Innate Immunity and the Skin

INNATE IMMUNITY AND THE SKIN

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Throughout evolution, the immune system has developed complex and intricate mechanisms to defend the host against infection and cancer. The ability of the immune system to distinguish self from non-self is critical in determining when a response will be elicited. The immune system of higher vertebrates uses both rapid (innate) and sustained (adaptive) immune responses, which differ in the way they recognize foreign antigens, and the speed in which they respond. The innate immune system uses germ-line–encoded pattern recognition receptors to respond to biochemical structures commonly shared by a number of different pathogens and elicits a rapid response against encountered pathogens, although no lasting immunity is generated. In contrast, cells of the adaptive immune system recognize molecules specific to particular pathogens, and clonally expand cells bearing antigen-specific receptors. A few of these cells become memory cells when the infection subsides, and thus have the capability of providing long-lasting protective immunity. Together, the innate and adaptive immune systems defend the host against infections and disease, making survival possible. This chapter describes the distinct role of the innate immune responses in generating host defense mechanisms in skin.

While adaptive immunity occurs only in vertebrates, the innate immune system exists in all multicellular organisms.

Defense mechanisms that are used by the host immediately after encountering a foreign ligand are referred to as innate immunity. These include physical barriers such as the skin and mucosal epithelium, soluble factors such as complement, antimicrobial peptides, chemokines, and cytokines, and cells of the innate immune system including monocytes/macrophages, dendritic cells, and polymorphonuclear leukocytes (PMNs).

Physical structures prevent most pathogens and environmental toxins from harming the host. The skin and the epithelial lining of the respiratory, gastrointestinal, and the genitourinary tract provide physical barriers between the host and the external world. Skin, once thought to be an inert structure, plays a vital role in protecting the individual from the external environment. The epidermis impedes penetration of microbial organisms, chemical irritation, and toxins, absorbs and blocks solar and ionized radiation, and inhibits water loss.

The stratum corneum, the outermost layer of the epidermis that results from the terminal differentiation of the keratinocytes, forms the primary layer of protection from the external environment. This layer of anucleated keratinocytes is composed of highly cross-linked proteinaceous cellular envelopes with extracellular lipid lamellae consisting of ceramides, free fatty acids, and cholesterol. The free fatty acids create an acidic environment that inhibits colonization by certain bacteria such as Staphylococcus aureus, providing further protection.

The oral mucosa, gastrointestinal, and respiratory linings have special features to protect the body from microbes and other foreign materials. In the mouth and in the upper gastrointestinal tract, chemical substances such as digestive enzymes and acidic secretions inhibit microbial growth. Furthermore, nonpathogenic microbes colonize the epithelium of the gut and prevent invasion by pathogenic microbes. The protective role of the normal flora becomes clear when the use of antibiotics destroys these nonpathogenic bacteria, opening up a window of opportunity for invasion by pathogenic bacteria. Finally, the upper respiratory tract is lined by ciliated columnar epithelium, and the movement of cilia protects inhaled foreign particles from entering the alveolar space. Given these vital functions, it is not surprising that a break in this physical barrier, as often occurs in patients with severe burn or large wounds, results in high morbidity and mortality.

One of the first innate defense mechanisms awaiting pathogens that overcome the epithelial barrier is the alternative pathway of complement. Unlike the classical complement pathway that requires antibody triggering, the alternative pathway of complement activation can be activated by microbial surfaces in the absence of specific antibody. In this way, the host defense mechanism comes into play immediately after encountering the pathogen without the 5 to 7 days required for antibody production.

Initially, the complement component C3 undergoes spontaneous hydrolysis to give C3(H2O), which binds to factor B, allowing it to be cleaved by factor D into Ba and Bb. The C3(H2O)Bb complex, which is a C3 convertase, forms cleaving C3 to C3a and C3b. C3b can attach covalently through its reactive thioester group to the surfaces of host cells or to pathogens. The bound C3b is able to bind factor B, which is then cleaved by factor D to yield Ba and the active protease Bb. If C3bBb forms on the surface of the host cells, it is rapidly inactivated by complement regulatory proteins expressed on the host cell such as CR1 (complement receptor 1), DAF (decay-accelerating factor), factor H, and MCP (membrane cofactor of proteolysis). However, bacterial surfaces do not express complement regulatory proteins, and properidin (factor P) binds and stabilizes the C3bBb complex. C3bBb complex also is a C3 convertase and initiates the cleavage of further molecules of C3 leading to opsonization by C3b and the generation of C3b2Bb, the alternative pathway C5 convertase, leading to activation of the terminal complement components.

Not all microbial surfaces allow for activation of the alternative complement pathway. Although the content of sialic acid (high levels are present on the surfaces of vertebrate cells) has been implicated as one of the factors that determine the ability to trigger the alternative complement pathway, it is not clear exactly what distinguishes surfaces that allow the complement cascade to proceed.

Once thought to be inert, keratinocytes, the predominant cells in the epidermis, can mount an immune response through secretion of antimicrobial peptides. Human epithelial cells produce β-defensins, cysteine-rich cationic low molecular weight antimicrobial peptides. Antimicrobial peptides are an important evolutionarily conserved innate host-defense mechanism in many organisms. The first human β-defensin, HBD-1, was isolated from human hemofiltrate obtained from a patient with end-stage renal disease. HBD-1 is constitutively expressed in the epidermis, is not transcriptionally regulated by inflammatory agents, and has antimicrobial activity against gram-negative bacteria. In addition, recent findings suggest that HBD-1 plays a role in keratinocyte differentiation. A second human β-defensin, HBD-2, was discovered in extracts of lesions from psoriasis patients. Unlike HBD-1, HBD-2 expression is inducible by microbes including Pseudomonas aeruginosa, S. aureus, and Candida albicans. In addition to stimulation by microbes, proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1 can also induce HBD-2 transcription in keratinocytes. When tested for antimicrobial activity, HBD-2 also showed effective activity against gram-negative bacteria such as Escherichia coli and P. aeruginosa, but not against gram-positive bacteria such as S. aureus. Recently, a third β-defensin, HBD-3, was isolated and characterized. Contact with TNF-α and with bacteria were found to induce HBD-3 mRNA expression. In addition, HBD-3 demonstrated potent antimicrobial activity against S. aureus and vancomycin-resistant Enterococcus faecium. Therefore, HBD-3 is among the first human β-defensins in skin to demonstrate effective antimicrobial activity against a gram-positive bacteria. The localization of human β-defensins to the outer layer of the skin and the fact the β-defensins have antimicrobial activity against a variety of microbes suggest that human β-defensins are essential parts of cutaneous innate immunity. Furthermore, evidence indicating that human β-defensins attract dendritic cells and memory T cells via the chemokine receptor CCR6, provide a link between the innate and the adaptive immunity is skin.

Other innate antimicrobial peptides, called cathelicidins, have also been identified in skin. Animal studies show that these cathelicidins are an important component of innate host defense in mice and protect against necrotic skin infections produced by group A streptococci. These peptides are produced in increasing amounts following skin wounding due to their release by neutrophils and increased synthesis by keratinocytes.

Keratinocytes can also mount an immune response through the secretion of inflammatory cytokines. Keratinocytes constitutively release very low levels of cytokines; however, upon injury or stimulation with exogenous factors such as lipopolysaccharides (LPS), silica, poison ivy catechols, Staphylococcus toxins, and UV radiation, keratinocytes secrete high levels of IL-1, IL-6, IL-8, IL-10, and TNF-α. These cytokines can induce differentiation and growth of keratinocytes and other resident or migrating cells in the epidermis, dermis, and vessels. Furthermore, they are important mediators of both local and systemic inflammatory and immune responses.

In addition to cytokines, keratinocytes secrete other factors such as neuropeptides, eicosanoids, and reactive oxygen species. These mediators have potent inflammatory and immunomodulatory properties and play an important role in the pathogenesis of cutaneous inflammatory and infectious diseases as well as in aging.

Effective host defense against invading microorganisms requires detection of the foreign pathogens and the rapid deployment of an effective antimicrobial response. This function is imparted to the innate immune system that recognizes the molecular structures of microbial invaders. The microbes are rich with molecular arrays or patterns that are shared among groups of pathogens. Examples include LPS of gram-negative bacteria, lipoteichoic acids of gram-positive bacteria, lipoproteins of bacteria and parasites, glycolipids of mycobacteria, mannans of yeast, bacterial DNA sequences, and double-stranded RNA of viruses. Phagocytes, such as macrophages and PMNs, are the major cellular component of the innate immune system and have the capacity to detect these microbial patterns using complement receptors on their surface.

In the epidermis, there are skin-specific cells known as Langerhans cells. After encountering a pathogen in the epidermis, Langerhans cells take up the pathogen by an endocytotic process and migrate to the draining lymph nodes where they develop into mature dendritic cells. These migrating cells lose the ability to take up and process antigen but they upregulate MHC molecules and costimulatory molecules to activate naive T cells.
Neutrophils are normally not present in skin; however, during inflammatory processes, these cells migrate to sites of infection and inflammation where they are the earliest phagocytic cells to be recruited. PMNs can phagocytose microbes coated with antibody and with the complement component C3b. In addition, PMNs have specific receptors that recognize pathogens directly.

Phagocytes express pattern recognition receptors that function to recognize pathogen-associated molecular patterns. Examples of pattern recognition receptors include the mannose receptor that recognizes mannan and CD14 that recognizes the lipid A portion of LPS. Pattern recognition receptors are nonclonal receptors that are present on all cells of a class, e.g., macrophages, and do not depend on immunologic memory because they are germ-line encoded. Recently, a new mammalian pattern recognition receptor, the Toll-like receptor, has been identified and important studies are emerging to provide new insights into the mechanisms by which the innate response recognizes and combats microbial invaders.

How do phagocytes recognize foreign pathogens? One way that pathogens can be recognized and destroyed by the innate immune system is via receptors on phagocytic cells. Unlike adaptive immunity, the innate immune response relies on a relatively small set of germ-line–encoded receptors that recognize conserved molecular patterns found only on microorganisms. The phagocytic cells recognize pathogen-associated molecular patterns that are shared by a large group of pathogens. These pathogen-associated molecular patterns are usually conserved molecular structures required for survival of the microbes and therefore are not subject to selective pressure. In addition, pathogen-associated molecular patterns are specific to microbes and are not expressed in the host system. Consequently, the innate immune system has mastered a clever way of distinguishing between self and non-self and of relaying this message to the adaptive immune system.

Molecules that recognize pathogen-associated molecular patterns are known as pattern-recognition receptors. Pattern-recognition receptors can be divided into several families of proteins by their structure. For example, Toll and CD14 are pattern-recognition receptors that contain leucine-rich repeats, collectins have calcium-dependent lectin domains, and macrophage scavenger receptors contain scavenger-receptor protein domains. These pattern-recognition receptors can also be divided into three different classes by their function: secreted, endocytic, and signaling. Secreted pattern-recognition receptors function as opsonins by binding to microbial cell walls and flagging them for recognition by the classical complement system and phagocytes. The best-characterized receptor of this class is the mannan-binding lectin. The macrophage mannose receptor is an endocytic pattern recognition receptor that functions by recognizing carbohydrates with a large number of mannose residues that are characteristic of microorganisms, and mediating their phagocytosis by macrophages. Signaling receptors recognize pathogen-associated molecular patterns and activate signal transduction pathways that lead to the expression of a number of immune response genes, including cytokine genes. The recently identified family of Toll-like receptors is a pattern-recognition receptor family.

There is now substantial evidence to support a role for mammalian Toll-like receptors in innate immunity. First, Toll-like receptors are pattern-recognition receptors that recognize pathogen-associated molecular patterns present on a variety of bacteria and fungi. Second, Toll-like receptors are expressed at the interface with the environment where the host must defend against microbial threats. Third, the activation of Toll-like receptors induces expression of costimulatory molecules and the release of cytokines that instruct the adaptive immune response. Fourth, Toll-like receptors directly activate host defense mechanisms that then combat the foreign invader.

Toll was first identified in dorsoventral patterning in the Drosophila melanogaster embryo and in antifungal defense in adult flies. Since then, at least 10 mammalian Toll homologues, Toll-like receptors, have been identified. The extracellular domain has leucine-rich repeats and recognizes a spectrum of microbial products. Studies suggest that mammalian Toll homologues, Toll-like receptors, mediate responsiveness to specific molecular structures from both gram-positive and gram-negative organisms. For example, LPS activates Toll-like receptor 4; microbial lipoproteins, peptidoglycans, and S. aureus lipoteichoic acid activate Toll-like receptor 2; a specific bacterial DNA, an unmethylated cytidine-phosphate-guanosine (CpG) activates Toll-like receptor 9; bacterial flagellin activates Toll-like receptor 5; and double-stranded RNA activates Toll-like receptor 3.

The intracellular domain of Toll-like receptors has homology to the IL-1 receptor and shares common signaling molecules of the Rel/NF-?B pathway. Considerable progress has been made toward understanding the cell-signaling events that occur after Toll-like receptor activation with emphasis on the activation of NF-?B, a transcription factor involved in the expression of many proinflammatory cytokines. The Toll-like receptor signaling pathway has been analyzed and found to share features in common with Toll signaling in Drosophila. LPS activation via Toll-like receptors has been shown to induce an intracellular signaling cascade involving MyD88, IL-1 receptor accessory protein kinase (IRAK), TNF-receptor associated factor (TRAF-6), and NF-?B–inducing kinase (NIK) leading to activation of NF-?B and subsequent immunoregulatory gene transcription. Further study of mammalian Toll-like receptor signaling pathways could identify different downstream events according to the nature of the microbial ligand and Toll-like receptor family member that has been activated.

Toll-like receptors were initially found to be expressed in all lymphoid tissue but were most highly expressed in peripheral blood leukocytes. Expression of Toll-like receptor mRNA has been found in monocytes, B cells, T cells, granulocytes, and dendritic cells. Protein expression of toll family members has been verified by using monoclonal antibodies for B cells, monocytes, and dendritic cells. Underhill et al. suggest that following phagocytosis, Toll-like receptors are recruited to the pathogen-containing phagosomes and discriminate between gram-positive and gram-negative bacteria, thus surveying the intracellular compartments of the cells for microbial invaders. The expression of Toll-like receptors on cells of the monocyte/macrophage lineage is consistent with the role of Toll-like receptors in modulating inflammatory responses via cytokine release. Because these cells migrate into sites that interface with the environment, lung, skin, and gut, the location of Toll-like receptor expressing cells would be situated to defend against invading microbes. Toll-like receptor expression by adipocytes, intestinal epithelial cells, and dermal endothelial cells supports the notion that Toll-like receptors serve a sentinel role for invading microorganisms. The regulation of Toll-like receptor expression is critical to their role in host defense, yet few factors have been identified that modulate their expression. IL-4 acts to downregulate Toll-like receptor expression, suggesting that T H2 cell adaptive immune responses might inhibit Toll-like receptor activation.

LPS and lipoproteins have historically been known to be potent inducers of cytokine production. However, not until the identification of Toll-like receptors was a receptor linked to this response. Medzhitov et al. first demonstrated that constitutive activation of Toll-like receptor 4, using a dominant active construct transfected in a human monocytoid line, induced cytokine production and upregulation of costimulatory molecules. Since then, critical proinflammatory and immunomodulatory cytokines, such as IL-1, IL-6, IL-8, IL-10, IL-12, and TNF-α, have been shown to be induced following activation of Toll-like receptors by microbial ligands. Evidence suggests that monocyte-derived dendritic cells produce the proinflammatory IL-12, and not the anti-inflammatory IL-10, upon activation with lipoproteins. Activation of Toll-like receptors on dendritic cells triggers their maturation, in terms of expression of CD83, MHC class II, as well as the costimulatory molecules CD80 and CD86. In this manner, the activation of dendritic cells via Toll-like receptors enhances their ability to present antigen to T cells and generate T H1 cytokine responses critical for cell-mediated immunity (CMI). Consequently, activation of Toll-like receptors, as part of the innate response, can lead to instruct the nature of the adaptive T cell response.

In Drosophila, Toll is critical for host defense. Flies with a mutation in Toll are highly susceptible to fungal infection. It is now evident that mammalian Toll-like receptors play a prominent role in directly activating host defense mechanisms. Activation of Toll-like receptor 2 by microbial lipoproteins induces activation of the inducible nitric oxide (iNOS) promoter, which leads to the production of nitric oxide (NO), a known antimicrobial agent. There is strong evidence that Toll-like receptor 2 activation leads to killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages. 35 In mouse macrophages, bacterial lipoprotein activation of Toll-like receptor 2 leads to a nitric oxide–dependent killing of intracellular tubercle bacilli. In human monocytes and alveolar macrophages, bacterial lipoproteins similarly activated Toll-like receptor 2 to kill intracellular M. tuberculosis; however, this occurred by an antimicrobial pathway that is nitric oxide independent. These data provide evidence that mammalian Toll-like receptors have retained not only the structural features of Drosophila Toll that allow them to respond to microbial ligands, but also the ability directly to activate antimicrobial effector pathways at the site of infection. In Drosophila, activation of Toll leads to the NF-?B–dependent induction of a variety of antimicrobial peptides, including metchnikowan, defensins, cecropins, and drosomycin. The clues from Drosophila suggest that exploring the mechanisms of induction of antimicrobial peptides in mammalian cells is warranted. In this regard, it was recently shown that LPS induces β-defensin-2 in tracheobronchial epithelium, suggesting the conservation of the Drosophila Toll pathway of antimicrobial peptide induction.

The activation of Toll-like receptors can also be detrimental, leading to tissue injury. The administration of lipopolysaccharides to mice can result in manifestations of septic shock, which is dependent on Toll-like receptor 4. Evidence suggests that Toll-like receptor 2 activation by Propionibacterium acnes induces inflammatory responses in acne vulgaris leading to tissue injury (J. Kim and R.L. Modlin, unpublished). Aliprantis et al. demonstrated that microbial lipoproteins induced features of apoptosis via Toll-like receptor 2. Thus, microbial lipoproteins have the ability to induce both Toll-like receptor–dependent activation of host defense and tissue pathology. This dual signaling pathway is similar to TNFR and CD40 signaling, which can induce both NF-?B activation and apoptosis. In this manner, it is possible for the immune system to activate host defense mechanisms, and then, by apoptosis, to downregulate the response to prevent it from causing tissue injury. Activation of Toll-like receptors can lead to the inhibition of MHC class II antigen presentation pathway, which can downregulate immune responses leading to tissue injury but could also contribute to immunosuppression.

The susceptibility of mice with spontaneous mutations in Toll-like receptors to bacterial infection implicates Toll-like receptors as critical receptors in mammalian host defense.Toll-like receptor 4 mutations are associated with lipopolysaccharide hyporesponsiveness in humans. By inference, we anticipate that humans with genetic alterations in other Toll-like receptors may have increased susceptibility to certain microbial infections. Furthermore, it should be possible to exploit the pathway of Toll-like receptor activation as a means to adjuvant immune responses in vaccines and treatments for infectious diseases, as well as to abrogate responses detrimental to the host.

Activation of phagocytes by pathogens induces several important effector mechanisms. One such mechanism is the triggering of cytokine production. A number of important cytokines are secreted by macrophages in response to microbes, including IL-1, IL-6, TNF-α, IL-8, IL-12, and IL-10. IL-1, IL-6, and TNF-α play critical roles in inducing the acute-phase response in the liver and in inducing fever for effective host defense. TNF-α induces a potent inflammatory response to contain infection; IL-8 is important as a mediator of PMN chemotaxis to the site of infection.

One of the most important cytokines produced by monocytes is IL-12. Both in murine models of infection and in human infectious disease, susceptibility or resistance to infection is often determined by the T H1 and T H2 T cell cytokine patterns. It has become increasingly evident that IL-12 is a pivotal regulator of T H1 responses and hence is essential for promoting CMI against intracellular microbial pathogens. In this manner, the ability of the innate immune system to release IL-12 influences the nature of the adaptive immune response.

Phagocytes also secrete an inhibitory cytokine, IL-10, that has many anti-inflammatory activities. The importance of IL-10 is underscored in both human disease and murine infection models. IL-4 and IL-10 production is associated with the progressive forms of leishmanial, schistosomal, and trypanosomal infections. In leprosy, IL-10 expression in lesions correlates with susceptibility to infection. Thus, IL-10 appears to inhibit aspects of CMI required for the effective elimination of intracellular pathogens.

There is strong evidence that dysregulation of IL-10 is associated with human allergic diseases. In addition, a number of studies have reported the production of IL-10 in association with malignant cell types including carcinomas of skin, breast, colon, kidney, bladder, ovary, and lung. The production of IL-10 by tumors has been implicated as one of the mechanisms that neoplastic cells use to evade the local immune response.

Another important defense mechanism triggered in phagocytes in response to pathogens is the induction of a direct antimicrobial responses. Phagocytic cells such as PMNs and macrophages recognize pathogens, engulf them, and induce antimicrobial effector mechanisms to kill the pathogens. PMNs generate oxygen-dependent or oxygen-independent killing. The release of toxic oxygen radicals, lysosomal enzymes, and antimicrobial peptides such as the human neutrophil defensins, leads to direct killing of the microbial organisms. Similarly, activation of Toll-like receptors on macrophages by microbial ligands upregulates iNOS, which results in rapid generation of NO and powerful microbicidal activity. Macrophages use this mechanism to contain some infectious organisms not susceptible to PMN attack, such as mycobacteria, certain fungi, and parasites. In addition, stimulation of monocytes by activated T cells also leads to the generation of NO.

Finally, Langerhans cells, monocytes, and macrophages present antigens to T and B lymphocytes, the adaptive immune cells. Upon pathogen recognition, monocytes are able to discriminate self from non-self molecules and foreign antigens are presented to lymphocytes for initiation of adaptive immune response.

Phagocytic cells of the innate immune system can also be activated by cells of the adaptive immune system. CD40 is a 50-kDa glycoprotein present on the surface of B cells, monocytes, dendritic cells, and endothelial cells, and its ligand is CD40L. CD40–CD40L interactions play a crucial role in the development of effector functions. CD40–CD40L interactions between T cells and macrophages play a role in maintenance of T H1 type cellular responses and mediation of inflammatory responses. Other studies have established a role for CD40–CD40L interactions in B cell activation, differentiation, and in Ig class switching. In addition, CD40–CD40L interaction leads to upregulation of B7.1 and B7.2 on B cells. This costimulatory activity induced on B cells then acts to amplify the response of the T cells. These mechanisms underscore the importance of the interplay between the innate and the adaptive immune system in generating an effective host response.

Thus, the innate immune response is a critical first line of defense. It is rapid, allowing for early detection of microbial pathogens and control of infection. Using germ-line–encoded receptors, the innate immune system distinguishes pathogens from self-antigens and activates appropriate effector mechanisms. Furthermore, the innate immune response controls the initiation of the adaptive immune response by regulating costimulatory molecules and releasing effector cytokines. In contrast, the adaptive immune response is delayed in onset, is characterized by highly specific receptors that are distributed clonally on subsets of particular cells, and involves immunologic memory. Not only do the innate and adaptive immune response complement each other, they are interactive in that the innate immune response influences the type of adaptive response and the adaptive immune response influences the function of innate cells.