The Integument

Hair Follicles

The growth of hair occurs within hair follicles in a sequence of stages (Fig. 17-6). These stages include hair genesis, growth, maturation, and loss. In the anagen stage of the hair cycle, mitotic activity and growth occur. The catagen stage is a transitional phase during which cellular proliferation ceases. The follicle then enters a resting stage, telogen, after which mitotic activity and new hair production resumes. The exogen stage is the phase in which the old hair is shed. In many animals hair follicle growth occurs in cycles, resulting in periodic loss or shedding of the hair coat. The reason the hair growth occurs in cycles is not clear. Some hypotheses include that the cycle provides the ability to: (1) shed fur to cleanse the body surface, (2) adapt and change body cover in response to changing environment (winter to summer) or social conditions, or (3) protect against malignant transformation that might occur in a rapidly dividing tissue. The regulation of hair cycling is exceedingly complex and incompletely understood. Factors that play a role include genetics, photoperiod, temperature, nutrition, hormones, health status, and neural mechanisms. Genetic factors determine the hair shaft length (e.g., short-haired versus long-haired breeds of dogs). The exact signals that control the hair cycle have not been identified; however, growth factors, such as epidermal growth factor, fibroblast growth factor (FGF), hepatocyte growth factor, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and insulin-like growth factor (ILGF), have been localized to the skin and hair follicles. These growth factors probably play a crucial role in regulation of the hair cycle and follicle growth.

Photoperiod acts via the hypothalamus, pituitary, and pineal glands, which secrete tropic hormones, such as melatonin and the gonadal, thyroid, and adrenal hormones, that also influence hair growth. Some hormones, such as thyroid and growth hormone, stimulate hair growth, whereas excessive levels of estrogen or glucocorticoids suppress hair growth. The cells in the follicular papilla (sometimes called dermal papilla) regulate epithelial growth and are probably the target of the tropic hormones. Nutrition and health status have a significant influence on hair growth and quality. Hair is largely composed of protein. Thus diets low in protein or disease states associated with severe protein loss result in poor quality hair coat. Animals in poor health have larger numbers of hair follicles in telogen, and because telogen hairs are more easily shed than anagen hairs, animals in poor health tend to shed more heavily than healthy animals. Also, in disease states, cuticle formation can be defective, resulting in a dull or dry hair coat. It is also thought that the hair cycle is influenced by the nervous system. Evidence of such is implied by the abundant nerve supply of the hair follicle, the high density of Merkel cells in the hair follicle epithelium, and facts that keratinocytes express several neurotransmitter and neuropeptide receptors whose stimulation can alter keratinocyte proliferation and differentiation. Indirect influence by autonomic nerves could also be mediated by alteration of hair follicle blood flow and thus of oxygen and nutrient supply.

Forms of hair follicles vary in different animals (Fig. 17-7). Horses and cattle have evenly distributed simple follicles with one large (i.e., primary) follicle, usually with sebaceous and apocrine glands and arrector pili muscles. Pigs have simple follicles grouped in clusters. Goats, dogs, and cats have compound follicles that consist of primary follicles and smaller secondary follicles. Sheep have simple follicles in hair-growing areas and compound follicles in wool-growing areas. Primary follicles have the hair bulb rooted more deeply in the dermis than secondary follicles. The depth of the hair bulbs varies with species. In dogs and cats, the anagen hair bulbs of primary follicles are at the dermal-subcutaneous junction, whereas in horses and cattle the anagen hair bulbs are in the mid-dermis. In all species, the bases of telogen follicles are more superficially located than the bases of anagen follicles. Typically, primary and secondary hair shafts emerge through a common follicular opening. Tactile hairs include sinus and tylotrich hairs. Sinus hairs, also termed vibrissae, arise in simple follicles with a blood-filled sinus located between the inner and outer layers of the dermal sheath. Sinus hairs generally occur on the nose, above the eyes, on the lips and throat, and on the palmar aspect of the carpus of cats. Sinus hairs function as mechanoreceptors (i.e., touch receptors). Tylotrich hairs also function as mechanoreceptors and are scattered among the regular body hairs. The arrector pili muscles extend from the connective tissue sheath of the hair follicles at the junction of the middle and inferior portion of the follicle and attach to the superficial dermis. The arrector pili smooth muscles are oriented almost perpendicularly to the wall of the follicle and are well developed on the back of animals, especially dogs. Muscle contraction causes erection of hairs and expression of the contents of sebaceous glands.

Sweat Glands

There are two basic types of sweat glands: apocrine glands and eccrine glands. Apocrine glands are located throughout haired areas of skin in domestic animals and are tubular- or saccular-coiled glands (see Fig. 17-7). The ducts of the apocrine glands open in the superficial portion of the hair follicle; thus these glands are also called epitrichial glands. The glands are lined by secretory cuboidal to low columnar epithelium surrounded by contractile myoepithelial cells. Other apocrine glands include the interdigital glands of small ruminants, glands of the external ear canal and eyelids of domestic animals, anal sac glands of dogs and cats, and the mental organ of pigs. Eccrine glands are merocrine in secretion and in contrast to ducts of apocrine glands, the ducts open directly onto the surface of the epidermis. Thus eccrine glands are also called atrichial glands. They are tubular glands lined by cuboidal epithelium surrounded by myoepithelium and are confined mainly to pawpads of dogs and cats, frog region of ungulates, carpus of pigs, and nasolabial region of ruminants and pigs.

Sebaceous Glands

Sebaceous glands are simple, branched, or compound alveolar glands that undergo holocrine secretion, with ducts opening into hair follicles except at some mucocutaneous junctions where the glands open on the surface of the skin (e.g., meibomian gland/also known as tarsal gland). Well-developed sebaceous glands are found in the supracaudal gland of dogs and cats; infraorbital, inguinal, and interdigital regions of sheep; the base of the horn of goats; the anal sac glands of cats; the preputial glands of horses; and the submental organ of cats.

Specialized Structures

Anal sacs are specialized cutaneous structures that are especially prone to develop lesions. Anal sacs are bilateral diverticula located between internal and external anal sphincter muscles in dogs and cats and have ducts that open onto the anus at the level of the anocutaneous junction. Ducts and sacs are lined by stratified squamous epithelium. In cats, the sac wall has sebaceous and apocrine glands, but in dogs, the wall has only apocrine glands. The anal sacs can become distended with secretory products, rupture after trauma, and cause bacterial infection and chronic inflammation (foreign body reaction) in contiguous tissues. Carcinomas of the apocrine gland of the anal sac in dogs are often associated with tumor cell production of parathyroid hormone-related protein (PTHrP) and humoral hypercalcemia of malignancy.

Hepatoid (i.e., circumanal or perianal) glands occur most commonly in the skin around the anus and are also present in skin near the prepuce, tail, flank, and groin. These glands are modified sebaceous gland that have nonpatent ducts and are composed of peripheral reserve cells that surround lobules of differentiated cells resembling hepatocytes, resulting in the name “hepatoid” glands. Adenomas of the perianal glands in male dogs are often testosterone dependent.

The claws of dogs and cats shield the third phalanx and consist of a wall (i.e., dorsal and lateral sides) and sole (i.e., ventral side), both of which are stratified squamous keratinizing epithelium. The wall consists of hard keratin and the sole of softer keratin. The dermis of the claw consists of dense collagen, elastic tissue, and blood vessels that can bleed profusely if the claw is trimmed too short. The claw fold is a fold of skin that covers the wall laterally and dorsally for a short distance. Hooves consist of the wall, sole, and frog in solipeds; and a wall, sole, and prominent bulb in ruminants and pigs. The hoof wall comprises three structurally distinct layers (i.e., stratum externum, stratum medium, and stratum internum), which are formed by the proliferation and downward movement of epidermal cells arising from a specialized junction of the epidermis and dermis, a region known as the coronary band or coronet. The stratum internum of the inner hoof wall interdigitates with the dermal lamina of the corium, thereby anchoring the inner hoof wall to the dermis that covers the third phalanx. If the attachment of the stratum internum to the dermal lamina fails, the shearing forces of body weight and movement lead to vascular damage in this region and ultimate damage to the corium of the sole and coronet. The separation of the third phalanx from the inner hoof wall is the primary pathologic process leading to the painful condition of laminitis most often seen in horses and cattle.

The digital pads of dogs and cats have a thick epidermis composed of all layers, including the stratum lucidum. The surface is covered by compacted layers of stratum corneum and is smooth in the cat; however, in the dog, the surface is covered by conical papillae that conform to the outline of the epidermal surface (see Fig. 17-2). The epidermis and dermis interdigitate via rete ridges and dermal papillae, thus providing resistance to shear forces. Eccrine (atrichial) glands are present in the dermis and the adipose tissue. Lobules of adipose tissue that act as a cushion are subdivided by collagenous stroma and elastic tissue.

The chestnuts and ergots of the horse are considered to be vestiges of the first, second, and fourth digits. Chestnuts are located in the supracarpal and tarsal area on the medial surface of the limbs, and the ergot is located at the flexion of the fetlocks (metacarpophalangeal articulation). Chestnuts and ergots are histologically similar and consist of compact stratum corneum covering thick cellular layers of the epidermis. The rete ridges are long and interdigitate with long dermal papillae.

Barrier Functions

Barriers of the skin against physical injury are listed in Box 17-3. The hair coat, particularly the long dense hair coat of some dogs and cats, serves as a physical barrier to temperature extremes, UVR, and minor trauma. The hair coat also sheds water as a result of the lipids provided by sebaceous gland secretion. Vibrissae, or tactile hairs, and sensory neurons provide awareness of the physical environment, allowing the animal to make appropriate reactions for survival such as reflex responses to heat and other noxious stimuli. Claws, especially on cats, serve as a quite effective barrier against predators by providing traction for climbing and serve as weapons to be used against aggressors. Horns of cattle, sheep, and goats also provide some physical defense capabilities.

The stratum corneum is an exceedingly important component of the barrier, imparting protection from the exterior and preventing water loss from the interior. The stratum corneum is composed largely of keratins, a family of proteins called intermediate filaments. Keratin proteins are the major structural proteins of the skin, hair, and claws. The stratum corneum is considered to be the “bricks and mortar” of the barrier. The bricks are the flattened cornified cells (corneocytes) with their resistant cell envelopes and keratin microfibrils, and the mortar consists of intercorneocyte lipids.

The bricks are formed at the level of the stratum granulosum when the keratinocytes are transformed into the flattened corneocytes. The transformation occurs when (1) the nucleus is digested, (2) the keratin intermediate filaments aggregate into microfibrils oriented parallel to the skin surface, (3) the lipids are released into the intercellular space, and (4) the cell membrane is replaced by a resilient cell envelope consisting of cross-linked protein with lipids covalently bound to its surface. Filaggrin (which is an acronym for filament-aggregating protein) from the keratohyalin granules in the stratum granulosum plays a significant role in formation of the bricks by participating in the aggregation of keratin filaments into tight bundles. The keratin intermediate filaments and filaggrin compose 80% to 90% of the protein mass of the epidermis. Later, filaggrin is digested by proteolytic enzymes to produce components of amino acids that form the “natural moisturizing factor” of the stratum corneum, which serves to help maintain hydration, flexibility, and proper desquamation. Concurrent with the aggregation of the keratin filaments, the resistant cornified envelope is transformed from the water-permeable phospholipid cell membrane of the keratinocyte when the membrane-bound enzymes (e.g., transglutaminases), cross-link proteins from the keratohyalin granules (e.g., loricrin) and the cytoplasm (e.g., involucrin) in isopeptide bonds. Other proteins (including trichohyalin and small proline-rich proteins) are similarly cross-linked and eventually the entire cell membrane consists of cross-linked proteins. The proteins of the cornified envelope compose 7% to 10% of the protein mass of the epidermis. The corneocytes are joined together by desmosomes that are modified from those joining keratinocytes in lower layers of the epidermis by the addition of a protein called corneodesmosine. These stratum corneum desmosomes are referred to as corneodesmosomes.

The mortar is formed when lipids, from the lamellar bodies in the stratum granulosum, are released into the intercellular space. These intercorneocyte lipids (glycosyl ceramides, cholesterol, cholesterol esters, and long-chain fatty acids) are hydrophobic and prevent transepidermal water loss. Lamellar bodies have other important functions: (1) they provide enzymes that generate ceramides and free fatty acids that are incorporated into the lipid membranes; (2) they provide proteases and antiproteases that regulate digestion of corneodesmosomes and shedding of cornified cells to the exterior; and (3) they secrete antimicrobial peptides including defensins into the intercellular compartment of the stratum corneum. The lipid component of the stratum corneum surrounds the protein component to which it is covalently bound and provides adhesion of the cornified cells (i.e., bricks) to the intercellular lipids (i.e., mortar). Layers of the corneocytes and their corneocyte envelope (i.e., bricks), and intercellular lipids (i.e., mortar) form a tough and resilient protective barrier. The waterproofing and repellency of the stratum corneum is in part provided by the keratinocyte and sebum-derived lipids.

Other barrier functions of the skin include defense against antioxidant injury provided by vitamin E in sebaceous gland secretion and defense against UV light provided by the hair coat and also by melanin pigment in keratinocytes. The cap of melanin pigment over the nucleus helps protect the nucleus (and its nucleic acid) against UV light–induced injury by scattering and absorbing UV light rays. The basement membrane zone serves as an initial barrier to invasion of the dermis by neoplastic epidermal cells. The panniculus through its insulating properties serves as a barrier against cold temperatures. Secretion from apocrine glands in cattle and horses provides defense against excessive heat.

Resistance to Mechanical Forces

Anatomic features of the skin that provide resistance to physical injury are listed in Box 17-3. Hair follicles help anchor the epidermis to the dermis, as do epidermal-dermal interdigitations, thus these interdigitations are most numerous in nasal planum and pawpad where hair follicles are absent and resistance to shearing force is necessary. Host defense against mechanical injury is also provided by the tightly bundled keratin filaments of the corneocyte, the resilience of the cornified envelope, the adhesion of the cornified envelope and intercellular lipids, and the corneodesmosomes. In addition, the keratinocytes contain keratin filaments and form desmosomal junctions with adjacent cells (see Fig. 17-4). The keratin filaments perform a structural role (i.e., cytoskeletal) in the cells, and the desmosomes promote adhesion of epidermal cells and resistance to mechanical stresses. The basement membrane anchors the epidermis to the dermis via hemidesmosomes providing structural integrity against trauma. Dermal collagen and elastic tissue provide resilience and strength to the skin and support for the vessels, nerves, and adnexa. The panniculus protects against surface trauma by providing some shock absorption (e.g., pawpads), by facilitating movement, and by anchoring the dermis to fascia. Thus the various components of the epidermis, dermis, adnexa, and panniculus provide a flexible interconnecting framework to protect the host against mechanical injury.

Innate Immunity

Innate immunity is a primitive, highly conserved response that quickly detects and impairs pathogens and harmful environmental stimuli encountered daily in life and does not require antigen-specific receptors. Innate immunity protects the host during the first 7 days of exposure to a pathogen before development of an adaptive immune response, and also initiates and assists the adaptive immune response (see Web Table 17-1). The diversity of microorganisms requires equal diversity in host defense responses. The first phase of innate host defense consists of the barrier of the stratum corneum, which prevents pathogen adherence and provides an antimicrobial surface consisting of antimicrobial peptides and fatty acids. The antimicrobial peptides (e.g., β-defensins and cathelicidins) are effective against many organisms, including viruses, bacteria, protozoa, and insects, and probably kill some of these pathogens by damaging their lipid membranes. The surface barrier also includes normal flora of nonpathogenic bacteria that competes with pathogenic microorganisms for nutrients and for attachment sites on cells. The normal flora also produces antimicrobial substances that prevent pathogen colonization. Because the dry cornified surface is such an effective barrier to pathogens, a wound or abrasion is usually necessary for a pathogen to gain entrance. Web Fig. 17-1 provides an illustration of how the innate and adaptive (acquired) immune systems participate in host defense against bacterial pathogens that have gained entrance into the skin through a wound in the epidermis. When injured, keratinocytes release a variety of antimicrobial peptides, chemokines, and cytokines, which activate endothelial cells and attract macrophages, neutrophils, and lymphocytes to the site of injury. Early key cells that play a role in innate immunity are the tissue macrophages that contact, bind, phagocytose, and thereby eliminate many types of pathogens. Pathogen recognition is mediated by pattern-recognition receptors (PRRs), including Toll-like receptors and others, that recognize repeating patterns of molecular structures common to broad classes of pathogens and efficiently differentiate pathogen antigens from self-antigens. The repeating patterns of molecular structures on pathogens are called pathogen-associated molecular patterns (PAMPs).

A few examples of PAMPs include lipopolysaccharide (most Gram-negative bacteria), peptidoglycan (Gram-positive bacteria), CpG motifs (mostly bacterial pathogens), lipoarabinomannan (mycobacteria), mannans and zymosan (yeast), double and single stranded RNA (viruses), and heat shock proteins (bacteria, fungi, algae, protozoa). These PAMPs have been conserved during evolution and allow the innate immune system to broadly distinguish between self-antigens and pathogen antigens. The important outcomes of pathogen-receptor binding include the activation of phagocytic and other immune effector cells and release of cytokines, chemokines, adhesion molecules, and other inflammatory mediators initiating an acute-phase response. The acute-phase response proteins can opsonize a broad range of pathogens and can also activate the complement cascade, making pathogens more susceptible to phagocytosis and killing by macrophages and neutrophils. Once initiated, the innate immune response helps to start the antigen-specific immune response (i.e., adaptive immunity). Innate immunity is crucial to protecting the host in the early days of infection; however, pathogens can evade innate immunity and innate immunity does not lead to immunologic memory characteristic of adaptive immunity.

Acquired (Adaptive) Immunity

Whereas innate immunity works immediately to detect and destroy microorganisms, acquired or adaptive immunity develops later because the lymphocytes that contribute to adaptive immunity specific for the invading pathogen must increase in number by clonal expansion (see Web Table 17-2). The major components of the cutaneous adaptive immune system include keratinocytes, dendritic antigen-presenting cells (Langerhans’ cells and dermal dendritic cells), lymphocytes, and endothelial cells (see Web Fig. 17-1).

The adaptive-immune response is initiated by a stimulus (in this example, the epidermal injury, microbial invasion, and signals provided from the innate immune system), at which time the bone marrow–derived antigen processing and antigen-presenting cells (Langerhans’ cells in the epidermis and dendritic cells in the perivascular dermis) ingest and process the antigen. Pathogen recognition is mediated through PAMPs as described previously. The major function of Langerhans’ cells and dermal dendritic cells is antigen processing and presentation; thus these cells are referred to as “professional antigen processing and presenting cells.” Langerhans’ and dermal dendritic cells reexpress the ingested and processed antigen on their cell surfaces and migrate via afferent lymphatic vessels to the paracortical areas of skin-associated lymph nodes, where they arrive as mature and powerful antigen-presenting cells. These skin-derived dendritic cells then initiate a pathogen-specific protective immune response by presenting antigen to the naïve T lymphocytes. Langerhans’ cells also produce cytokines (e.g., IL-1 and TNF-α), thus participating in upregulation of inflammatory and immune responses in the skin.

The T lymphocytes activated by antigen presentation in the skin-associated lymph nodes are also known as sensitized or memory T lymphocytes. These memory T lymphocytes are subdivided into two types, central memory and effector memory T lymphocytes. The central memory cells generally circulate between the blood and lymph nodes, serve mostly as long-term reservoirs of immunologic memory, and when stimulated by antigen give rise to both central memory and effector memory T lymphocytes. The effector memory T lymphocytes express skin-associated homing receptors (e.g., cutaneous lymphocyte antigen [CLA]) that interact with adhesion molecules (E-selectin, P-selectin, vascular cell adhesion molecule 1 [VCAM-1], and intercellular adhesion molecule 1 [ICAM-1]) on cytokine-activated endothelial cells in the dermal vessels at the site of initial injury, thus providing a way for the effector memory T lymphocytes to find their way back to the site of the injury and pathogen entrance. Once in the skin and after receipt of a renewed antigenic stimulus by the professional antigen-presenting cells, the effector memory T lymphocytes undergo clonal expansion, resulting in the generation of protective effector mechanisms. Most of the lymphocytes in the skin are T-helper lymphocytes, but various types of T and B lymphocytes contribute to adaptive immunity.

Lymphocytes recognize pathogens (i.e., antigens) via cell surface receptors. The B lymphocytes have immunoglobulin molecules as the receptors for antigen, and on activation, B lymphocytes secrete immunoglobulin, which provides defense against pathogens (often bacteria) in the extracellular spaces. Antibody facilitates pathogen neutralization, complement activation, and enhanced endocytosis by phagocytes. In contrast, T lymphocytes have receptors that recognize foreign antigens expressed as peptide fragments bound to major histocompatibility complex (MHC) proteins (see Chapter 5 for a review). One class of T lymphocyte expresses the CD8 molecule on the surface (i.e., CD8⁺ T lymphocytes). These CD8⁺ T lymphocytes recognize peptide fragments bound to MHC I, then kill the cell, and thus are also called cytotoxic T lymphocytes.

Another class of T lymphocytes expresses the CD4 molecule on their surface. This class of T lymphocyte is divided into subclasses. One subclass, the CD4⁺ T lymphocyte subset (T_H1 [helper]), recognizes peptide fragments (e.g., microbial antigen) bound to MHC II and releases cytokines, including interferon-γ (IFN-γ), resulting in an inflammatory response via macrophage activation. A second subclass, CD4⁺ T lymphocyte subset (T_H2 [helper]), recognizes peptides (including allergens) bound to MHC II and releases cytokines, including IL-4, IL-5, and IL-13, resulting in inflammatory responses in which eosinophils predominate and stimulates B lymphocytes to secrete immunoglobulin. Another subclass, the T regulatory lymphocyte (Treg), acts to suppress responses of other T lymphocytes. Most antigens require an accompanying signal from helper T lymphocytes before they can stimulate B lymphocytes to proliferate and differentiate into antibody-secreting plasma cells. Thus T lymphocytes are crucial to adaptive immunity by destroying pathogen-infected cells, by activating macrophages, and by activating B lymphocytes.

Thus complex interactions between host cells, pathogens or other antigens, and inflammatory mediators of the innate and adaptive immune system typically result in appropriate host defenses, the removal of the inciting pathogen, and the generation of differentiated memory lymphocytes through clonal expansion, allowing faster specific immune responses in future encounters with the offending antigen. Impaired host defense mechanisms can lead to increased susceptibility to infection, to development of neoplasia, or to chronic inflammatory or autoreactive disorders such as atopic dermatitis, contact hypersensitivity, or lupus erythematosus.

Disease Example of Barrier Dysfunction

Atopic dermatitis (atopy) serves as an example of a common disease associated with impaired function of the epidermal barrier and immunity. It is a multifactorial, chronic and relapsing, often severely pruritic skin disease that affects humans, horses, dogs, and cats. It can cause severe discomfort including sleeplessness from pruritus, and is associated with secondary skin infections. Although the etiopathogenesis of atopic dermatitis has been studied most extensively in humans, many similarities with the human disease have been identified in atopic dogs. Advances in the knowledge of canine atopic dermatitis have occurred through the recent development and validation of animal models of the disease; these models have allowed more in-depth investigative studies in dogs and more precise comparisons of the disease between humans and dogs. Atopic dermatitis is a common problem in dogs; it is estimated to affect 10% to 15% of the population (Web Fig. 17-2). It is also a common human disease estimated to affect 5% to 20% of children. Similarities in the disease in humans and dogs include young age of onset; genetic inheritance; similar clinical lesion distribution; similar histopathologic lesions, including infiltration of immunoglobulin E (IgE⁺) CD1c⁺ dendritic cells; dry skin with increased transepidermal water loss; decreased stratum corneum ceramides (lipids); decreased epidermal filaggrin; increased colonization of surface staphylococci; positive atopy patch test; increased IgE-specific responses; and T_H2-dominated immune responses. A major difference in the disease is that children with atopic dermatitis often develop asthma and allergic rhinitis, whereas dogs do not; the reason for this difference is currently unknown.

Atopic dermatitis in humans is a multifactorial, heterogeneous genetic disease that arises in association with environmental factors. Defects in three groups of genes important in epidermal barrier function in atopic humans have suggested that in most cases alterations in the epidermal barrier contribute to the development of atopic dermatitis and may represent the primary event. The type and degree of genetic defect plus interaction with environmental factors appear to influence the severity or probability of developing the disease. A defective epidermal barrier facilitates penetration of allergens though the skin, as well as the interaction of the allergens with the local antigen-presenting and immune effector cells (Web Fig. 17-3). Changes in genes encoding for structural proteins (especially filaggrin), epidermal proteases, and protease inhibitors have been identified in humans with atopic dermatitis. These defects serve to reduce stratum corneum hydration and increase transepidermal water loss, increase cleavage of corneodesmosomes junctions, reduce lamellar body secretion and thus stratum corneum lipids, increase pH, and reduce antimicrobial properties of the stratum corneum. These alterations to the barrier favor population by pathogenic versus nonpathogenic bacteria and sustain allergen entrance through the barrier. The allergens are phagocytized by dendritic cells, which present the allergen to T_H lymphocytes and recruit other CD4⁺ T lymphocytes to the site. The activated dendritic cells and the cytokines produced by the CD4⁺ T lymphocyte (particularly IL-4) lead to switching from a T_H1 to T_H2 response (in early lesions) and release of proinflammatory cytokines and production of IgE, which in the presence of allergen, activates mast cells. There is evidence suggesting that at least one of these proinflammatory cytokines, IL-31 produced by T lymphocytes that express CLA and localize in the skin, significantly contributes to pruritus, a hallmark of atopic dermatitis. Langerhans’ cells also contribute in early lesions as they prime naïve T lymphocytes into the T_H2 type (with high IL-4 production). IL-4 may also be produced by mast cells, basophils, or eosinophils. Exogenous factors also contribute. For example, proteases from house dust mites can facilitate cleavage of corneodesmosomes and additionally contribute to epidermal barrier damage. The resulting inflammation causes pruritus, which stimulates scratching, further damaging the epidermal barrier (and keratinocytes resulting in release of additional proinflammatory cytokines, including IL-1), and creating a vicious cycle that perpetuates the disease and that is difficult to control. About 25% of humans with severe atopic dermatitis have IgE antibody directed toward self-proteins. These antibodies may develop after intracellular proteins from keratinocytes are released when the keratinocytes are damaged by scratching. These keratinocyte proteins may mimic microbial structure and induce IgE autoantibodies that further perpetuate the disease. In summary, atopic dermatitis is a complex, multifactorial, heterogeneous disease in which altered epidermal barrier function appears to contribute to the pathogenesis by facilitating penetration of allergens though the skin, as well as the interaction of the allergens with the local antigen-presenting and immune effector cells. It serves as an example of the importance of the epidermal barrier in protecting the host against injury from allergens, microbial infections, and autoreactive disorders.

Healing of Wounds with Opposed Edges

The simplest healing involves the clean, uninfected surgical incision in which the edges of the wound are closely opposed by sutures so that the wound space is narrow (see Web Fig. 17-4). Healing of these wounds is called primary union or healing by first intention. These wounds cause minimal necrosis of cells of the epidermis, dermis, and adnexa and minimal disruption of the basement membrane. Thus they heal quickly without significant architectural change, although a thin scar remains and the adnexa destroyed by the incision are permanently lost.

The first stage of wound healing is blood clotting and inflammation and begins within the first 12 to 24 hours of injury. The process begins with blood vessel disruption, platelet aggregation, blood coagulation, and clot formation in the vessel and wound space. Dehydration of the surface of the clot forms the scab that covers the wound. The clot in the wound space provides the matrix for migration of inflammatory cells, endothelial cells, and fibroblasts. Platelet-derived factors and factors associated with the coagulation and complement cascades facilitate recruitment of inflammatory cells, such as neutrophils and macrophages, to phagocytose pathogens, foreign particles, and debris. Many of the cytokines and chemokines that govern inflammation in regeneration and repair are the same as those participating in inflammatory processes of other causes. Neutrophils, the first cells to arrive at the margins of the incision, phagocytose pathogens and debris, then either slough with desiccated tissue, are phagocytosed by macrophages, or undergo apoptosis. Macrophages replace neutrophils, secrete collagenase (facilitates tissue debridement), and release growth factors initiating the formation of granulation tissue.

The second stage of wound healing consists of reepithelialization, fibroplasia, and angiogenesis and is maximal between 3 and 7 days of injury. The process of reepithelialization begins within hours of injury from basal cell keratinocytes adjacent to the incision. For the basal cells to become mobile, they retract intercellular tonofilaments, dissolve desmosomes and hemidesmosomes, and form cytoplasmic actin filaments. In addition to mobility, within 24 to 48 hours from injury, there is mitosis of basal cells at the edges of the wound. The mitosis is induced by growth factors from epidermal cells, macrophages, and dermal parenchymal cells. Basal cells from both sides of the wound migrate along the cut edges of the dermis separating nonviable tissue from viable tissue by using the expression of various surface integrin receptors on viable cells and through the production of collagenase, which debrides dead tissue. The basal cells join at the midline of the wound, and as reepithelialization develops, the nonviable tissue above this newly united epidermis, is sloughed. Basal cells also produce extracellular matrix, such as fibronectin, to facilitate reestablishment of the basement membrane. The basal cells revert to a nonmigratory normal phenotype, form hemidesmosomes, and firmly reattach to the newly formed basement membrane, thus beginning the reestablishment of the epidermis over the wounded skin.

Fibroplasia and angiogenesis begin with the formation of granulation tissue, which is the term applied to a specialized type of tissue composed of proliferative fibroblasts and vascular endothelial cells produced in healing of soft tissue injury. Granulation tissue is the hallmark of healing and is named for its clinical appearance of soft pink to tan tissue with minute red foci consisting of capillaries (granules). However, it is the histologic appearance that is diagnostic, a latticework array of proliferative capillaries (angiogenesis) oriented perpendicular to proliferative fibroblasts (fibroplasia). The fibroblasts produce extracellular matrix, which is remodeled, ultimately resulting in scar formation (i.e., cicatrix formation).

The process of forming granulation tissue begins about 3 days after injury. Fibroplasia (fibrosis) is induced when cytokines (IL-1, TNF-α) and growth factors (TGF-β, PDGF, endothelial growth factor [EGF], FGF) from inflammatory cells (especially macrophages), platelets, and endothelial cells stimulate fibroblasts to proliferate, migrate, and ultimately produce extracellular matrix. As in reepithelialization, the ability of fibroblasts to migrate into the clot or provisional matrix requires alteration in expression of surface receptors and production of proteolytic enzymes to provide a path for migration. When fibroblasts have migrated into and filled the wound space, growth factors and cytokines (TGF-β, PDGF, FGF, IL-1, IL-13) direct fibroblasts to switch from migration to protein (collagen) production, providing the structural protein that eventually contributes to wound strength. Collagen production begins by days 3 to 5 and persists for several weeks, depending on the size of the wound.

Concurrent with fibroblast migration into the wound space, angiogenesis, which is the formation of new blood vessels in an area of tissue injury, is also occurring (see Chapters 2 and 3). Angiogenesis develops from preexisting vessels and also from endothelial precursor cells (angioblast-like cells) from the bone marrow. Many growth factors play a role in angiogenesis, but vascular endothelial growth factor (VEGF) is considered to be the most important. In angiogenesis from preexisting vessels, macrophages and injured cells in the wound site release cytokines (such as FGF and VEGF), causing endothelial cells to release proteinases (e.g., procollagenase). The proteinases degrade components of the endothelial cell basement membrane. The disruption of the endothelial cell basement membrane removes the barrier otherwise confining endothelial cells, thereby permitting endothelial cells to migrate into the injured site in response to cytokines and growth factors released from injured or stimulated cells (e.g., FGF from macrophages, VEGF from keratinocytes, and heparin from mast cells). The migratory endothelial cells form tubes that express α_vβ₃ integrin, facilitating endothelial cell adhesion and migration. The endothelial cells deposit provisional matrix of fibronectin and proteoglycans and eventually form new basement membrane.

The growth factors continue to stimulate endothelial cell proliferation, ensuring a supply of endothelial cells for the extension of capillary tubes so that blood flow to the area of soft tissue injury can be reestablished. In angiogenesis from endothelial precursor cells, VEGF stimulates the mobilization of endothelial cell precursors from the bone marrow and proliferation and differentiation of these cells at the site of injury; however, the mechanism whereby these cells are directed to the site of injury is unclear. Once at the site of injury, these endothelial cell precursors form a delicate capillary plexus that eventually links to existing capillaries, facilitating formation of a capillary network. The newly formed vessels, whether produced by sprouting from preexisting capillaries or from endothelial precursor cells, are provided structural support by pericytes (recruited by angiopoietin 1 interacting with Tie 2, an endothelial cell receptor) and smooth muscle cells (recruited by PDGF), and by production of extracellular matrix proteins (stimulated by TGF-β).

Angiogenesis is a well-regulated process. More than 20 molecules present in tissue act as angiogenesis inhibitors and modulate the reparative process. By the end of the second stage of wound healing, the incision is filled with granulation tissue, neovascularization is maximal, collagen fibrils bridge the incision, and reepithelialization has been completed. By day 7, the usual time to remove sutures, the tensile strength of the incision site is about 10% that of unwounded skin.

The third and final phase of wound repair involves collagen production and wound contraction. However, wound contraction principally affects large wounds that heal by second intention and is described later. Production of collagen and proliferation of fibroblasts (directed by cytokines, such as TGF-β) progress fairly rapidly in this phase of wound repair. In contrast, inflammation, edema, and increased vascularity disappear. The proliferation and maturation of endothelial cells, fibroblasts, and inflammatory cells that contribute to wound repair depend on complex feedback control mechanisms between cells, cytokines, enzymes, and the extracellular matrix microenvironment. This complex interaction has been called dynamic reciprocity. As the wound space is bridged by granulation tissue and fibroblasts produce collagen, the endothelial cells undergo apoptosis, thus reducing capillary numbers. Similarly, fibroblasts and macrophages also undergo apoptosis. The reduction of endothelial cells, fibroblasts, and inflammatory cells leads to formation of an acellular scar. By the third week of wound repair, the wound has about 20% the tensile strength of normal skin. Over the ensuing months, collagen production is reduced, but there is continued slow remodeling of the extracellular matrix leading to a healed wound that at maximum strength is only 70% to 80% that of unwounded skin.

Healing of Wounds with Separated Edges

Healing of wounds with separated edges (healing by second intention) occurs when there is more extensive loss of skin tissue. Healing by this process is more difficult and time consuming, and larger scars result and replace the cutaneous architecture. The principal differences between healing by primary intention and healing by second intention include the following: (1) in healing by second intention, inflammation is usually more extensive because there is more tissue damage that must be removed and secondary infection is more likely; (2) granulation tissue is more extensive because the wound has wider edges so there is a larger gap to fill; and (3) wound contraction occurs, reducing the size of the wound to a fraction of the original size (experimentally, it has been shown that large wounds in rabbits are reduced to 5% to 10% of their original size in about 6 weeks).

Wound contraction begins during the second week of healing when fibroblasts develop phenotypic characteristics of smooth muscle cells. These consist of cytoplasmic bundles of actin-containing microfilaments, and formation of cell-cell and cell-matrix linkages. Fibroblasts link to the extracellular fibronectin matrix and collagen matrix by fibronectin (e.g., α₅β₁) and collagen (e.g., α₁β₁, α₂β₁) receptors and to each other through direct adherens junctions. Also, newly synthesized collagen bundles form covalent cross-links with themselves and with collagen bundles at the wound edge. These linkages thus provide a conduit across the wound space so that the traction of fibroblasts on the matrix can contract the wound, substantially reducing its size and facilitating healing by second intention.

Responses of the Epidermis to Injury

Responses of the Dermis to Injury

Responses of the Adnexa to Injury

The term adnexa refers to appendages or adjunct parts, which in the skin includes hair follicles and glands. The major clinical and histologic changes involve the hair follicles, and thus follicular lesions are emphasized in the next discussion.

Responses of the Vessels to Injury

Vasculitis, inflammation of vessels (see Fig. 17-22, B) in which the vessels are the primary target of injury, can be the result of infection by microbes, immunologic injury, toxins, photodynamic chemicals, or disseminated intravascular coagulation (DIC) or can be idiopathic. The species most commonly presenting with vasculitis are the horse and the dog, and most cases are idiopathic in that a specific cause cannot be determined. Histologic diagnosis of vasculitis is often challenging because it is difficult to differentiate between vessels taking part in inflammation simply by providing a conduit for inflammatory cells to reach a site of injury in the epidermis or dermis from those vessels that are actually the target of injury. Also, the type of inflammatory cell that participates in the inflammatory process can vary more with the age of the vascular lesion than with the type of disease process. Histologic lesions in vasculitis include damage to the vessel wall such as the presence of few necrotic cells or foci of fibrinoid necrosis, mural infiltrates of leukocytes, and intramural or perivascular edema, hemorrhage, or fibrin exudation. Vascular injury leads to the clinical lesions of edema and hemorrhage and if severe, can include ischemic necrosis and infarction. Ulceration with or without sloughing of the skin can occur. Partial ischemia can lead to atrophic changes in the components of the skin. Classic examples include immune-complex deposition in vessel walls (systemic lupus erythematosus and equine purpura hemorrhagica), rabies vaccination–associated vasculitis and ischemic dermatopathy, infection with an endotheliotropic organism (Rickettsia rickettsii), and septicemia with bacterial embolism and infarction in pigs (Erysipelothrix rhusiopathiae).

Responses of the Panniculus to Injury

Pathologic Reactions of the Entire Cutaneous Unit

Disease processes involving the skin uncommonly affect just one component of the skin (i.e., only the epidermis or only the lumens of hair follicles). More often, multiple components of the skin are involved in the disease process. In addition, lesions evolve through different stages; some lesions can resolve, and there can be secondary lesions, such as self-induced trauma, complicating the initial lesions. Therefore multiple biopsy samples collected from different areas of the skin are often necessary to help illustrate the range of lesions necessary to reach a diagnosis. Multiple biopsy samples provide a more representative picture of the disease process than could a single biopsy sample. Even so, evaluation of multiple biopsy samples does not always lead to a specific diagnosis, but the pattern of lesions identified often suggests categories of disease and rules out other differential diagnoses.

The involvement of the multiple components of the skin in a disease process can be illustrated by a poxvirus infection (Fig. 17-31). When a poxvirus invades the epidermis, the virus replicates in the cells of the stratum spinosum and causes cytoplasmic swelling (ballooning degeneration) and rupture (reticular degeneration) of some of the epidermal cells. Cytoplasmic viral inclusions form in some cells. Cellular constituents released from damaged epidermal cells act as chemical mediators of the acute inflammatory response and are chemotactic for leukocytes. These chemical mediators and chemotactic factors (1) increase blood flow to the site of viral invasion by dilation of arterioles, (2) cause margination of leukocytes in capillaries and postcapillary venules in the dermis, (3) increase vascular permeability (dermal edema), and (4) cause migration of leukocytes out of vessels into the tissue, creating the formation of macular lesions. The epidermal degeneration, dermal edema, and perivascular inflammation can progress to exudative lesions. Ballooning and reticular degeneration of keratinocytes result in the formation of intraepidermal vesicles. Leukocytes in perivascular sites under the influence of inflammatory mediators from the epidermis migrate to the epidermis and enter the vesicle to form pustules. Some poxviruses also cause epidermal hyperplasia by stimulating host cell DNA synthesis, presumably by a viral gene product similar to epidermal growth factor, resulting in pseudocarcinomatous hyperplasia. The pustule enlarges and eventually ruptures, releasing the exudates onto the skin surface. The exudates dry and form a crust (scab). The primary lesions of vesicles and pustules are fragile and often transient, lasting only hours, and so are difficult to identify and collect in biopsy samples. The secondary lesions of crusting and scarring are more long-standing. In this way, multiple components of the skin participate in the development of the lesions and are responsible for the clinical stages of macule, papule, vesicle, pustule, crust, and scar.

When to Collect Biopsy Samples

Knowing when to collect biopsy samples helps obtain the most diagnostic samples; facilitates obtaining samples early so acute, serious, or neoplastic disorders are diagnosed quickly; and prevents the frustration and economic loss when samples are inappropriately collected, such as when concurrent therapy might alter diagnostic lesions, when lesions are in a quiescent stage and might not be diagnostic, and when clinical dermatologic evaluation would have been a better method of achieving a diagnosis. Biopsy sampling is recommended when the following are present:

Site Selection

Multiple cutaneous sites representative of the range of lesions should be selected for biopsy. Fully developed nontreated primary lesions, such as macules, papules, pustules, nodules, neoplasms, vesicles, and wheals, are often the most useful for diagnosis (see Table 17-3). However, primary lesions may not be present at the time the animal is examined, so secondary lesions, such as scales, crusts, ulcers, comedones, or scars then need to be sampled and evaluated (see Table 17-3). These secondary lesions can be diagnostic or contribute substantially to the diagnosis when multiple cutaneous sites are selected for biopsy. One of the most useful secondary lesions is the crust because acantholytic cells from drying pustules in pemphigus foliaceous and organisms, such as Dermatophilus congolensis or dermatophytes, may be identified in crusts, providing the information necessary for diagnosis. Also, the margin of a chronic ulcer may represent a squamous cell carcinoma, or the scale at the edge of an epidermal collarette (i.e., peripherally expanding ring of epidermal scales) may represent superficial spreading pyoderma, thus providing the key to diagnosis.

Methods

Excisional biopsy samples (entire lesions) are recommended for large pustules or vesicles that can be damaged by use of a smaller punch biopsy instrument. Deeper excisional biopsy samples are generally necessary for diagnosis of lesions, such as panniculitis, that are deep to the epidermis and dermis. Digital amputation can be required, particularly in dogs, for the diagnosis of claw bed lesions. Electrocautery or laser should not be used for small biopsy samples because the samples can be damaged and rendered nondiagnostic. Tissue forceps (small toothed) should grasp, if at all, only one nonaffected margin, preferably in the subcutis.

Site Preparation

Generally, the skin at a punch biopsy site should not be surgically prepared because the procedure may remove a diagnostic portion of the sample. Gentle clipping of hair is acceptable, and surgical preparation of the skin is acceptable for an excisional biopsy of lesions deep to the epidermis. For collection of biopsy samples in areas of alopecia, drawing a line with a fine-tipped permanent marking pen in the direction of the hair coat helps laboratory personnel orient the sample (Fig. 17-32, A). In the laboratory, the sample is cut along the line so that the hair follicles are oriented longitudinally (Fig. 17-32, B). If the line is not drawn on the sample, the sample might be cut so that the follicles are in cross or tangential section rather than longitudinal section (Fig. 17-32, C), which reduces histologic value of the sample when evaluating for follicular disease. For collection of ulcers, depigmented lesions, or other lesions where the interface between normal and affected skin is critical to diagnosis, use of incisional or excisional samples collected from affected skin and contiguous normal skin is often preferable. However, a large (8 mm) biopsy punch instrument can be used to collect the junction between normal and affected skin, if a line with a fine-tipped permanent marking pen is drawn perpendicular to the junction between the normal and affected tissue before sample collection (Fig. 17-33). The line instructs laboratory personnel how to trim the sample to ensure that the critical areas are present for dermatopathologic evaluation. For lesions suspected of being invasive tumors, complete excision of the mass, including a 3-cm margin of clinically normal skin around all borders, is recommended (Fig. 17-34).

Fixation

Punch biopsy specimens should be placed in 10 times the volume of 10% neutral buffered formalin (NBF). To prevent warping in the fixative, thin excisional biopsy specimens should be gently attached to a flat object, such as a piece of cardboard or tongue depressor, and permitted to dry for 20 to 30 seconds. Twenty to 30 seconds is all that is necessary for the sample to adhere to the flat object. The specimen and flat object are then immediately immersed in formalin. Care should be taken not to let the sample become dehydrated (i.e., remain unfixed for longer than 20 to 30 seconds), which could damage the morphology of small samples and the lesions. In cold climates during winter months, adding 1 part alcohol to 9 parts 10% NBF reduces the chance of freezing of specimens during transport.

History

Accurate histopathologic diagnosis and interpretation require knowledge of the gross features of the lesions. Therefore it is essential to include with biopsy samples the following information: (1) age, breed, and sex of the animal; (2) location, gross appearance, and duration of the lesions; (3) presence or absence of lesion symmetry; and (4) presence or absence of pruritus affecting the animal (Box 17-6). Clinical information, including results of laboratory evaluations (i.e., hemograms, serum biochemical analyses), results of skin cultures, scrapings, or cytology, current medications, and response to therapy, should be included along with a list of clinical differential diagnoses. History can be critical to reaching a diagnosis. For example, presence of luminal and mural folliculitis with no apparent follicular infectious agents in H&E-stained sections in conjunction with the history of lack of response to appropriate antibiotic therapy would suggest to the pathologist that a fungal stain to evaluate for occult dermatophyte infection should be performed. Without the history of lack of response to appropriate antibiotic therapy, the folliculitis could easily be presumed to be of bacterial origin, and a fungal infection missed.

Ancillary Procedures

Other diagnostic procedures can supplement information gained from histologic examination of biopsy samples. These procedures include aspiration of pustule contents for cytologic evaluation, performing touch imprints of the cut surface of suspected neoplastic or infectious lesions for cytologic evaluation, aseptically collecting a tissue sample for microbiologic culture, and collecting biopsy samples of suspected immune-mediated diseases or poorly differentiated tumors for immunostaining. Use of immunostaining for identification of cell surface or cytoplasmic proteins (to aid in diagnosis of tumors) or for identification of immunoglobulin, complement, or other antigens (to aid in the diagnosis of immune-mediated skin disease, such as pemphigus) can be helpful in some cases. The formalin-fixed tissue submitted for histopathology also can be used for most routine immunohistochemical procedures, since most employ immunoperoxidase staining rather than immunofluorescence.

Unfortunately, immunostaining techniques for immune-mediated skin diseases can give false-positive or false-negative results, thus they must be done in conjunction with standard histopathology. For autoimmune diseases, if immunofluorescence evaluation is desired, specimens can be fixed in Michel’s medium, which preserves immunoglobulin and complement. If immunohistochemistry (immunoperoxidase) staining is desired, formalin-fixed samples are used; however, for best results, samples should not remain in formalin longer than 48 hours. Prolonged fixation in formalin results in cross-linking of proteins and false-negative results. For autoimmune disease, newer techniques that detect more specific antigens, such as desmoglein (transmembrane glycoproteins found in desmosomes that provide physical connections between keratinocytes), use of “salt-split” skin sections (a technique used in immunostaining of the skin where sodium chloride splits the epidermis from the dermis through the lamina lucida, allowing better differentiation of subepidermal bullous diseases), use of better substrates for indirect immunostaining, and use of immunoblotting and enzyme-linked immunosorbent assay (ELISA) techniques may improve diagnostic accuracy of immune-mediated skin diseases in the future.

Identification of cell surface or cytoplasmic proteins can help identify the cell type in poorly differentiated tumors (Table 17-5). However, there can be anomalous expression of proteins in some tumors (e.g., anomalous expression of cytokeratin in melanoma); thus evaluation of a series (panel) of antibodies is preferred because the pattern of staining with a panel of antibodies is more reliable than staining with one or two antibodies. Formalin-fixed specimens are acceptable for most procedures, but for others, fresh or frozen specimens are better. Discussion with a pathologist is recommended regarding when to use immunostaining procedures.