Immunohistochemistry of normal human skin

ARTICLE

The skin is the largest organ of the body and consists of various cell types (epithelial, mesenchymal, vascular, neural, muscular). The cellular and extracellular components of the skin express an ever-growing number of antigens that can be recognised by specific monoclonal or polyclonal antibodies. Whereas several antigens are widely distributed, some differentiation antigens are sufficiently specific and sensitive to particular components of the skin and can therefore be used as "immunohistochemical markers". Immunohistochemical techniques nowadays allow us to detect with confidence a large variety of various cell types and extracellular molecules in normal skin. Knowledge of the immunohistological features of the various skin components is an absolute prerequisite for the application of these techniques in diagnostic dermatopathology, particularly in the field of tumoural and bullous skin diseases [1-4]. This review will present the most important immunohistochemical features of the cellular and extracellular components of the skin, with particular emphasis on those markers that are important for diagnostic purposes and which can be detected on routinely-processed tissue specimens (fixation in formalin & embedding in paraffin). The quality of immunohistochemical labelling depends largely on technical conditions pertaining to fixation (type of fixative and duration of fixation), tissue embedding, antigen retrieval (microwave or proteolytic pretreatment) and the protocol for the technique itself (direct, indirect, amplification, etc.), but dealing in detail with these conditions is beyond the scope of this review; relevant data can be found elsewhere [5-7]. Let us simply recall here that the meaningful interpretation of immunohistochemically-labelled slides requires both sound histological knowledge of the skin and a particular skill so as to recognise false-positive or negative results, since the pitfalls are numerous [6, 8].

The epidermis

The epidermis is a stratified, keratinizing epithelium consisting of epithelial cells (keratinocytes) (85-90%) and symbiotic non-keratinocyte cells usually with a dendritic morphology, representing 10-15% of the total cell population; these include Langerhans cells, melanocytes and Merkel cells. Furthermore, recent immunohistochemical studies have revealed that the epidermis contains free nerve endings (see about cutaneous nerves).

Keratinocytes (KC)

KC constitute the epithelial component of the epidermis and its adnexae; they express keratins (K), which represent the pan-epithelial marker. K are a multigenic family of fibrous proteins belonging to the class of intermediate filaments (Table I), that participate in the formation of the cytoskeleton. Individual K polypeptides are classified according to their MW and pI into low MW (40-56.5 kD) or acidic (pI < 5.5) or type I K, and high-MW (52-67 kD) or basic (pI > 6) or type II K [9-12]. The catalogue of Moll [12], based on this classification, comprises 20 different polypeptides (K1-K9 or type II K and K10-K20 or type I K). Each KC expresses K in pairs made of an acidic- and a basic-type filament, in a pattern that depends on the level of cell differentiation (Table II). In normal epidermis, basal KC express K5 and K14 whereas suprabasal KC express K1 and K10 (Fig. 1). Stratum granulosum cells express K2 and K11; KC from the plantar region express K9. Activated or hyperproliferative KC further express K6 and K16. Both broad-spectrum and polypeptide-specific anti-K monoclonal antibodies exist that allow the detection of individual K polypeptides. Upper-layer KC further express some other antigens that are highly specific for epithelial differentiation but not sensitive enough since they are only expressed at a late (terminal) stage of differentiation; these include (pro)filaggrin, a 37 kDa component of keratohyalin [13], as well as soluble cytoplasmic proteins precursors of the cornified envelope (involucrin, loricrin, keratolinin, envoplakin,...) [14, 15]. Similarly, the surface of KC bears carbohydrate residues that are expressed in a differentiation-specific pattern; they may be recognised with the use of lectins (e.g. PNA, UEA-I, etc.) but are not epithelial-specific. Finally, all KC express desmosomal proteins (desmoplakins, desmogleins, desmocollins, desmocalmin, plakoglobin, plakophilin,...) and E-cadherin [16]; most of these antibodies can only be used on frozen-skin specimens, but reagents are now available that recognise some of these antigens (e.g. desmogleins 1 & 3, E-cadherin) in paraffin-embedded material (Fig. 2).

Langerhans cells (LC)

LC are resident cells of all stratified epithelia, and are occasionally observed in the dermis. They originate from CD34⁺ bone-marrow precursors and make up 2-8% of the total cell population of normal human epidermis [16]. They can be recognised by electron-microscopy thanks to the presence of the Birbeck granule, and immunohistochemically by virtue of expression of a set of markers. The most widely used of these is the CD1a antigen, a 49kDa surface glycoprotein also expressed on cortical thymocytes. Whereas for a long time this antigen could be detected only on frozen tissue specimens, a monoclonal antibody now exists (010) that allows detection of CD1a⁺ LC in routinely-processed tissue specimens (Fig. 3) [17]. Another apparently very specific antibody for LC is Lag, reacting with Birbeck granules [18]. LC can also be detected on routinely-processed tissues by the expression of S100 protein (Fig. 4), belonging to a family of acidic calcium-binding proteins expressed also in several other cell types (melanocytes, chondrocytes, adipocytes, eccrine secretory cells, Schwann cells...) [19, 20]. Other antigens expressed by LC (but not very specific for these cells) include vimentin (Fig. 5), and the class II major histocompatibility complex antigens (recognised by antibody LN3).

Melanocytes (MC)

MC are cells with a dendritic morphology, of neural crest origin located within the basal cell layer. They label on routinely-processed tissue sections with antibodies to S100 protein (Fig. 4) [19, 20]. HMB-45 is a monoclonal antibody recognising a 100 kDa premelanosome-associated antigen expressed in foetal MC and is widely used for the diagnosis of melanoma [21, 22]. Whereas adult MC do not usually express HMB-45 immunoreactivity, MC of the normal hair bulb as well as epidermal MC overlying inflammatory dermatoses may label with HMB-45. MC also constitutively express bcl-2 oncoprotein, a 25 kDa cytoplasmic protein encoded by an oncogene preventing cells from undergoing apoptosis [23, 24], the c-kit protein [25], tyrosinase, the MART1/Melan-A antigen and the pigmentation-associated glycoprotein (75 kDa). On the other hand, MC (along with LC) are the only intraepidermal cells expressing vimentin (Fig. 5).

Merkel cells (MKC)

MKC are neuroendocrine cells located within the basal layer of the epidermis and the outer root sheath. Once thought to be derived from the neural crest, they could in fact be derived from epidermal stem cells. MKC express a set of epithelial and neural markers. The former include the low MW (simple epithelium type) keratins K8, 18, 19 and 20, the latter being by far the most specific/sensitive marker of these cells in the skin [26, 27] (Fig. 6). Fewer than 10% of MKC express the 200 kDa (but not the 68, 70 and 160 kDa MW) neurofilaments. Other MKC markers (of variable sensitivity) include: chromogranin A, a 68 kD proteic component of neurosecretory granules and neuron specific enolase (alpha/gamma or gamma/gamma dimer), a glycolytic enzyme expressed in neuronal and neuroendocrine cells; synaptophysin, a 38 kDa acidic synaptic vesicle-associated protein; various neuropeptides, such as protein gene product 9.5, met-enkephalin, calcitonin-gene related peptide and vasoactive intestinal polypeptide; neural cell adhesion molecule (N-CAM). According to some studies, (a subset of) MKC may also express epithelial membrane antigen, a glycoprotein usually used as a marker of glandular epithelia (see below).

Epidermal appendages

These are made of KC that express to various degrees keratins and the other antigens of epithelial differentiation (involucrin, filaggrin, desmosomal antigens, etc.). Adnexal KC however express particular keratin polypeptides as well as some additional specific markers that allow their distinction from epidermal KC [28].

The pilosebaceous unit

Apart from "soft" keratins expressed within epidermal keratinocytes, 10 other polypeptides of "hard" or pilar K have been described that are expressed by hair-follicle KC. These are classified as type I (acidic) K (polypeptides Ha1-4 and Hax) and type II (basic/neutral) (polypeptides Hb1-4 and Hbx) [29]. Recently, it was found that cells of the hair bulge express a cytoplasmic antigen recognised (most likely as a result of cross-reactivity) by the monoclonal antibody CD8/144B directed against the CD8 molecule of suppressor/cytotoxic T lymphocytes [30].

Within sebaceous glands, sebocytes express detectable amounts of K (namely K14). Mature sebocytes label for epithelial membrane antigen, a highly glycosylated 70 kDa protein complex constituent of human milk fat globules [31], and for biliary glycoprotein, a 160 kDa glycoprotein member of the carcinoembryonic antigen family [32]. Mature sebocytes also label strongly with antibodies to the Thomsen-Friedenreich antigen and, to a lesser degree, to its precursor, the Tn antigen (the latter but not the former being expressed by Demodex folliculorum mites) [33]. Other, less frequently used sebaceous markers include Leu-M1, OM-1 (an ovarian cystadenocarcinoma antigen) and lipase.

Sweat glands

The most useful marker of sweat gland differentiation is carcinoembryonic antigen (CEA), a heterogeneous high molecular weight (180-200 kDa) oncofetal glycoprotein comprising several members (NCA-1, NCA-2, NFA-2, biliary glycoprotein...). CEA can be readily detected on formalin-fixed specimens by polyclonal and monoclonal antibodies mainly on the luminal border of cells of the secretory and excretory part of eccrine sweat glands (ESG), including the acrosyringium, and, to a lesser extent, on apocrine sweat glands (ASG) [34, 35]. Epithelial membrane antigen is detected on the apical pole of secretory cells of ESG & ASG, showing well the intercellular canaliculi of ESG (Fig. 7); it is also less intensely expressed on the apical pole of cells of the excretory duct. Secretory cells of both ESG & ASG express cytoplasmic reactivity for simple-epithelia type keratins (n° 8, 18, 19). Excretory ESG cells show a pattern of K expression similar to that of epidermal keratinocytes. A subset of secretory cells of ESG (but not ASG) label for S100 protein [36] and bcl-2 oncoprotein [37]. The Leu-7 monoclonal antibody, raised against the CD57 surface antigen of natural-killer cells, crossreacts with a cytoplasmic antigen of a subset of secretory cells of ESG & ASG [36]. Gross cystic disease fluid protein-15 (GCDFP-15) is the best-known member of a family of proteins isolated from breast cyst fluid, originally considered as a specific marker of apocrine epithelia; however this antigen seems to be expressed by the secretory cells of both ESG and ASG [38, 39].

Myoepithelial cells lining the secretory acini of sweat glands express the "contractile" K17. They can be readily visualised on routinely-processed tissue specimens with antibodies to muscle-specific actin (Fig. 8).

The dermal-epidermal junction

The dermal-epidermal junction (DEJ) is a complex basement membrane mediating the cohesion of the epidermis to the underlying dermis. Ultrastructurally it divides into four levels, comprising (from the surface to the depth) the plasma membrane of keratinocytes and the associated hemidesmosomes, the lamina lucida (LL) (including anchoring filaments), the lamina densa (LD) and the sub-lamina densa fibrillar zone (anchoring fibres). To date, about twenty biochemical components have been well characterised, and many of them have been precisely localised within the morphological compartments of the DEJ [16, 40, 41]. The two bullous pemphigoid antigens (BPAG1 of 230 kD and BPAG2 or type XVII collagen of 180 kDa), the alpha6ß4 integrin and plectin are associated with hemidesmosomes. Laminins are a family of glycoproteins comprising 11 known isoforms; each laminin molecule is made up of three chains (alpha, ß and gamma). The DEJ contains laminin isoforms 1 (alpha1ß1gamma1), 5 (alpha3ß3gamma2) and 6 (alpha3ß1gamma1), associated with the LL and partly also with the LD. Laminins 5 (formerly known as BM-600/nicein, kalinin or epiligrin) and 6 as well as uncein (19-DEJ-1 antigen) seem to be associated with the anchoring filaments of the LL. Type IV collagen, the backbone of all basement membranes, is the major component of the LD whereas type VII-collagen (290 kDa) is the major constituent of anchoring fibrils. A number of other antigens or immunoreactivities have been identified but not fully characterised at the biochemical and molecular level. These include: the cicatricial pemphigoid antigen, probably related to anchoring filaments; the NU-T2 antigen, localised within the LL; the LD-associated antigens KF1, LDA1, nidogen, heparan-sulfate proteoglycan and chondroitin-sulfate proteoglycan; the anchoring fiber-associated antigens AF1 and AF2; thrombospondin and BM-40/osteonectin. A 97 kD antigen (tentatively termed "ladinin") recognised by sera from patients suffering from linear IgA bullous dermatosis was described some years ago, but this could be a degradation product of the 180 kDa BPAG2. The basement membrane surrounding epidermal appendages shows a similar antigenic composition with only minor differences. The DEJ antigens can be detected on frozen tissue specimens by specific antibodies. A limited number of them (e.g. type IV collagen and laminin) can also be detected on routinely-processed tissue specimens, namely after proteolytic pretreatment (Fig. 9).

The dermis

The dermis is an ordered connective tissue consisting of an extracellular matrix made up of fibrous and non-fibrous proteins, resident and trafficking cells, vessels, nerves and muscles.

Resident cells of the dermis

These include fibroblasts, a network of mesenchymal cells with a dendritic morphology and mast cells.

Fibroblasts. Fibroblasts (and fibrocytes) can be recognised with antibodies to the pan-mesenchymal markers vimentin (Fig. 5) and Te7 and to proline-4-hydroxylase, but these markers are not very specific. Recently, antibodies were produced (FibAS) and were claimed to be specific for human fibroblasts [42], but these are applicable only on frozen specimens. Myofibroblasts are cells probably derived from fibroblasts; they appear during the process of wound healing and express the pan-mesenchymal markers, but can be differentiated from ordinary fibroblasts by virtue of the expression of muscle-specific actin (and less frequently desmin) [43].

Mast cells. These are bone-marrow-derived resident cells of the dermis found in a perivascular location. They are usually recognised thanks to metachromatic histochemical (toluidine blue) or histoenzymatic stains. Immunohistochemically, mast cells can be identified on routinely-processed tissue specimens with monoclonal antibodies to tryptase (AA1) and to chymase [44]; they also express some cytoplasmic and membrane antigens shared with mononuclear phagocytes and basophils. Recently it was found that mast cells express the c-kit oncoprotein [45], also expressed by melanocytes [25].

Dermal dendrocytes (DD). These include a heterogeneous network of dendritic cells that became known thanks to immunohistochemistry [46]. In vivo, at least two immunohistochemically distinct types of DD seem to exist in human dermis. The first one, sometimes referred to as "DD of type I", specifically expresses the coagulation factor XIIIa [47], the intracellular form of a protransglutaminase stabilising fibrin. DD I are found mainly around capillary vessels of the papillary dermis (Fig. 10), and (in smaller numbers) around sweat glands and within fibrous septa of the hypodermis. They express panmesenchymal markers and some surface antigens shared with antigen-presenting cells (HLe1, HLA DR/DQ, CD14, CD36) but are CD1a and prot. S100-negative. The second type of DD cells, referred to as "DD type II", is characterised by the expression of CD34 (or "human progenitor cell antigen", HPCA-1), a transmembrane glycoprotein of 105-120 kDa. DD II also express the panmesenchymal antigens but are fact. XIIIa, CD1a and S100 prot.-negative [48]. They are found predominantly within the reticular and deep dermis, namely around the secretory portion of eccrine sweat glands (Fig. 11) and around the bulge of hair follicles. At least in vivo, DD I, DD II and (dermal) Langerhans cells seem to represent distinct cell types, recognised by the mutually exclusive expresssion of fact. XIIIa, CD34 and CD1a, respectively.

Lymphocytes. Normal human skin may occasionally harbour lymphocytes within the dermis and rarely the epidermis. These can be characterised thanks to the expression of the CD (cluster of differentiation) antigens, usually (but not invariably) cell-membrane molecules specifically expressed by leukocytes at various stages of their differentiation [49]. More than 200 CD antigens have now been well characterised; although most of them are recognisable only on frozen specimens, some can now be detected on routinely-processed histological sections with commercially available antibodies (e.g. CD3, CD8, CD15, CD20, CD30, CD45, CD68...) and are therefore used for the immunophenotyping of cutaneous lymphoproliferative disoders [50].

Cutaneous vessels

The most specific marker of (blood) endothelial cells is von Willebrand factor (vWf), also known as "fact. VIII-related antigen"; this is a 260 kDa glycoprotein (also present in megacaryocytes and platelets), readily detectable on formalin-fixed specimens by mono- or polyclonal antibodies (Fig. 12) [51]. CD31/platelet-endothelial cell adhesion molecule 1 (PECAM-1 or EndoCAM), a 130 kDa glycoprotein involved in platelet adhesion to endothelium is another specific and sensitive endothelial marker, also expressed by platelets, megacaryocytes and B lymphocytes. CD34 is expressed on the plasma membrane of vascular endothelial cells within interdigitations of intercellular junctions [52] (but is less specific for vessels, being also expressed by DD II). Thrombomodulin was recently reported to be a sensitive marker of blood and lymphatic endothelial cells [53]. The H antigen (comprising alpha-L-fucose residues), recognised by the lectin ulex europaeus agglutinin-I has been widely used in the past as an endothelial marker, but is not very specific, being expressed also on differentiated keratinocytes. The antibody PAL-E recognises endothelial cell vesicles. Finally, endothelial cells express cytoplasmic vimentin and some surface antigens (HLA-DR, CD36, M241...) shared with antigen-presenting cells. Endothelial lymphatic vessels express vimentin but lower amounts of vWf than blood endothelial cells; therefore, lymphatics usually do not stain with antibodies to vWf, namely when formalin-fixed specimens are examined. Similarly, they do not express the CD34 antigen.

The medium wall of larger vessels contains smooth muscle cells expressing muscular antigens (see below). Finally, blood vessels are surrounded by basement membrane macromolecules (laminin, type IV collagen) (Fig. 9) and by pericytes expressing vimentin and muscle-specific actin (Fig. 8).

Cutaneous nerves

Axons of dermal nerves label with antibodies to neurofilaments (68, 160, 200 kDa) and to peripherin, two neuron-specific types of intermediate filaments [54, 55]. They also express neuron-specific enolase (gamma/gamma), an enzyme that seems however to be not strictly neuron-specific. Recently, it was documented that dermal axons enter the epidermis and associate with LC; these axons label for calcitonin gene-related peptide [56]. Schwann cells express: glial fibrillary acidic protein (GFAP), the 55 kDa glial-specific intermediate filament [57]; S100 protein (Fig. 13); a 110 kDa myelin-associated glycoprotein recognised (as a result of cross-reactivity) by the anti-CD57/HNK-1 monoclonal antibody Leu-7 [36]; myelin basic protein; and the Schwann cell-associated antigen AHMY1. The specialised terminal nerve structures, such as the Wagner-Meissner and Pacini corpuscles, also label strongly for Schwann-cell antigens (such as S100 protein). Perineural fibroblasts express vimentin and epithelial membrane antigen (Fig. 14).

Cutaneous muscles

Smooth muscle cells are encountered within the arrector pili muscles, the muscular wall of vessels, in the tunica dartos of genitalia and in areola of the nipples. Striated muscle cells may occasionally be disclosed in deep skin biopsies. Both smooth and striated muscle cells express desmin, the 53 kDa intermediate filament protein specific of muscle tissue [57], and muscle-specific actin, a very sensitive cytoplasmic marker of muscle cells (also expressed in pericytes, myofibroblasts and myoepithelial cells) [58]. A smooth-muscle-specific isoform (alpha-actin) also exists. Myoglobin, a single-chain protein of 153 amino acids and titin [59] are cytoplasmic antigens expressed only by striated muscle cells.

Extracellular matrix

Several antibodies are available recognising the various macromolecules of the dermal connective tissue (various types of collagen, fibrillin, elastin, fibronectin, tenascin, epimorphin...); however these work better on frozen tissue sections and are not often used in diagnostic dermatopathology. An exception to this rule is the antibody to type IV collagen of basement membranes (see about the dermal-epidermal junction), used for the diagnosis of bullous dermatoses.

The hypodermis

Adipocytes, the characteristic cells of the hypodermis, loose their (lipidic) cytoplasmic content during processing for routine histology, and therefore appear optically empty; however, they express detectable amounts of vimentin and S100 protein, the latter expressed in a pericellular/membrane pattern (Fig. 15). The remaining components of the hypodermis (vessels, resident cells of the connective septa, etc.) do not substantially differ from an antigenic point of view from their dermal counterparts.

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