Keratinocytes

Introduction

In this chapter, the roles of keratinocytes from an internal factor perspective are analyzed in relationship to the disease atopic dermatitis. Situated at the very surface of the human body, epidermis, the functional products of keratinocyte, is logically the first line of defense to ensure the body’s safety against invading enemies, whatever they may be. Daily, the human body is being attacked by countless invaders: bacteria, fungi, viruses, parasites, chemicals, allergens, pollutants, and the like. For most of the invading incidences, the body manages them handily by physically blocking their entry inside, thanks to skin defense. From time to time, however, the first line of defense, the skin, is weakened or damaged either by internal defects or by external factors. These unwanted invaders enter the body through the skin portal, causing harm and disease. Atopic dermatitis, manifested as itchy, chronic, inflammatory lesions primarily in exposed skin areas, is generally recognized to be due in part to the compromise of skin integrity, an internal factor. We have thoroughly discussed the external factors contributing to the disease when the skin barrier is weakened in the previous section of this book. In this section, we will examine what internal factors could contribute to the disease development. Since keratinocyte is the major cell type that forms the skeleton of the epidermis, it is therefore prudent to examine the role of keratinocyte in relationship to the development of atopic dermatitis. In this chapter, the structural and functional roles of keratinocyte in the skin integrity are first discussed, followed by examination of sensation, wound healing, and immunologic roles of keratinocyte in the skin, and then by delineation of the structural and immunologic effects on keratinocyte by three of the prominent atopic dermatitis-related cytokines, interleukin-10 (IL10), IL13, and particularly IL4. Studies have documented that these three cytokines are upregulated in the skin of patients affected by atopic dermatitis. In addition, IL4 is determined to be the sole initiating factor in an animal model of atopic dermatitis ( ).

Keratinocyte: Roles in skin structure and integrity

It is best to demonstrate the structural and functional roles of keratinocyte when we view them from a histologic (tissue level) perspective (i.e., to see the location and the relative abundance of keratinocytes in the epidermis and the proximal relationship between keratinocytes and other cell types that are involved in the immune functions). Fig. 11.1 depicts this information visually. As illustrated, keratinocyte is the principle cell type of the epidermis. From findings of accumulative research studies, we now know that keratinocytes are responsible for the structural integrity of the epidermis and to some extent the entire skin. It is now clear that the basal (lowest) layer of the keratinocytes are the epidermal stem cells, which proliferate and give birth to keratinocytes that will differentiate and move to the upper layers of the epidermis. With proper calcium condition, the keratinocytes differentiate. As the differentiated keratinocytes move up toward the superficial portion of the skin, they form the suprabasal cell layer, then the granular layer, and die and convert into a keratin layer, the stratum corneum of the skin. In Fig. 11.1 , one can also observe two common immune cells present in the skin, the lymphocytes and the Langerhans cells.

Roles in epidermal structure

Looking at the epidermis from a brick-and-mortar model, the keratinocyte, being the major (90%) cellular component of the epidermis, has the right to be considered the bricks of the epidermis (see Fig. 11.1 ). What about the mortar aspect of the equation? Between adjacent cells, keratinocyte-produced intercellular proteins (desmogleins and desmocollins) and intracellular proteins (plakoglobin, desmoplakins, plakophilins, intermediate filaments, etc.) form intercellular structures called desmosomes and other adherence components that act as strong bonding to glue the epidermis together ( Fig. 11.2 ). When one of these intercellular components (e.g., desmoglein) is weakened either by external factors such as bacterial toxin or by internal causes such as autoantibodies, the adherence and coherence of epidermis are lost, resulting in keratinocyte cell-cell separation (acantholysis), leading to intraepidermal blister formation and epidermal loss. An excellent clinical example in support of the epidermal coherence functions of keratinocyte is staphylococcal scalded skin syndrome, which occurs when the bacterial endotoxin breaks down desmoglein-1, leading to blister formation ( ). Pemphigus vulgaris, a life-threatening form of intraepidermal blistering skin disease due to autoantibodies against desmogleins-1 and -3 and other nondesmoglein adhering proteins, provides another strong clinical evidence for the important role of keratinocyte in epidermal integrity ( ).

Roles in barrier proteins: Stratum corneum

To prevent undesirable substance or pathogens from entering the skin, keratinocytes produce proteins and form the corresponding protein-dominant physical barriers between the skin and the outside world in a form of keratin layer (i.e., the stratum corneum). Keratinocytes are the sole cell type that provides these proteins, which include filaggrin, involucrin, and loricrin ( ). Filaggrin, a protein with an apparent molecular weight of 37 kD, is derived from a high-molecular-weight precursor profilaggrin ( ). Filaggrin gene mutations have been documented in European patients with ichthyosis vulgaris and atopic dermatitis initially ( ) and have subsequently been identified in other populations in a worldwide distribution ( ). Clinical evidence supports their skin barrier functional roles. For example, filaggrin gene loss-of-function mutations, particularly the R501X mutation, has been significantly linked to allergen polysensitivity (defined as positivity to three or more allergic compounds) in patch testing, supporting the notion that a defect of filaggrin allows the easy penetration of allergen into the skin to trigger allergic reactions ( ). In the filaggrin-deficient flaky tail (ft/ft) mice, skin barrier defect was evident from clinically visible dry skin and from electron microscopy-visualized reduction of stratum corneum contents ( ). Similar to polysensitivity in human patients with atopic dermatitis, these flaky tail mice also have been observed with increased immune responses to allergens (nickel, 2, 4-dinitrofluorobenzene, and cinnamal) ( ). In an experimental mouse line where the mice were double deficient for filaggrin and hornerin (a gene shares similar structure and function with filaggrin), marked reductions of skin granular layer and a condensed cornified layer were observed, with predisposition to develop allergic contact dermatitis ( ). In a study performed in the Indian subcontinent, hand dermatitis patients were found to have a much higher percentage of filaggrin gene mutation (33.7% vs. 3.5% in control group), predominantly in the S2889X polymorphism ( ). Furthermore, filaggrin mutations are associated with increased risk of infection by poxviruses ( ). In addition, colonization of Staphylococcus aureus is significantly increased in atopic dermatitis patients with filaggrin mutations ( ). Elimination of filaggrin gene expression in keratinocytes (knocked down by lentivirus) not only led to loss of skin barrier protein filaggrin production but also resulted in inhibition of keratinocyte cell adhesion, migration, and proliferation and in promotion of apoptosis and altered cell cycle progression ( ).

Normal skin barrier functions could also be weakened by external substances. For example, exposure to lipoteichoic acid, a cell wall product of a common skin-located pathogen S. aureus , could lead to decreased expression of skin barrier proteins filaggrin and loricrin ( ).

Roles in barrier proteins: Epidermal tight junction

The second type of physical skin barrier structure, besides the stratum corneum mentioned already, is the tight junction—the intercellular junction sealing assembly between adjacent keratinocytes in the stratum granulosum, just below the stratum corneum ( ). Keratinocytes are the providers of these tight junction proteins. A schematic presentation of an epidermal tight junction is shown in Fig. 11.3 . Several epidermal tight junction–associated mRNAs have been identified in human keratinocytes, including claudins-1, -4, -7, -8, -11, -12, -17, -23, ZO (zonula occludens), and occludin ( ). While claudin-1 protein has been identified in all epidermal cell layers, ZO-1 protein is found primarily in the uppermost cell layers, and occludin is detected only in the stratum granulosum ( ). The role of claudin-1 in epidermal barrier protection is documented by the construction of a claudin-1–deficient mouse model. Experimentally, the caludin-1–deficient mouse skin allowed the subcutaneously administered tracer diffuse toward the skin surface, whereas such diffusion was blocked by the skin of wild type mice with intact claudin-1. These claudin-1–deficient mice died within 1 day of birth with wrinkled skin from dehydration because of severe transepidermal water loss ( ). From this perspective, claudin-1 is a far more essential skin barrier protein than filaggrin in terms of animal survival, as filaggrin deficiency is not life threatening in flaky tail mice ( ). In addition, tight junction proteins claudin-1 and occludin are important for cutaneous wound healing ( ), and tight junction is a physical blocker for viral entry into the skin ( ). Patients with atopic dermatitis significantly have epidermal tight junction defects in that their expressions of the claudin-1 and -23, in both mRNA and protein levels, are strikingly lower compared to normal individuals. The functional result of this defect is supported by experimental silencing claudin-1 expression in mice, which leads to diminishing tight junction function ( ). The reduction of claudin-1 expressions occurs only in lesional skin (not in nonlesional skin) of atopic dermatitis patients, suggesting the reduction is related to the inflammatory process and not because of genetic mutation ( ). This inflammation-induced reduction of lesional skin claudin-1 in atopic dermatitis is supported by a study conducted in human epidermal equivalent, documenting the suppression of keratinocytes’ claudin-1 by a trio of atopic dermatitis-related cytokines, IL4, IL13, and IL31 ( ). The tight junction protein expression, formation, and function are also influenced and regulated by a variety of proteins, enzymes, and cytokines, including IL1, CD44, somatostatin, kinase-directed phosphorylation, a Rho-family protein GTPases Rac, activation of toll-like receptor-2 (TLR2), epidermal growth factor receptor (EGFR), adenosine triphosphate (ATP)–powered calcium-pump protein 2C1, histamine, IL33, IL17, enolase-1, antimicrobial peptide LL37, keratin-76, integrin-linked kinase, activated protease-activated receptor-2, and receptor tyrosine kinase EphA2 ( ).

Roles in epidermal-dermal adhesion

Keratinocytes are essential providers not only for structure and proteins needed for the integrity of the epidermis but also for the proteins that contribute to the adherence between the epidermis and dermis, the dermal-epidermal junction, or skin basement membrane zone. This role is accomplished through the keratinocytes’ synthesis of several important skin basement membrane anchoring proteins, α6β4-integrin ( ), type XVII collagen (or bullous pemphigoid antigen II) ( ), type VII collagen ( ), and laminin 5/laminin 332 ( ), which are essential connecting components of the skin basement membrane at the junction between epidermis and dermis ( Fig. 11.4 ) ( ). The functional importance of these keratinocyte-produced skin basement membrane components is vividly illustrated in both genetic skin diseases (epidermolysis bullosa group of diseases) and autoimmune skin diseases. When these keratinocyte-produced skin basement membrane components are weakened either by genetic defect ( ) or by assault from autoantibodies in autoimmune diseases ( ), the whole skin integrity is compromised, leading to dermal-epidermal separation, subepidermal blister formation, and even epidermis loss. In addition, keratinocytes synthesize another lower lamina lucida 105-kD protein that has yet to be fully characterized. When this 105-kD protein was targeted by autoantibodies from a patient, skin weakness occurred and subepidermal blisters developed, further supporting a role of keratinocytes in skin basement membrane stability ( ).

Keratinocyte: Roles in cutaneous sensation

Recently, the traditional concept that intraepidermal nerve fibers are the sole transducers of neural signal has been challenged, and the role of keratinocytes in neural signaling is proposed. The putative sensory role of keratinocyte was proposed based on the findings that keratinocytes express diverse sensory receptors that are present in sensory neurons such as transient receptor potential vallinoid-1 (TRPV1) and TRPV4 ( ). Since TRPV1 is a known transducer for pain, heat, and to a less extent itch, and TRPV4 is a heat transducer, keratinocytes’ roles for cutaneous sensation cannot be neglected. Moreover, the findings that specific and selective activation of TRPV1 in keratinocytes can induce pain and that targeted stimulation of TRPV4 in keratinocytes can result in itch sensation leading to scratch behavior support a potential role of keratinocytes in skin sensation ( ). Moreover, lineage-specific deletion of keratinocyte TRPV4 reduced chronic itch in animal model ( ). In addition, the findings that keratinocytes possess neuropeptide substance P and that keratinocytes express nerve growth factor, in response to neuropeptide activation of the ERK1/2 and JNK MAPK transcriptional pathways, also support their roles in cutaneous sensation ( ). The relevance of this keratinocyte’s role in skin sensation in the disease of atopic dermatitis is that eczema is well characterized for its pruritic clinical presentation ( ).