Key points
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Epidermis is the outermost layer of skin surface that forms the first line of defense against invaders from the environment. Keratinocyte is the epidermis’s principle cell type responsible for generating and maintaining the integrity of the epidermis.
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Besides being the structural foundation of the epidermis, keratinocytes also possess functions capable of interacting with their environment immunologically.
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Keratinocytes are capable of expressing neurologic factors that contribute to the itch sensation, which is a characteristic clinical finding in atopic dermatitis.
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Weakness of skin integrity at the epidermal level would allow easy entry of environmental pathogens or allergens into the skin, leading to chronic inflammatory processes as observed in atopic dermatitis.
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Internally altered immune milieu enhancing cytokines of helper T-cell subset 2 could trigger the release of proinflammatory components from epidermal keratinocytes, lessen keratinocyte’s contribution to epidermal barrier, reduce keratinocyte’s ability in wound healing, decrease keratinocyte’s role in host defense, and thereby diminish the integrity of the skin and promote an inflammatory milieu of the skin and beyond.
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 ( ).
Keratinocyte: Role in wound healing
When skin is injured, the repair process will be initiated. The sequence of events in wound healing starts with hemostasis (to stop blood loss), inflammation (to stop invaders), angiogenesis (to repair blood supply for nutrient and oxygen delivery), growth (to restore dermal structures), reepithelialization (to close epidermal gap and seal off external barrier), and remodeling (to fine-tune structure and function). On a cellular level, the wound healing process mobilizes a variety of cells and involves spatial and temporal synchronization of many cellular and molecular actions. The near last step of wound healing is the process of reepithelialization where keratinocytes play the most important role. In essence, this process will involve proliferation of epidermal stem cells at the basal layer of the epidermal wound edge and differentiation of hair follicle unipotent stem cells to keratinocytes. The subsequent formation of epithelial tongues (migration front), migration of these keratinocytes across the dermal wound bed, and differentiation of these keratinocytes into suprabasal, granular, and stratum corneum layers will accomplish the epidermal barrier repair ( ). IL4, the major cytokine that positively influences the development of atopic dermatitis, also seems to play a negative role in wound healing. In an animal model of atopic dermatitis triggered by an overexpression of IL4 in the basal epidermis, reepithelization and final wound healing were delayed compared to that of wild type mice; and such delay would likely have negative consequence for atopic dermatitis, as it will prolong the skin barrier defect that allows external invaders to enter the skin, thus flaming the ongoing inflammatory responses ( ).
Keratinocyte: Roles in immune regulation
As depicted in Fig. 11.1 , at the tissue level, keratinocytes are in physical contact with immune cells such as lymphocytes and Langerhans cells. Since lymphocytes are the major immune cells in the adaptive immune system responsible for immune memory and the effector cells for defense against previously recognized pathogens, their proximal relationship to keratinocytes is worth noting. The importance of this keratinocyte-lymphocyte relationship is even more pronounced given the facts that there are nearly twice the amount of lymphocytes present in the skin than those present in the circulation and that most CLA+ memory effector T cells are located in the skin, as recently reported in the scientific literature (Clark et al., 2006). Langerhans cells are the professional antigen-presenting cells responsible for immune recognition and immune activation. Vaccination through epidermis-restricted microperforation can effectively activate keratinocytes and Langerhans cells, resulting in excellent antibody responses ( ). In addition, Langerhans cells are also responsible for maintaining skin homeostasis by activating resident regulatory T cells ( ). In the following paragraphs, the roles of keratinocytes in both innate and adaptive immune responses will be discussed.
Role in innate immune responses
Keratinocytes produce skin-localized antimicrobial substances, which can be a good and immediately available defense against invading microorganism, by mounting their innate immune responses.
Normal human keratinocytes regularly produce, usually in response to injury or infection challenge, many antimicrobial peptides (also described as host defense peptides) known for their innate immune defense functions. These peptides include two major families: human β-defensins and cathelicidins, and others ( ). Besides maintaining a homeostasis with commensal microorganisms in a symbiosis manner, these peptides function to defend against invading bacteria, viruses, fungi, and to a less extent parasites. They can also activate surrounding cells, keratinocytes or immune cells, to regulate many biologic functions, including angiogenesis, epithelialization, and immunity. Sometimes the roles of these peptides are also implicated in allergic disease, including atopic dermatitis ( ). In atopic dermatitis patients, some of these peptides failed to be properly induced, thus allowing infection to set in ( ). Although cathelicidins are also produced by neutrophils and other immune cells, besides keratinocytes, their strategic location at the front layer of the skin (therefore being the first to encounter pathogens) makes keratinocytes’ roles in host defense particularly important. β-defensins are primarily located in epithelial surfaces, including the skin ( ).
Keratinocytes, when encountering pathogens, can respond by releasing immune substances. In the presence of West Nile virus infection, human keratinocytes release type I and type III interferon inflammatory responses, including mRNAs of CXCL10 and interferon-induced proteins, as well other proinflammatory cytokines and chemokines, including tumor necrosis factor-α (TNF-α), IL6, CXCL1, CXCL2, CXCL8, and CCL20 ( ).
One of the common skin infections encountered by humans is from fungi. When exposed to dermatophytes in culture, human keratinocytes upregulate their mRNAs of many cytokines and chemokines, including IL1, IL2, IL4, IL6, IL8, IL10, IL13, IL15, IL16, IL17, and interferon-gamma (IFN-γ), and significantly increase secretion, compared to control, of many cytokines and chemokines, including eotaxin, eotaxin-2, G-CSF, GMCSF, ICAM-1, IFN-γ, IL1, IL2, IL3, IL4, IL6, IL7, IL8, IL13, IL15, IL16, IL17, IP10, and MCP-1 ( ).
Besides the cytokines and chemokines reported in human keratinocytes in responding to pathogen exposure, other cytokine present in keratinocyte includes IL20 ( ). Furthermore, keratinocytes express many adhesion, activation, or cytokine receptors on their cell surfaces, including IL4R, IL20R, IL31R, CD95, IFN-γR1/CD119, IL2Rγ/CD132, ICAM-1, HLA-DR, and B7/BB1 ( ). Therefore, with a variety of immune receptors, keratinocytes can respond to immune signals, cytokines, and other stimulatory signals released by other cells. Their responses in turn would result in changing of immune milieu.
One of the most recent findings implicating keratinocyte’s roles in atopic dermatitis is the acryl hydrocarbon receptor (AhR) on the surface of keratinocytes ( ). The expression of this receptor, critical for the effects of major environmental pollutants on the human body, is highly upregulated in the skin of patients with atopic dermatitis ( ). Consistent to this observation is the documentation that a transgenic mouse line constitutively expressing AhR in keratinocytes developed severe inflammatory skin lesions resembling atopic dermatitis in human patients ( ). Experimentally, the link between AhR and atopic dermatitis is determined to be in the activation of AhR, leading to induced expression of artemin, allokness, epidermal hyperinnervation, severe pruritus, and the subsequent inflammatory process ( ). Artemin is a member of the glial cell line–derived neurotrophic factor family with a variety of neuronal functions ( ). Alloknesis is a term describing abnormal itch sensation that can be induced by innocuous stimuli and by the absence of a keratinocyte-expressing and endogenous corticosteroid-activating enzyme 11β-hydroxysteroid dehydrogenase-1 ( ).
Role in adaptive immune responses
In addition to their innate immune functions, studies have pointed to a bigger role of keratinocytes in skin adaptive immunity. Keratinocytes can also orchestrate T-cell immunity in various adaptive immune responses ( ). In a study aiming to examine the antigen-presenting abilities of keratinocyte, investigators generated a transgenic mouse model in which these epidermal cells exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II β-chain, under inflammatory conditions. These researchers found that inflammation induced by epicutaneous contact sensitization led to an expansion of MBP-specific CD4+ T cells in the skin. In addition, repeated applications of contact sensitizer to these mice preceding a systemic MBP immune administration, a classic method of immune desensitization, reduced the reactivity of these MBP-specific T cells and lessened the symptoms of the resulting experimental autoimmune encephalitis. Thus keratinocytes are shown that they can present antigen to T cells, and presenting neo-self antigen under the inflamed condition by keratinocyte can modulate CD4+ T cells’ autoimmune aggressiveness at a distant organ ( ). In another experimental study, coculture of epidermal professional antigen-presenting Langerhans cells with T cells in the presence of keratinocytes resulted in an enhanced acquired immunity compared to the condition in the absence of keratinocytes, suggesting a role of keratinocyte in augmenting antigen presentation ( ). The proximity of keratinocytes to Langerhans cells and lymphocytes in the epidermis also supports this role from a histologic perspective (see Fig. 11.1 ). In another experiment where human CD40 was transgenically expressed in mice under the control of keratin-14 promoter, which drives CD40 expression in basal keratinocytes exclusively, selective engagement of keratinocyte CD40 resulted in enhanced cell-mediated immune responses, supporting a notion that CD40 engagement by keratinocyte could amplify cutaneous and regional T-cell immune responses in vivo ( ). In an animal model of vitiligo using a conditional STAT-1–knockout mouse, investigators have discovered that the keratinocyte-generated IFN-γ signaling was essential for proper T-cell homing to the epidermis and for the disease progression, without the need of participation from professional immune cells, endogenous T cells, Langerhans cells, or γδ T cells ( ). A 2019 publication documented that the MHCII expressions in keratinocytes could be induced by association with commensal bacteria ( Staphylococcus epidermidis ), thus these friendly bugs could promote the antigen-presenting abilities of keratinocytes for the purpose of host defense ( ).
Keratinocyte’s barrier proteins: Impacts of IL4 and other Th2 cytokines
IL4, the most important cytokine in the helper T-cell subset 2 (Th2) family, has significant involvement in the development of atopic dermatitis ( ). In its absence, the IL4-knockout mice showed a substantial reduction of serum levels of immunoglobulin E (IgE), which is essential for allergic skin reaction and typically elevated in human patients affected by atopic dermatitis ( ). In fact, the most significant way IL4 exhibits in relationship to atopic dermatitis is in a mouse model of atopic dermatitis, in which IL4 has been transgenically inserted and expressed in basal epidermis through a promoter of keratin-14, a basal keratinocyte-located keratin ( ). The keratin-14/IL4 transgenic mice (but not the nontransgenic mice) spontaneously developed a very itchy and chronically inflammatory skin disease primarily affecting the hairless skin of mice ( Fig. 11.5 ), with high total serum IgE, staphylococcal skin infection, and prominent skin infiltration of lymphocytes, mast cells, and eosinophils ( Fig. 11.6 ) ( ). Using clinical criteria for human atopic dermatitis, the manifestations of these diseased mice fulfill the diagnostic criteria for human atopic dermatitis ( ). From a different angle, a recently successful treatment of atopic dermatitis by the biologic medication dupilumab, a humanized antibody directly against IL4 receptor alpha, provides another strong support for the essential role of IL4 in atopic dermatitis development ( ). Therefore it is both academically interesting and theoretically important to examine what the results of interaction between IL4 and keratinocyte would show and how these results would affect the skin environment in a way that is contributing to the development or sustaining the disease atopic dermatitis. Toward that end, we seek to answer the question, How are epidermal keratinocytes impacted when the internally altered immune system produces and releases an excessive amount of IL4?
Th2 cytokines and keratinocyte’s physical barrier proteins
Experimental data now clearly show that IL4 negatively impacts keratinocyte’s abilities to produce the essential epidermal barrier protein production ( ). In the following paragraphs the details of these impacts are depicted.
IL4 suppresses involucrin mRNA and protein production
Involucrin, one of the skin barrier proteins synthesized by keratinocyte, showed reduced expression in the lesional skin of atopic dermatitis patients, using ELISA on human skin extracts ( ). In the nonlesional skin of the keratin-14/IL4 transgenic mice, we found that the mRNA and protein levels of involucrin are substantially reduced, compared to the wild type mice, suggesting that the involucrin reduction was likely due to the impact of IL4, not by an inflammatory process per se ( ). To determine if IL4 could affect the expression of this protein, immortalized human keratinocyte HaCaT cells exposed to IL4 in different concentrations for 24 hours were extracted for examination of the mRNA expressions of involucrin by reverse transcription and then real-time polymerase chain reaction (PCR) in our laboratory. IL4 suppresses the involucrin expressions in a dose-dependent manner. This downregulation of involucrin in the protein level by IL4 was also confirmed by Western blot analysis ( Fig. 11.7 ) ( ). In addition, this IL4 suppression mechanism has been attributed to the Stat6 signaling pathway experimentally ( ). This finding supports the notion that internal immune dysregulation with IL4 upregulation can by itself lead to a reduction of skin barrier protein involucrin, thus impairing the skin barrier function.
