Anatomy, histology and immunohistochemistry of normal human skin

ARTICLE

The skin is a complex organ covering the whole surface of the body, and is continuous with the mucous membranes lining the body's orifices. It accounts for about 15% of the total adult body weight, and is therefore the largest organ of the body. It exerts multiple vital functions (namely of protection against external physical, chemical and biological aggressions), rendered possible thanks to an elaborate structure, associating tissues of various origins (epithelial, connective, vascular, muscular and nervous). These are organised in three layers, including from top to bottom: a) the epidermis (and its associated appendages, pilosebaceous follicles and sweat glands); b) the dermis, separated from the epidermis by the dermal-epidermal junction, and c) the hypodermis. The fine structure of the skin shows considerable regional variations, concerning its thickness (varying from 1 to 4 mm), distribution of epidermal appendages, density of melanocytes, to name but a few. Glabrous (hairless) skin is found on the palms and soles, whereas hair-bearing skin covers the rest of the body. Embryologically, the epidermis and its appendages are of ectodermal origin whereas the dermis and hypodermis are of mesodermal origin [1-3].

The epidermis

General architecture

The epidermis is a malpighian (stratified) epithelium that renews itself continuously. It is made of various cell types, the majority of which (90-95%) are keratinocytes (KC): these are epithelial, ectodermally-derived cells undergoing a specific differentiation process, resulting in the production of flattened, anucleate cells (corneocytes) that eventually shed from the skin surface. Five to ten per cent of epidermal cells are non-keratinocytes, including mainly Langerhans cells, melanocytes and Merkel cells. Epidermal cells are arranged in continuous layers, comprising (from bottom to top): the basal layer (single layer), the malpighian or prickle-cell layer or stratum spinosum (5-15 layers), the granular layer (1-3 layers) and the horny (or cornified) layer (5-10 layers), further subdivided into a deep, compact layer (stratum compactum) and a superficial, loose part (stratum disjunctum). In some body areas (namely the palmoplantar region) an additional layer, the stratum lucidum, can be seen between the granular and the horny layers (Fig. 1). Epidermal KC originate from mitotic divisions of stem cells of the basal layer; the daughter KC produced there migrate towards the skin surface while undergoing morphological and biochemical differentiation (keratinisation), and shed there-from in about 30 days. The epidermis has an average thickness of 100 mum, but this varies considerably with the body area considered (50 mum on the eyelids to 1 mm on the palms and soles). The epidermis also contains two distinct structural units, i.e. the superficial part of eccrine sweat glands (acrosyringium) and hair follicles (acrotrichium); these are also made of epithelial cells, but have their own biological properties (concerning namely renewal, differentiation, and response to external stimuli), reflected by a particular histologic aspect.

Keratinocytes

The morphology of KC varies with the epidermal layer where they are observed. Basal layer KC are columnar or cubical, measuring 6-10 mum; they possess a basophilic cytoplasm and a rather large nucleus. They are aligned perpendicular to the underlying basement membrane to which they are anchored by special attachment structures, the hemidesmosomes. Electronmicroscopically, basal KC contain melanosomes over the nucleus and loose bundles of tonofilaments, electron-dense cytoplasmic bundles of intermediate filaments (tonofibrils) consisting biochemically of keratin polypeptides; these insert to the attachment plaques of desmosomes and contribute to the formation of the cytoskeleton, conferring mechanical resistance to the KC and the epidermis as a whole. Basal KC are connected to adjacent cells by several types of intercellular junctions (including gap and adherens junctions), the most characteristic of which are the desmosomes. The basal cell layer contains stem cells and cycling KC, expressing proliferation antigens (such as Ki67 and PCNA): they correspond to the germinative or proliferative compartment, which is responsible for the regeneration of the epidermis through mitotic divisions. Malpighian layer KC are larger (10-15 mum), polygonal, with an eosinophilic cytoplasm and a vesicular nucleus with one or two conspicuous nucleoli. Ultrastructurally they contain coarse bundles of tonofilaments. Granular layer KC are flattened, lying parallel to the skin surface, with a diameter of 25 mum; they contain keratohyalin granules, highly basophilic polygonal grains made of an assembly of histidin-rich proteins (mainly profilaggrin) and keratin. KC of the uppermost malpighian and granular layers contain also lamellar bodies (also known as Odland bodies, keratinosomes or membrane-coating granules), round 100-300 nm organelles with a characteristic lamellar internal structure; these are involved in the process of desquamation and the formation of a lipidic pericellular coat acting as a penetration barrier against foreign (hydrophilic) substances. The horny layer is made of corneocytes, highly flattened, eosinophilic KC that eventually shed from the skin surface, thus contributing to the barrier function of the skin. Viewed from above, corneocytes are hexagonal in shape and measure 30-40 mum in diameter, corresponding to the surface occupied by 5-10 basal KC. Corneocytes are devoid of nucleus and of cytoplasmic organelles; they are made of a dense filamentous keratin matrix and of a thick cornified envelope deep to the cell membrane, consisting of cross-linked proteins (namely involucrin, loricrin, SPRRs, elafin and cystatin A) [4].

The cytoskeleton of KC is essentially made of keratins (K), fibrous proteins belonging to the family of intermediate filaments. At least 20 distinct polypeptides of "soft" or epidermal K have been well-characterised and classified according to their MW and their pI; they include low MW (40-56.5 kDa) or acidic (pI < 5.5) or type I K (K9-K20), and high MW (52-67 kDa) or neutral/basic (pI > 6) or type II K (K1-K8) [5-8]. Each KC expresses K polypeptides in pairs, made of an acidic and a basic polypeptide, according to a pattern depending on the differentiation of the cell considered. Within normal epidermis, basal KC express K5 and K14/K15 (Fig. 2), suprabasal ones express K1 and K10, and granular layer ones express K2e and K11. Plantar KC express K9. KC also express desmosomal proteins (desmoplakins, desmogleins, desmocollins...), and corneocytes express corneodesmosin [9], a protein stored in lamellar bodies. Upper layer KC also express epithelial-specific antigens such as (pro)filaggrin (37 kDa), the main component of keratohyalin granules, and soluble cytoplasmic protein precursors of the cornified envelope [4].

Langerhans cells (LC)

LC are mobile, dendritic, antigen presenting cells present in all stratified epithelia, originating from CD34⁺ haemopoietic precursors of the bone marrow. LC are capable of uptaking exogenous antigens (namely infectious) deposited on the skin, processing them and presenting them to naive T-cells; they represent 3-6% of all cells in the epidermis, where they are considered immature, becoming mature after contact with the antigen [10].

LC have a rounded cell body with dendritic processes extending between adjacent KC, to which they adhere via homophilic binding between molecules of E-cadherin, present on the surface of both cell types. Ultrastructurally, LC have an electron-lucent cytoplasm, devoid of tonofilaments and melanosomes, and an indented or lobulated nucleus. They contain a specific cytoplasmic marker, the Birbeck granule, originating from the cell membrane and involved in the process of endocytosis. This would have the form of a disk, and in transmission electronmicroscopy it presents as a 300 nm-1 mum long rod, with an internal trilaminar structure reminiscent of a zipper (Fig. 3); it occasionally terminates with a vesicle, thus having a tennis-racquet shape.

Light-microscopically, LC can be demonstrated by histoenzymatic (ATPase) and especially immunohistochemical techniques, thanks to the expression of a set of antigens, the most specific of which are the CD1a antigen (Fig. 4) and above all antigens associated with the Birbeck granules, CD207/langerin [11] (Fig. 5) and Lag [12]. Other (less specific) antigens include vimentin, HLA class II antigens, S100 protein (Fig. 6), receptors of Fcgamma Fcepsilon and C3.

The term "indeterminate cell" has been used to describe intraepidermal dendritic non-keratinocytes with no specific ultrastructural organelles. Studies of serial sections have shown that these cells are in fact LC containing few Birbeck granules, necessitating the examination of multiple sections.

Melanocytes (MC)

MC originate from the neural crest and migrate into the epidermis where they produce melanin, the main natural pigment of the skin. They are distributed regularly among basal KC, at a ratio of 1 MC for 4-10 basal KC. Their density reaches 500-2,000 cells per mm² of cutaneous surface, with regional variations (maximal density on genital skin). Melanin (eumelanin or phaeomelanin) is produced through the enzymatic activity of tyrosinase from the substrate tyrosine and stored in melanosomes, ovoid or spherical organelles undergoing 4 maturation stages (I to IV). Mature melanosomes are subsequently transported along the dendritic processes of MC and are eventually transferred, by an as yet poorly-understood mechanism, to adjacent KC where they form an umbrella-like cap over the nucleus, protecting it from injurious effects of UV light. The "epidermal melanin unit" consists of one MC and 36 associated KC to which the MC delivers melanosomes. The ethnic variations in pigmentation are due to differences in activity of MC and distribution of melanosomes within the epidermis, and not to a different number of MC. In routinely-stained skin sections (haematoxylin-eosin), MC appear as basal cells with a basophilic nucleus and a clear cytoplasm (Masson's clear cells). Ultrastructurally, they have an electron-lucent cytoplasm containing loose intermediate filaments (vimentin) and melanosomes at various maturation stages. MC can be recognised by histochemical stains that reveal melanin (such as the ammoniacal silver nitrate reaction of Fontana-Masson), or by the DOPA histoenzymatic reaction. They can also be confidently identified with antibodies recognising MC-specific antigens such as the MART1/Melan-A antigen, a 118 amino-acid protein, tyrosinase and pigmentation-associated glycoprotein (75 kDa). MC also express constitutively the bcl-2 oncoprotein, S100ab protein (Fig. 6) and vimentin. The premelanosome-associated glycoprotein gp100, recognised by the monoclonal antibody HMB-45, is expressed by embryonic MC, hair bulb MC and activated (but not resting) MC of adult epidermis [13].

Merkel cells (MKC)

MKC display both neuroendocrine and epithelial features, and their ontogenesis is not unequivocally settled (neural crest or epidermal stem cells). MKC seem to function as mechanoreceptors, as suggested by their frequent contact with dermal sensory axons with whom they form synaptic junctions.

MKC are localised in the basal layer of the epidermis and the epithelial sheath of hair follicles. Their density shows regional variations (being maximal in palmoplantar skin), but is generally low [14]. MKC are easily recognisable electronmicroscopically thanks to the presence of cytoplasmic neurosecretory granules, round 80-120 nm granules with a central electron-dense core surrounded by a clear halo and a single limiting membrane. The plasma membrane of MKC occasionally contains small desmosomes linking them to adjacent KC (Fig. 7). MKC can also be identified with histochemical (uranaffin) stains and more confidently by immunohistochemistry, thanks to the expression of specific antigens, including keratin n° 20 [15], neuron-specific enolase (glycolytic enzyme of neuronal/neuroendocrine cells), chromogranin (68 kDa protein component of neurosecretory granules), synaptophysin (acid 38 kDa protein associated to synaptic vesicles), neural cell adhesion molecule (N-CAM) and various neuropeptides (protein gene product 9.5, met-enkephalin, calcitonin-gene related peptide, vasoactive intestinal polypeptide).

Lymphocytes

The normal human epidermis contains a low percentage (< 1.3%) of lymphocytes, that are usually inconspicuous by immunohistochemical techniques. These cells would be present mainly in the basal layer, and have been studied by flow-cytometry. They express predominantly a T-memory/effector phenotype (TCR alphabeta⁺, CD3⁺, CD4⁺ CD8^- CD45RO⁺, Fas⁺, aeb7 integrin), with regional phenotypic variations [16].

Toker cells

These are cells with a clear cytoplasm, first described in 1970 in the epidermis of 10% of nipple skin in both sexes. Their precise nature and role in normal and diseased skin remain poorly understood. According to recent studies, these cells express keratin n° 7 [17] as well as some mucins (MUC1, MUC2, MUC5AC) also present in cells of mammary Paget's disease.

Epidermal appendages

These are specialised epithelial structures, connected to the surface epidermis but located mainly within the dermis and hypodermis. They comprise sweat glands and pilosebaceous follicles.

Sweat glands

They are tubular exocrine glands, consisting of a secretory coil and an excretory duct; two main types of sweat glands exist in humans, eccrine (ESG) and apocrine (ASG).

ESG are the main sweat glands in humans, playing a vital role in the process of thermoregulation. They are present almost everywhere on the skin (but not on mucous membranes), with a maximal density over the palms, soles (300/cm²), axillae and forehead. Their secretory coil is a blind tube surrounded by dermal dendrocytes and non-myelinated cholinergic nerve fibres, and lies in the deep dermis and the dermal-hypodermal junction. It is composed histologically of 1-2 rows of secretory, clear or dark cells. The former produce sweat, evacuated toward the central lumen through intercellular canaliculi. Dark cells are found toward the lumen of the ESG coil and contain acid PAS-positive mucopolysaccharides, but their exact role is not known with certainty. In some people the secretory coil of ESG is made uniquely of clear cells with a foamy cytoplasm. The secretory cells are lined peripherally by a discontinuous layer of flat myoepithelial cells, containing myofilaments, visible electron-microscopically and consisting of muscle-specific actin. The excretory duct traverses the dermis vertically and enters the epidermis, where it forms an individual, coiled structure (acrosyringium), opening to the skin surface by a small orifice, the pore. The dermal excretory canal of ESG is made of a double layer of cubical cells, expressing keratins n^os 6 and 16 [7]. The basal layer ones are mitotically active and are responsible for the renewal of acrosyringeal cells [18]. The cells of the inner layer have a villous luminal surface, corresponding light-microscopically to a PAS-positive cuticle.

ASG (rare in humans but abundant in all animal species) derive embryologically from the primary epithelial germ that also produces the pilosebaceous follicle; accordingly, ASG are invariably associated with a pilar apparatus, and are hence called "epitrichial", contrasting with ESG (that are "atrichial"). In human skin, ASG are found in the axillary and anogenital region and the nipple. They consist of a secretory coil which is larger and more irregular in shape than that of ESG, lying in the deep dermis. Secretory cells are columnar or pyramidal and show decapitation secretion, characteristic of apocrine epithelia. A peripheral layer of myoepithelial cells exists, as in ESG. The excretory canal is short, lined by a double cell layer, and empties into the pilar canal, at a site above the sebaceous gland.

A third type of sweat gland was more recently described in the axillary region, the so-called "apoeccrine" glands. These are atrichial glands, opening directly to the skin surface (like ESG), but their secretory coil is similar to that of ASG; they are not present in children but develop during puberty from ESG [19].

The various cells of sweat glands express specific antigens that allow their immunohistochemical identification: the Carcinoembryonic antigen (a 180-200 kDa oncofetal glycoprotein representing a heterogeneous group including several members such as NCA-1, NCA-2, NFA-2, biliary glycoprotein...) is expressed on the apical pole of cells of both the secretory and excretory part of ESG and, to a lesser degree, ASG. Epithelial membrane antigen is detected on the apical pole of secretory cells of ESG and ASG, namely at the level of the intercellular canaliculi of ESG; it is also expressed, although more weakly, on the apical pole of the cells of the excretory canal [20]. Secretory cells of ESG and ASG express low MW keratins (n^os 8, 18, 19), characteristic of simple (glandular) epithelia. A sub-population of cells of secretory ESG (but not of ASG) express S100 protein and bcl-2 oncoprotein [21]. Gross cystic disease fluid protein 15 (GCDFP-15), the best characterised member of a family of proteins isolated from the liquid of fibrocystic disease of the breast, was initially considered specific of apocrine secretory epithelia, although it seems now to be also occasionally expressed by ESG. Myoepithelial cells lining the secretory coil of ESG and ASG express the "contractile" keratin K17 and smooth muscle actin.

Pilosebaceous follicles

They develop embryologically from the primary epithelial germ and lie obliquely in the dermis, their deepest part reaching the hypodermis. They are distributed throughout the integument, with the exception of palms, soles and portions of the genitalia (the so-called glabrous skin). Their size and morphology is variable (terminal, vellus, lanugo and intermediary hair). Their growth is cyclic and proceeds through three distinct phases of uneven duration (anagen, catagen, telogen) during which their histological aspect varies considerably.

Hair follicles comprise several segments, including from top to bottom: a) an intraepidermal part (acrotrichium); b) the infundibulum, extending down to the opening of the sebaceous duct; c) the isthmus, extending down to the bulge, the site of insertion of the arrector pili muscle. The bulge contains the stem cells of the hair follicle, that express keratin K15 [22]; d) the lower follicle, extending down to the top of the hair bulb; e) the hair bulb, a terminal bell-like extremity containing highly basophilic matrix cells responsible for hair growth, and melanocytes responsible for the pigmentation of the hair. The hair bulb forms an invagination that envelops the follicular (or dermal) papilla, a richly vascularised and innervated connective tissue containing papillary fibroblasts, important for the growth of the hair.

The upper segments of the hair follicle (acrotrichium and infundibulum) undergo an epidermal-type differentiation, and their morphology varies little with time. By contrast, the structure of the deeper part of hair follicles varies with the phase of the hair cycle. This part is made of an epithelial sheath, consisting of several concentric layers. The outer root sheath (or trichilemma) is made of large clear PAS⁺, glycogen-rich cells and is surrounded by a thick, vascular connective sheath. The inner root sheath is made of three concentric layers (Henle, Huxley and cuticle).

The pilar canal contains the hair shaft, product of the terminal keratinisation of the hair follicle. In transverse section the hair is made of three concentric layers, the cuticle, the cortex (forming the bulk of the hair shaft) and the medulla (present only in terminal hair). It consists of hard or pilar keratins, comprising two types, acid or type I (polypeptides hHa1-9) and basic/neutral or type II (polypeptides hHb1-6) [23].

The hair or pilar disk (Haarscheibe) is a specific structure measuring 0.5 mm, found in the vicinity of hairs and considered as a slow-adapting mechanoreceptor. Histologically it presents as an acanthotic elevation of the epidermis, limited laterally by two elongated rete ridges. The underlying dermis is richly vascularised and innervated. Electronmicroscopically, the epidermis contains several Merkel cells.

Sebaceous glands are multilobated holocrine glands associated with hair follicles. They are located in the space delimited by the skin surface, the hair follicle and the arrector pili muscle. They are made of an outer layer of basal cells and of several layers of mature, lipid-laden cells with a foamy cytoplasm (sebocytes); these cells disintegrate toward the centre of the gland and thus form the secretion product (sebum), that empties into the pilar canal through a short duct. Sebaceous glands are more abundant and large in facial skin, associated with small vellus hairs.

Basal sebocytes express appreciable amounts of keratins. Mature sebocytes express cytoplasmic reactivity for high MW keratins, Epithelial membrane antigen (a complex, highly glycosylated protein of 70 kDa), Biliary glycoprotein (160 kDa member of the family of the Carcinoembryonic antigen) [20], the Thomsen-Friedenreich (T) and, to a lesser degree, its precursor, the Tn antigen [24], the ovarian cystadenocarcinoma antigen OM-1 and lipase. Mites of the Demodex folliculorum species, often found within hair follicles of the face, are recognised by antibodies to the Tn antigen [25].

The arrector pili muscle is a smooth muscle arising in the papillary dermis and extending obliquely to the hair follicle, into which it inserts at the level of the bulge. Its positioning could be guided during foetal life by epidermal Merkel cells. It is composed of elongated cells with an eosinophilic cytoplasm, expressing desmin (the 53 kDa intermediate filament specific of muscle cells) and (smooth) muscle specific actin. When contracted, stimulated by sympathetic nerve fibres, this muscle makes hair "stand on end", thus increasing the thermal barrier.

The nails

They cover the dorsal aspect of the last phalanges of the fingers and toes, and belong to the cutaneous appendages. They consist anatomically of three parts: a) the root, overlapped by the proximal nail fold, in continuity with the lateral nail folds; b) the nail plate, hard keratin plate consisting of "hard" (pilar) keratins, and c) the free edge, overlying the hyponychium, a thickened epidermis. The nail lies on the nail bed, a richly vascular connective tissue containing numerous arteriovenous shunts. The proximal part of the nail bed is continuous with the nail matrix, responsible for nail growth. This undergoes an epidermal-type keratinisation, but (like hair) is devoid of granular layer.

The dermal-epidermal junction (DEJ)

The DEJ is a complex basement membrane synthesised by basal KC and dermal fibroblasts. It plays a fundamental role as a mechanical support for the adhesion of the epidermis to the dermis and regulates the exchanges of metabolic products between these two compartments; besides, it serves as a support for KC migration during wound healing, and is traversed by various cell types (LC, lymphocytes...) during immunologic and inflammatory processes.

By light microscopy, the DEJ presents as an undulating zone, hardly visible with routine stains, highlighted after PAS staining (due to the presence of neutral mucopolysaccharides). Its fine structure was revealed thanks to electronmicroscopy. The DEJ is made of four distinct layers, comprising (from top to bottom): a) the cell membrane of basal KC with their hemidesmosomes, to which cytoskeletal filaments (keratin and plectin) attach; b) the lamina lucida, an electron-lucent space (ca 40 nm wide) traversed by anchoring filaments (2-4 nm); c) the osmiophilic lamina densa, (50-70 nm thick) and d) the sub-basal lamina filamentous zone, mainly made of anchoring fibres (Fig. 8).

The antigenic composition of the DEJ is complex; presently over twenty macromolecules have been well characterised at the biochemical and genomic level [26]. Hemidesmosomes contain: a) the bullous pemphigoid antigen of 230 kDa (BPAG1), member of the plakin family b) plectin, a 500 kDa phosphoprotein, also of the plakin family; b) the alpha6beta4 integrin, specific of epithelia; c) the bullous pemphigoid antigen of 180 kDa (BPAG2), also known as collagen XVII, transmembrane molecule spanning the lamina lucida and anchored in the lamina densa. The lamina lucida contains various laminin isoforms, namely laminin 5, formerly known as nicein, kalinin or epiligrin (alpha3beta3gamma2), laminin 6 (alpha3beta1gamma1) and laminin 10 (alpha5beta1gamma1), contributing, along with BPAG2, to the formation of anchoring filaments. The lamina densa is essentially made of type IV collagen, the structural scaffold of all basement membranes, and of laminin 5. Anchoring fibres are made of type VII collagen (290 kDa) (formerly known as the epidermolysis bullosa acquisita antigen).

Some other antigens have been detected within the DEJ, but not yet adequately characterised, such as uncein (19-DEJ-1 antigen), NU-T2 antigen, antigens KF1, LDA1, nidogen, heparan-sulfate proteoglycan, chondroitin sulphate proteoglycan, antigens AF1 and AF2, thrombospondin, type V collagen, osteonectin/BM-40. "Ladinin" is a 97 kDa antigen recognised by sera of patients with linear IgA dermatosis; this was considered as a distinct molecule, but it seems now that this antigen is produced by alternative splicing of the gene of BPAG2.

The dermis

Architecture

The dermis (or corium) is a supportive, compressible and elastic connective tissue protecting the epidermis, its appendages and the vascular and nervous plexuses running through it. It consists of cells, fibrous molecules and a ground substance. It undergoes a continuous turnover, regulated by mechanisms controlling the synthesis and degradation of its protein components. The thickness of the dermis varies considerably with the anatomic location (being much thicker on the back or palms and soles than on the eyelids), and its fine structure varies somewhat depending on depth (superficial or papillary dermis, reticular or deep dermis).

The papillary (superficial) dermis forms conic upward projections (dermal papillae) alternating with epidermal rete ridges, thus increasing the surface of contact between the dermis and epidermis and allowing for a better adhesion between these layers. It contains several cells (fibroblasts, dermal dendrocytes, mast cells), vessels and nerve endings. It is made of collagen fibres arranged in loose bundles and of thin elastic fibres stretching perpendicularly to the dermal-epidermal junction. At the level of the extremities (namely fingers), dermal papillae contain tactile corpuscles, specialised nerve endings acting as mechanoreceptors. The reticular (deep) dermis is made of coarser collagen bundles, tending to lie parallel to the skin surface. The elastic network is also thicker. The reticular dermis contains the deep part of cutaneous appendages, vascular and nerve plexuses.

Fibres

The great majority (> 90%) of dermal fibres are made of interstitial collagen, mainly of types I and III, responsible for the mechanical resistance of the skin. Collagen accounts for 98% of the total mass of the dried dermis. Ultrastructurally, collagen fibres have a diameter of 100 nm, and show in longitudinal section a characteristic cross-striation with a periodicity of 64 nm (Fig. 9). These fibres are arranged in bundles that are loose in the papillary dermis and become thicker in the deep dermis. Other collagens found in the dermis include type IV collagen (DEJ and basement membranes of cutaneous appendages, vessels, muscles and nerves) and type VII collagen (anchoring fibres of the DEJ).

Elastic fibres, responsible for the retractile properties of the skin, can be visualised with histochemical stains (orcein). In the papillary dermis they are thin (elaunin-oxytalan fibres) but become thicker in the reticular dermis, where they tend to run horizontally. By electron microscopy, elastic fibres show variations depending on age and the area studied (sun-exposed or not). They have irregular contours and are made of a central amorphous matrix of variable electron density, showing irregular densifications and composed of elastin, an insoluble protein; this is surrounded by a varying number of microfibrils, made of fibrillin.

Reticulin fibres, visualised after special histochemical silver stains, consist biochemically of an assembly of thin collagen fibres (types I & III) and fibronectin.

The ground substance

This consists of macromolecules filling the space between fibres and dermal cells, and is more abundant in the papillary dermis and around cutaneous appendages. It is not visible in routine histologic sections, but is lightly stained by histochemical stains (namely alcian blue). Biochemically it consists of glycoproteins and proteoglycans (hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, fibronectin, tenascin, epimorphin...) interacting with the fibrous and cellular components of the dermis. These molecules can be revealed with specific antibodies.

Resident cells of the dermis

Fibroblasts are the fundamental cells of the dermis and all connective tissues, synthesising all types of fibres and the ground substance. They appear as spindle-shaped or stellate cells, containing a well-developed rough endoplasmic reticulum. They can be identified with antibodies recognising the pan-mesenchymal markers (vimentin, Te7 antigen), enzymes involved in collagen synthesis (proline-4-hydroxylase) or as yet poorly-characterised antigens (FibAS), allegedly specific of human fibroblasts [27]. Fibrocytes are small, quiescent fibroblasts, devoid of obvious metabolic activity, encountered in mature connective tissues. Fibroclasts are fibroblasts with phagocytic activity (toward collagen). Myofibroblasts are cells derived from fibroblasts, namely during the process of wound healing; they contain myofilaments, visible by electron microscopy, and express (smooth) muscle actin and more rarely desmin [28].

Dermal dendrocytes (DD) represent a heterogeneous population of mesenchymal dendritic cells, recognised mainly thanks to immunohistochemistry and previously most likely confused with fibroblasts [29]. At least two types of DD exist in human dermis. DD of type I are recognised thanks to the specific expression of the coagulation factor Xllla, an intracellular protransglutaminase that stabilises fibrin. They are present around capillaries of the papillary dermis (Fig. 10), around sweat gland coils and within the connective tissue septa of the hypodermis. They express mesenchymal markers (vimentin, Te7 antigen), as well as some surface molecules of antigen-presenting cells (HLe1, HLA DR/DQ, CD14, CD36), but do not express the Langerhans cell-specific antigens (CD1a, CD207, Lag, S100 protein) [30, 31]. DD type II are characterised by the expression of the CD34 antigen (or human progenitor cell antigen, HPCA-1), a 105-120 kDa transmembrane glycoprotein. They express mesenchymal antigens and occasionally HLA-DR antigens, but are factor XIIIa/CD1a/S100 protein-negative [32]. They are located in the mid- and deep dermis, namely around the eccrine secretory coils and hair follicles, at the level of the bulge. DD lack Birbeck granules but have no distinctive ultrastructural features.

Mast cells

These are mononuclear cells of bone marrow origin, sparsely distributed in the perivascular and periadnexal dermis. They stain metachromatically with toluidin blue stain, and immunohistochemically with antibodies to chymase and tryptase [33]. Similarly to melanocytes, they also express the c-kit protein [34]. Ultrastructurally, they have a villous cell membrane and contain characteristic cytoplasmic granules, with a tubuloreticular internal structure.

The mid- and deep dermis harbours a population of spindle-shaped cells expressing the CD10 antigen (neutral endopeptidase). The precise nature of these cells is not well known; they could represent a subset of fibroblasts or of deep DD [35].

Cutaneous vasculature

General architecture

Except epidermis, which is a non-vascular tissue, the skin possesses a rich vascular network, largely exceeding its nutritive requirements, involved in thermoregulation, wound healing, immune reactions and the control of blood pressure. Cutaneous vessels belong to the arterial, venous or the lymphatic system; they originate from perforating arteries originating from underlying vessels of the muscles, and form two distinct horizontal plexuses that communicate via vessels traversing the dermis vertically. The deep plexus lies close to the dermal-hypodermal junction and provides nutritional arteries to sweat glands and hair follicles. The superficial plexus, derived from terminal arterioles, lies at the interface between the papillary and reticular dermis, and provides to every dermal papilla a vascular loop ascending vertically toward the surface (except in the nail bed). This consists of an ascending precapillary arteriole, of arterial and venous capillaries forming a hairpin turn, and of a descending post-capillary veinule (these account for the majority of vessels in the papillary dermis) [36].

Arterioles and veinules communicate directly in some areas (fingers, nail bed, nose, ears) by specialised arterio-veinous shunts, the neuromyoarterial glomus bodies (Masson). These structures function like sphincters, allowing the capillary circulation to be short-circuited when they are open.

The lymphatic system plays an important role in the regulation of the pressure of the interstitial fluid, the removal of extracellular fluid and in immune reactions. It starts with blind lymphatic capillaries (or prelymphatic tubules) forming a superficial plexus in the sub-papillary dermis, and a deep plexus, below the deep arterial plexus. The lymphatic network is particularly well-developed in digital, palmoplantar and scrotal skin.

Histology of cutaneous vessels

Dermal arteries and arterioles (< 0.3 mm), recognisable by their round lumen, are composed of three layers: a) the intima, made of endothelial cells resting on an undulating internal elastic lamina, visible after orcein stain; b) the media, made of two layers of smooth muscle cells, lying longitudinally or concentrically, of increasing thickness in deep vessels; c) the adventitia, made of connective tissue, occasionally containing an outer elastic lamina. Veins and veinules have a similar structure, but have larger lumen and a thinner muscular wall occasionally containing valves; the internal elastic lamina is very thin or absent. The adventitia is thick, poor in elastic fibres. Arterioles and veinules of the deep dermis and hypodermis are larger than the corresponding vessels of the superficial plexus (diameter 50-100 versus 25 mum, wall thickness 10-15 versus 4-5 mum), and pericytes are more abundant.

Dermal capillaries (5-15 mum) consist of a single fenestrated endothelial cell layer, and of a (discontinuous) outer layer of pericytes, surrounded by a simple basement membrane. Post-capillary veinules have a similar structure, but have a slightly larger calibre, are surrounded by more pericytes and their basement membrane is multilayered. The pericapillary space of the superficial dermis contains factor XIIIa-positive dermal dendrocytes and mast cells; along with endothelial cells of the capillary loops, these cells constitute the dermal microvascular unit.

Neuromyoarterial glomus bodies are made of an arteriole (the Sucquet-Hoyer canal) measuring 20-40 mum, connecting directly to a veinule. The Sucquet-Hoyer canal is surrounded by 4-6 layers of glomus cells, specialised epithelioid-like muscle cells richly supplied with non-myelinated nerve fibres.

Lymphatic vessels are hardly visible in normal skin, being more readily apparent when they are dilated under the effect of an increased interstitial pressure (oedema). They are better seen after orcein staining, highlighting their elastic membrane, or by immunohistochemical techniques. They present as optically clear irregular, variously-shaped cavities, up to 60 mum in diameter. They are lined by a continuous layer of flat endothelial cells, having a scant cytoplasm and a prominent nucleus (up to 5 mum high). Except from the most superficial vessels, lymphatics contain multiple valves with intrinsic motility; they are devoid of basal lamina and of pericytes. Lymphatics of the deep dermis (namely of the lower limbs) occasionally have a muscular wall, but have no elastic lamina. Ultrastructurally, intercellular junctions are less conspicuous than in blood capillaries. Endothelial cells are attached to adjacent molecules of collagen/elastin by anchoring filaments that stretch under the effect of the increase of tissue pressure, thus favouring the removal of excess interstitial fluid [37].

Ultrastructurally, aside from the usual organelles, blood endothelial cells contain well-developed bundles of intermediate filaments (vimentin), pinocytotic vesicles, and specific organelles, the Weibel-Palade bodies. These are electron-dense, cigar-shaped bodies measuring 0.1-0.3 mum that contain 15 nm-thick tubules lying parallel to the long axis of the organelle; they are the storage site of the coagulation fact. von Willebrand ("fact. VIII"). These bodies are not seen in lymphatic endothelial cells (except in those of larger vessels). Endothelial cells of larger arterioles in the mid- and deep dermis contain also actin filaments (4-7 nm). Capillary endothelial cells possess fenestrations allowing the passage of intravascular fluid. Arteries are surrounded by a continuous and homogeneous basal lamina, whereas post-capillary veinules have a multilayered basal membrane. Pericytes have dendritic processes and are surrounded by a basement membrane; they contain cytoplasmic myofilaments (5 nm), also found in glomus cells.

Immunohistochemistry - histoenzymology

Blood endothelial cells express vimentin, von Willebrand factor ("factor VIII"), the CD34 (Fig. 11) and CD31 antigens [38], thrombomodulin [39], residues of a-L-fucose (recognised with the lectin UEA-I or with specific monoclonal antibodies such as BNH9), the PAL-E and EN-4 antigens, the adhesion molecules ICAM1 and VCAM1, and E-selectin. Lymphatic endothelial cells weakly express von Willebrand factor but are CD34-negative. Recently, some antigens were described that seem to be specific to lymphatic (as opposed to blood) endothelial cells; these include VEGFR-3 (VEGF-C receptor), podoplanin, and LYVE-1 (hyaluronic acid receptor) [40]. Muscle cells of the media express desmin, vimentin, and muscle-specific actin. Pericytes and glomus cells express vimentin and muscle actin. The vascular basement membrane contains laminin and type IV collagen, antigens weakly expressed around lymphatic vessels. Endothelial cells are endowed with some enzymatic activities (such as acid & alkaline phosphatase), that can be used to visualise the cutaneous vasculature.

Cutaneous innervation

The skin contains a rich and complex innervation, consisting of an afferent and an efferent limb. The afferent limb (centripetal innervation) belongs to the cerebrospinal system and is responsible for the perception of the variations and aggressions coming from the exterior (touch, pressure, vibration, pain, temperature, itch). This function is mediated by a network of sensory myelinated or non-myelinated fibres, free terminal nerve endings and tactile corpuscles (Wagner-Meissner, Vater-Pacini, Krause). The efferent limb (centrifugal innervation) is supported by non-myelinated fibres of the sympathetic system that regulate vasomotricity, sweat secretion and pilo-errection.

Histologically, the motor and sensitive endings are not visible on routinely-stained sections. Dermal nerves are readily recognisable thanks to their undulating appearance; they contain axons surrounded by Schwann cells and by perineural fibroblasts. These cells can be readily differentiated by immunohistochemistry: axons express neurofilaments (68, 160 and 200 kDa) (Fig. 12) and peripherin (the intermediate filaments of neuronal cells), neuron specific enolase and PGP9.5. Schwann cells express S100 protein, glial fibrillary acidic protein, myelin basic protein and the AHMY1 antigen. Perineural fibroblasts express vimentin and Epithelial membrane antigen [13].

Tactile corpuscles of Wagner-Meissner are ovoid organelles found in dermal papillae, namely in finger skin; they consist of a horizontal wrapping of Schwann cells contain-ing an axon, and function as rapidly-adapting mechanoreceptors. Mucocutaneous end-organs are found at the level of junction between the skin and mucous membranes. Vater-Pacini corpuscles function as receptors of deep pressure and vibration; they are found at the level of the dermal-hypodermal junction and consist of a concentric wrapping of Schwann cells and axons, with a characteristic, onion-like appearance.

The presence of nerve fibres within the epidermis has long been debated. Recent immunohistochemical studies have shown that dermal axons, expressing the N-CAM protein, PGP9.5 and Calcitonin Gene-Related Peptide, penetrate the epidermis and come in contact with Langerhans cells, possibly regulating their antigen-presenting capacity [41].

The hypodermis (subcutaneous fat, panniculus adiposus)

This is a fatty tissue representing the deepest part of the skin, separating it from the underlying aponevroses or the periosteum. It plays an important role in thermoregulation, insulation, provision of energy (nutritional store) and protection from mechanical injuries.

The main cells of the hypodermis are the adipocytes, large (up to 100 mum), rounded cells with a lipid-laden cytoplasm (triglycerides, fatty acids) compressing the nucleus against the cell membrane. In routinely-stained sections, adipocytes appear optically empty because their content is dissolved during fixation-dehydratation. Ultrastructurally, their cytoplasm is very weakly osmiophilic. Immunohistochemically, they show pericellular expression of S100 protein (Fig. 13) and vimentin [13].

Adipocytes are arranged in primary and secondary lobules, the morphology of which varies somewhat according to the sex and the body region considered. These lobules are separated by connective tissue septa containing cells (fibroblasts, dendrocytes, mast cells), the deepest part of sweat glands, as well as vessels and nerves contributing to the formation of the corresponding dermal plexuses.

Article accepted on 14/3/02

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