL-6-deficient mice restores the normal speed in permeability barrier repair (Fig. 47-10). In TNF-receptor 1-deficient mice, the generation of lipids for skin barrier repair was delayed and the activity of acid sphingomyelinase that generates ceramides for skin barrier repair was reduced.20 STAT 3 tyrosine phosphorylation was induced after barrier disruption in wild type, but markedly reduced in IL-6-deficient mice. The acute increase in TNF, IL-1, and IL-6 after barrier disruption is crucial for skin barrier repair. However, if barrier disruption is prolonged and a chronic increase in cytokine production occurs, it could have a harmful effect leading to inflammation and epidermal proliferation. This disrupted permeability barrier, epidermal hyperproliferation, and inflammation, and is well known in several diseases like irritant and allergic contact dermatitis, atopic dermatitis, and psoriasis, and could aggravate the disease.

Ionic Modulations: Epidermal Calcium and Potassium Levels

A perturbed barrier recovers normally when exposed to an isotonic, hypertonic, or hypotonic external solution instead of air. If the solution contains both calcium and potassium, the barrier recovery is inhibited. Inhibitor studies using both L-type calcium channels and calmodulin and ultrastructural examination by ion-capture cytochemistry showed that there is a calcium gradient in the epidermis. There is a relatively low calcium concentration in the basal epidermis, and an even lower concentration in the spinous layers, while the highest calcium concentrations are found in the granular layers. Calcium in the SC is very low because the relatively dry SC with extracellular lipids is not able to solve the high polar ions. After disruption of the permeability barrier there is influx of water in the SC and the ion gradient is lost (Fig. 47-11). This depletion of calcium regulates lamellar body exocytosis.67–69 Calcium is a very important regulator of protein synthesis in the epidermis, including regulation of transglutaminase 1 activity.70 Furthermore, extracellular calcium ions are important for cell-to-cell adhesion and epidermal differentiation. Intracellular calcium is controlled by more than one mechanism as demonstrated by the two genetic diseases discussed below.

Disturbed regulation of calcium metabolism and increased TEWL71 occur in Darier disease, characterized by loss of adhesion between suprabasal epidermal cells associated with abnormal keratinization, and in Hailey–Hailey disease which shows loss of epidermal cell-to-cell adhesion (see Chapter 51). The gene for Darier disease (ATP2A2) encodes a calcium transport ATPase of the sarco(endo)plasmic reticulum (SERCA2),72,73 while the gene for Hailey–Hailey disease (ATP2C1) codes for a secretory pathway for calcium and manganese transport ATPase of the Golgi apparatus (SPCA1).74

Neurotransmitters in the Keratinocytes: Common Origins of the Brain and Skin

Neurotransmitters are found in keratinocytes and may regulate skin permeability barrier function. The receptors can be categorized in two groups: (1) ionotropic (calcium or chloride ion) receptors and (2) G-protein-coupled receptors. Topical application of calcium channel agonists delays the barrier recovery while antagonists accelerate barrier repair.

The G-protein-coupled receptors modulate intracellular cAMP level, increase of intracellular cAMP in epidermal keratinocytes delays barrier recovery, while cAMP antagonists accelerate the barrier recovery. Activation of dopamine 2-like receptors, melatonin receptors, or serotonin receptor (type 5-HT 1) decreases intracellular cAMP and consequently accelerates barrier recovery, while activation of adrenergic β2 receptors increases intracellular cAMP and delays the barrier repair. Many agonists or antagonists of neurotransmitter receptors are used clinically to treat nervous disorders. Some of them might also be effective for treating skin diseases.75

Mild impairment of the skin barrier is found in monogenetic diseases expressing an impaired epidermal differentiation or lipid composition without inflammation, for example, ichthyosis vulgaris and X-linked recessive ichthyosis (XLRI).71 The diseases with a more pronounced barrier disruption are inflammatory diseases, for example, irritant and allergic contact dermatitis, atopic dermatitis, seborrheic dermatitis, psoriasis, and T-cell lymphoma. Also, blistering diseases, most of them inflammation-related, show an increase in TEWL, especially after loosening of the blister roof and the development of erosions (Table 47-4). Since HIV patients are known to display a xerotic phenotype, it seems likely that allergen penetration due to a disturbed skin barrier activates the Th2 pathway and consequently aggravates the underlying skin barrier disturbance (Table 47-4).76

Most inflammatory skin lesions are covered with dry scales or scale-crusts due to the disturbed epidermal differentiation and an SC with poor water-holding capacity. Inflammatory skin diseases can be produced by either exogenous or endogenous causes. In contact dermatitis, disruption of the barrier by irritants and allergens (which can also be irritants) is the primary event, followed by penetration of the chemicals into the living epidermal layers, irritation of the cell membrane, contact with immune cells, sensitization, inflammation, increased epidermal proliferation, and changes in differentiation. In T-cell lymphoma (mycosis fungoides), an endogenous cause for barrier disruption, changes in epidermal proliferation, and differentiation by expansion of clonal malignant CD4+ T-cells are obvious.77 In atopic dermatitis and in psoriasis, it is debatable whether permeability barrier disruption is followed by inflammation or whether inflammation leads to epidermal changes including barrier dysfunction. The vast majority of reports on the pathogenesis of atopic dermatitis and even more on psoriasis focused on the primary role of abnormalities in the immune system.78 However, others have proposed an “outside–inside” pathogenesis for atopic dermatitis and other inflammatory dermatoses with barrier abnormalities,79–81 as an alternative to the previous “inside–outside” paradigm.82

Atopic Dermatitis: The Consequence of a Chronically Disturbed Barrier

(See also Chapter 14)

The existence of a defective permeability barrier function in atopic dermatitis is now widely accepted. A genetically impaired skin barrier function is already present in nonlesional and more pronounced in lesional skin in atopic dermatitis. Increased epidermal proliferation and disturbed differentiation, including changes in keratins and cornified envelope proteins involucrin, loricrin, and filaggrin, and in lipid composition, cause impaired barrier function in atopic dermatitis (AD) (Fig. 47-12 and see eFig. 47-9.1).81 Mutations in the filaggrin gene have been described by several research groups.83–85 Two loss-of-function genetic variants in the gene encoding filaggrin are strong predisposing factors for atopic dermatitis in atopic kindreds of European origin.86 Slightly different mutations are found in Asian patients with atopic dermatitis and ichthyosis vulgaris.87 These mutations were also significantly associated with asthma, independent of atopic dermatitis, which means that genetic factors that compromise the epidermal barrier could also underlie mucosal atopic diseases (filaggrin is a protein that is unique to keratinizing epithelia). The atopic syndrome represents a genetically impaired skin barrier function as well as impaired nasal, bronchial, and intestinal mucous membrane barriers leading to atopic dermatitis, allergic rhinitis, bronchial asthma, or aggravation of atopic dermatitis. Defective permeability barrier function enables penetration of environmental allergens into the skin and initiates immunological reactions and inflammation (Fig. 47-13). Filaggrin mutation is the first strong genetic factor identified in this complex disease. Filaggrin hydrolysis generates amino acids in their deiminated products that probably serve, together with hornerin, as endogenous humectants.88,89 This may explain the dry skin of atopic dermatitis. Also, gene polymorphisms in the gene for SPINK5, which encodes the serine protease inhibitor LEKTI, have been reported90 and variations within two serine proteases of the kallikrein family, the SC chymotryptic enzyme that degrades corneodesmosomal proteins, involved in the cohesion between the corneocytes of the SC, have been found in some cohorts with AD.

The impaired skin barrier function in atopic dermatitis is also caused by a reduced lipid content or impaired lipid composition in the epidermis atopic dermatitis. In particular, a decreased content for the total amount and for certain types of ceramides has been described.80 A decrease in covalently bound ceramides91 and a reduced sphingomyelinase activity have been found in atopic dermatitis. Also, decreased lamellar body secretion, which is predominantly composed of lipids, with subsequent entombment of lamellar bodies within corneocytes, has been reported.92

Psoriasis: Epidermal Hyperproliferation and the Skin Barrier

(See also Chapter 18)

Psoriasis is a chronic, generalized, and scaly erythematous dermatosis that is primarily localized in the epidermis, showing highly enhanced proliferation and disturbed differentiation, which leads to hyperkeratosis and parakeratosis. In addition, there is a neutrophilic infiltrate in the beginning and in particular in severe cases of psoriasis; later on a moderate T-lymphocytic infiltrate is present. Because of this severely disturbed proliferation and epidermal differentiation, there is an impaired barrier function.93 The level of TEWL is directly related with the clinical severity of the lesion: high TEWL in acute exanthematous psoriasis; a moderate increase in TEWL in the chronic plaque type of the disease. Abnormalities in the SC intercellular lipids, especially a significant reduction in ceramide 1, have been found.94 Electron microscopy studies disclosed severe structural alteration of the intercellular lipid lamellae.95 A genetic linkage of psoriasis to the epidermal differentiation complex 1q21 has been found. Within the epidermal differentiation complex, the SPRRs are highly upregulated in psoriasis plaques.96 Also, the association of psoriasis with cytokeratin K17 has been discussed.

Ichthyosis: The Pathological Lack of Moisture in the Epidermis

(See Chapter 49)

Ichthyosis comprises a group of monogenetic diseases expressing a disturbed desquamation resulting in scales and a mild-to-moderate barrier defect. They are caused either by changes in epidermal lipids or by changes in epidermal differentiation. XLRI is a noncongenital ichthyosis, consisting of a generalized desquamation of large, adherent, and dark brown scales. The metabolic basis of XLRI is an enzymatic lysosomal deficiency of steroid sulfatase or arylsulphatase C. Complete deletions of the STS gene mapped to the Xp22.3-pter region have been found in up to 90% of patients. The reduced cholesterol sulfatase activity leads to accumulation of cholesterol sulfate and a reduction of cholesterol and consequent abnormality in the structural organization of the intercorneocyte lipid lamellae.97–99

Ichthyosis vulgaris is the most common monogenetic skin disease. Recently, loss-of-function mutations in the gene encoding filaggrin that cause ichthyosis vulgaris have been described. During terminal differentiation, profilaggrin is cleaved into multiple filaggrin peptides that aggregate keratin filaments. The resultant matrix is cross-linked to form a major component of the cornified cell envelope. Reduction of this major structural protein leads to an impaired keratinization and to a moderate defect in skin barrier function.100

Transglutaminase 1 is responsible for the cross-linking of several cornified envelope proteins. Therefore, deficiency in transglutaminases 157 leads to lamellar ichthyosis which is a more severe disease than ichthyosis vulgaris with a defect in filaggrin only.

Treatment strategies in inflammatory diseases often address immunogenic abnormalities and barrier function. Treatments with cyclosporine, tacrolimus, pimecrolimus, and UV light have been shown to reduce cell inflammation as well as to improve barrier function, thus helping to normalize proliferation and differentiation. Topical steroids, although clinically effective, do not lead to the repair of the disturbed skin barrier function seen in AD.101 However, because of their side effects, all of these treatments should be used for a short time only. In contrast, application of bland creams and ointments containing lipids and lipid-like substances, hydrocarbons, fatty acids, cholesterol esters, and triglycerides can be used without side effects for long-term treatment of mild-to-moderate inflammatory diseases. Creams and ointments partially correct or stimulate barrier repair and increase SC hydration,44,102–104 thus influencing epidermal proliferation and differentiation.15 It has been proposed that a lipid mixture containing the three key lipid groups [(1) ceramides, (2) cholesterol, and (3) free fatty acids] is able to improve skin barrier function and SC hydration in AD.105 Also, the efficacy of ceramide 3 in a nanoparticle cream in atopic dermatitis has been described.106 However, because several research groups and companies report that creams containing ceramides and a mixture of the three key lipids are not superior to “classical” cream or ointment preparations, such preparations have not yet been widely used.