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Department of Dermatology, IZKF Münster, and Boltzmann Institute for Cell and Immunobiology of the Skin, University of Münster, Münster, Germany; and Departments of Surgery and Physiology, University of California, San Francisco, California
ABSTRACT I. INTRODUCTION II. ANATOMY AND PHYSIOLOGY OF THE CUTANEOUS NERVOUS SYSTEM A. Neuroanatomy and Neurophysiology of Cutaneous Nerves B. The ''Skin-Sensory PNS-CNS Connection'' Exemplified by Itching C. Neuroanatomy and Neurophysiology of Autonomic Nerves III. BIOLOGICAL ACTIVITIES OF THE CUTANEOUS SENSORY NERVOUS SYSTEM A. Towards a Modern Concept of Neurogenic Inflammation B. Cutaneous Neuropeptides and Neuropeptide Receptor Biology 1. Tachykinins and neurokinin receptors 2. VIP 3. PACAP 4. VIP/ PACAP receptor family 5. CGRP 6. CGRP-like receptors 7. SST and receptors 8. Somatostatin receptors (sst) 9. Opioids, proopiomelanocortin (POMC) peptides, and receptors 10. POMC and endorphin peptides 11. beta-Endorphin 12. Enkephalins 13. Dynorphin 14. MSH 15. Melanocortin receptors 16. Cannabinoids and receptors 17. Cannabinoids are involved in T-cell regulation 18. CRH 19. CRH receptors 20. Secretoneurin 21. Other neurohormones and their receptors in the skin IV. ACETYLCHOLINE, CATECHOLAMINES, AND THEIR RECEPTORS A. ACh and Receptors 1. ACh receptors 2. Macrophages and the cholinergic anti-inflammatory pathway B. Catecholamines and Receptors 1. Catecholamines C. Adrenergic Receptors V. NEUROTROPHINS AND NEUROTROPHIN RECEPTORS A. Neurotrophins in the Skin B. NGF and NT Receptors C. NGF and Cutaneous Inflammation VI. ROLE OF CAPSAICIN AND TRANSIENT RECEPTOR POTENTIAL ION CHANNELS IN THE SKIN A. TRPV1 B. TRPV2 C. TRPV3 D. TRPV4 E. TRPM8 F. TRPA1 VII. ROLE OF PROTEINASE-ACTIVATED RECEPTORS IN CUTANEOUS NEUROGENIC INFLAMMATION AND PRURITUS VIII. CYTOKINES AND CHEMOKINES AS LIGANDS FOR SKIN SENSORY NERVES IX. MOLECULAR MECHANISMS REGULATING NEUROGENIC INFLAMMATION A. Synthesis, Posttranslational Processing, and Secretion of Neuropeptides B. Coexistence of Neurotransmitters C. Mechanisms Regulating Neuropeptide Receptor Function 1. Agonist removal by neuropeptide-degrading enzymes 2. Receptor desensitization and uncoupling of receptor from G proteins 3. Receptor endocytosis, trafficking, and recycling 4. Receptor downregulation 5. Decreased receptor synthesis X. ROLE OF THE NERVOUS SYSTEM IN SKIN PATHOPHYSIOLOGY A. Urticaria B. Psoriasis C. Atopic Dermatitis D. Immediate and Delayed-Type Hypersensitivity E. Wound Healing F. Pruritus XI. THERAPEUTIC APPROACHES FOR THE TREATMENT OF CUTANEOUS DISEASES WITH A NEUROINFLAMMATORY COMPONENT XII. CONCLUSIONS AND FUTURE DIRECTIONS GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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II. ANATOMY AND PHYSIOLOGY OF THE CUTANEOUS NERVOUS SYSTEM |
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The anatomy and classification of cutaneous sensory nerves has been extensively reviewed by Winkelmann (940). According to the classification of Halata, sensory nerves are based on two groups: the epidermal and the dermal skin-nerve organs. Both can be subdivided: the epidermal skin-nerve organs consist of "free" nerve endings or hederiform nerve organs (e.g., Merkel cells). The term free terminal nerve ending refers to a slight axon expansion that still contains perineural cells including cytoplasm of Schwann cells and multiple cell organelles (459, 881). In the dermal part, free sensory nerve endings, the hair nervous network (Pinkus discs), and the encapsulated endings [Ruffini, Meissner, Krause, Vater-Pacini (vibration), mucocutaneous end organ] have to be differentiated (Table 1). Neurophysiological studies have led to a more advanced functional classification of sensory nerves based on the type of cutaneous mechanoreceptor responses (Table 1).
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fibers (12–22 mm) are highly myelinated, show a fast conduction velocity (70–120 m/s), and are associated with muscular spindles and tendon organs. A
fibers are moderately myelinated (6–12 µm) and capture touch receptors. A
fibers constitute a thin myelin sheath (1–5 µm), an intermediate conduction velocity (4–30 m/s), and are generally polymodal. The slow-conducting C fibers (0.5–2 m/s) are unmyelinated and small (0.2–1.5 µm). A fibers constitute 
80% of primary sensory nerves sprouting from dorsal root ganglia, whereas C fibers make up 20% of the primary afferents (14, 470). Moreover, the activation threshold of A fibers is higher than that of C fibers.
In human peripheral nerves, 45% of the cutaneous afferent nerves belong to a subtype of sensory nerves that are mechano-heat responsive C fibers (C-m+h+) (729). However, only 13% of these nerves were found to be only mechanosensitive (C-m+), 6% were heat sensitive (C-h+), 24% were neither heat nor mechanoresponsive (C-m–h–), and 12% were of sympathetic origin; 58% of C-m+h+ responded to mustard oil, and 30% of C-m+ or C-m–h– did so (729).
Both C and A fibers respond to a variable range of stimuli such as physical (trauma, heat, cold, osmotic changes, distension or mechanical stimulation, ultraviolet light) as well as chemical (toxic agents, allergens, proteases, microbes) agents (reviewed in Ref. 811). However, although A fibers can also respond to chemical stimuli, their role in neurogenic inflammation and pruritus is still poorly understood.
On the molecular level, specific receptor distribution seems to be important for the various functions of sensory nerve subtypes. For example, mechanoreceptors exclusively express the T-type calcium channel Ca(v)3.2 in the dorsal root ganglion (DRG) of D-hair receptors. Pharmacological blockade indicates that this receptor is important for normal D-hair receptor excitability including mechanosensitivity (758). However, different mechanisms seem to underlie mechanosensory function in various tissues. In the gut and skin, for example, the degenerin/epithelial Na+ channel (DEG/ENaC) ion channel ASIC1 influences visceral but not skin mechanosensation (612).
Inflammation and trauma induce the activation and/or sensitization of nociceptors (769, 770). During chronic inflammation, pain, or pruritus, prolonged nociceptor activation may occur, thereby increasing the sensitivity of nociceptors which may lead to the perpetuation of neuronal stimulation and thus progression.
In the skin, cutaneous nerve fibers are principally sensory, with an additional complement of autonomic nerve fibers (114, 563). In contrast to sensory nerves, autonomic nerves never innervate the epidermis in mammals. Sensory nerves innervate the epidermis and dermis as well as the subcutaneous fatty tissue as a three-dimensional network (425, 881, 951). Most of the nerve fibers are found in the mid-dermis and the papillary dermis. The epidermis, blood vessels, and skin appendages such as hair follicles, sebaceous glands, sweat glands, and apocrine glands are innervated by several subtypes of sensory nerves (622, 811).
Regional-specific differences can be observed with respect to the mucocutaneous border, the glabrous skin, and hairy skin (940). With the use of electron microscopy (336), confocal laser scan microscopy (671), and immunohistochemistry (809), it is possible to demonstrate that the epidermis is also innervated by a three-dimensional network of unmyelinated C fibers with free-branching endings that arise in the dermis and their basement membrane apposed to epidermal cells such as keratinocytes, melanocytes, Langerhans cells, and Merkel cells, respectively. Increased epidermal innervation has been described in skin lesions of various inflammatory skin diseases (379, 383, 633, 640, 761, 809), wound repair (234), skin cancer (232, 447, 552, 567, 765), epithelial hyperplasia (702), after exposure to ultraviolet (UV) light, or during psoralen UVA therapy (525, 785).
B. The "Skin-Sensory PNS-CNS Connection" Exemplified by Itching
The skin is innervated by afferent somatic nerves with fine unmyelinated (C) or myelinated (A) primary afferent nerve fibers transmitting sensory stimuli (temperature changes, chemicals, inflammatory mediators, pH changes) via dorsal root ganglia and the spinal cord to specific areas of the CNS, resulting in the perception of pain, burning, burning pain, or itching (Table 1, Fig. 2) (see sect. IIB for details). Thus the skin "talks" to the brain via primary afferents thereby revealing information about the status of peripherally derived pain, pruritus, and local inflammation.
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Although pain and itch are different entities, a close relation exists between them. Both pain and itch can be reduced by soft rubbing which activates fast-conducting low-threshold fibers (117). However, the most characteristic response to itching is the scratch reflex: a more or less voluntary, often subconscious motoric activity, to counteract the itch by slightly painful stimuli. This itch reduction is based on a spinal antagonism between pain and itch-processing neurons (724). Thus itch appears to be under tonic inhibitory control of pain-related signals (21, 293, 724, 805). Indeed, itch and pain share the use of many neurophysiological tools and processing centers, and come along with similar autonomous skin reactions. Also, chronic pain and central sensitization to itch appear to be neurophysiologically closely related neurophysiological phenomena (71).
Neurophysiological recordings from the cat spinal cord support the concept of dedicated pruritoceptive neurons existing independently of pain fibers. Craig and Andrew (21) characterized a specialized class of mechanically insensitive, histamine-sensitive dorsal horn neurons projecting to the thalamus. Thus the combination of dedicated peripheral and central neurons with a unique response pattern to pruritogenic mediators and anatomically distinct projections to the thalamus provides the basis for a specialized neuronal itch pathway. The important role of the cutaneous PNS and CNS in the transmission of pain is reviewed elsewhere (397, 592).
C. Neuroanatomy and Neurophysiology of Autonomic Nerves
The anatomy of cutaneous autonomic nerves has been intensively reviewed by Brain (106). Autonomic nerve fibers in the skin almost completely derive from sympathetic (cholinergic) and, in the face, rarely parasympathetic (also cholinergic) neurons. Although very effective, they constitute only a minority of cutaneous nerve fibers compared with sensory nerves. Also in contrast to sensory nerve fibers, the distribution of autonomic nerves is restricted to the dermis, innervating blood vessels, arteriovenous anastomoses, lymphatic vessels, erector pili muscles, eccrine glands, apocrine glands, and hair follicles (902) (Figs. 1 and 2) (see sect. IIIB for details). Thus cutaneous autonomic nerves are involved in the regulation of blood circulation, lymphatic function, and the regulation of skin appendages (sweat glands, apocrine glands, hair follicles).
In general, cholinergic autonomic activity tends to be more pronounced in the dermis, although acetylcholine can be also produced by keratinocytes (155–157, 461). In addition, muscarinic and nicotinergic acetylcholine receptor expression has been described on keratinocytes in vivo and in vitro (285, 287–289).
Postganglionic autonomic nerves in the skin predominantly generate acetylcholine, although recent observations revealed an additional role for neuropeptides within the skin autonomic nervous system. Thus, in addition to classical neurotransmitters, autonomic nerves also release neuropeptides such as neuropeptide Y (NPY), galanin, calcitonin gene-related peptide (CGRP), or vasoactive intestinal polypeptide (VIP) (341, 536). Moreover, they generate neuromodulators such as tyrosine hydroxylase, which can be also used as a marker for autonomic nerves in the skin. Accordingly, immunoreactivity for NPY and atrial natriuretic peptide (ANP) (845) is only observed in autonomic nerve fibers, which differentiates them from sensory nerve fibers (73) (Table 2).
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Adult human sweat gland innervation, however, is not only cholinergic but coexpresses all of the proteins required for full noradrenergic function as well, including tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine -hydroxylase, and the vesicular monoamine transporter VMAT2. Thus cholinergic/noradrenergic cotransmission is apparently a unique feature of the primate autonomic sympathetic nervous system. Furthermore, sympathetic neurons innervating specifically the cutaneous arteriovenous anastomoses (Hoyer-Grosser organs) in humans also possess a full cholinergic/noradrenergic cophenotype (928).
Autonomic nerve fibers are also crucially involved in the regulation of vascular effects in the skin. Sympathetic nerve fibers release norepinephrine and/or NPY to innervate arterioles, arteriovenous anastomoses, and venous sinusoids which results in vasoconstriction, whereas parasympathetic nerves mediate vasodilatation through activation of venous sinusoides by the release of ACh and VIP/peptide histidine methionine (PHM) (5, 108, 407, 916). The occurrence of VIP within intradermal nerves is variable in different studies and appears to be species specific. The distribution of VIP, however, along with its ability to stimulate adenylate cyclase activity in vascular and glandular cells suggests an important role of VIP for the regulation of blood vessels as well as sweat gland function within the autonomic cutaneous nervous system (467, 743) (Table 2).
Effective heat exchange in the skin is controlled by terminal capillary loops that are regulated by shunt vessels, the arteriovenous anastomoses. Small arteries and arterioles as well as the arteriovenous anastomoses are richly supplied with noradrenergic nerves (316). The control of skin blood flow is maintained through two branches of the sympathic nervous system: a vasoconstrictor system and an active vasodilator system of unknown neurotransmitter. Previous studies suggest that this system is cholinergic and involves a cotransmitter, possibly VIP (52). Cholinergic sympathic nerves are also known to stimulate eccrine sweat glands via muscarinic receptors (106), whereas higher concentrations of acetylcholine induce an axon-reflex flare mediated via nicotinic receptors. In the vasoconstrictor system, the transmitter appears to be norepinephrine along with one or more cotransmitters.
The best characterized sympathic cotransmitters that participate in the regulation of blood flow include ATP (131) and NPY (818). NPY and norepinephrine were recently shown to be the major mediators of the reflex cutaneous vasoconstrictor response to body cooling. NPY acted mainly via the Y1 receptor and to a less extent via the Y2 receptor (160). Moreover, NPY was suggested to contribute to the nonnoradrenergic mechanism of reflex vasoconstriction (818). However, the role of NPY in the response to local cooling is subtle compared with its more pronounced role in the reflex responses to whole body cooling (391).
Local cooling stimulates cold-sensitive receptors that, in addition to conveying the thermal information centrally, also act on sympathetic vasoconstrictor nerves locally to stimulate release of norepinephrine to cause the initial vasoconstriction. This vasoconstriction masks a nonneuronal vasodilator response that may be present upon a more intense cooling (391, 632). Skin without intact sensory or autonomic function exhibits this vasodilator response, which is replaced by nonneurogenic vasoconstriction. The mechanisms for the nonneurogenic vasodilator and vasoconstrictor components of the response to direct cooling are unknown. In comparison, direct local warming of skin leads to vasodilation that involves nitric oxide (NO) and sensory nerves (424, 817) (Fig. 2, Table 2).
Both autonomic as well as sensory nerve fibers are reportedly involved in hair follicle cycling and inflammation (reviewed in Ref. 620). However, recent studies in denervated skin of C57BL/6 mice demonstrated that intact hair follicle innervation was not essential for anagen induction and development, although it had a minor modulatory role in depilation-induced hair growth (521). Various studies on the role of acetylcholinergic and adrenergic transmitters in cutaneous biology have been extensively reviewed elsewhere (680, 713, 769, 920, 933) (see also sect. IV, Fig. 3).
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III. BIOLOGICAL ACTIVITIES OF THE CUTANEOUS SENSORY NERVOUS SYSTEM |
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Stricker (823), and later Bayliss (47), described for the first time that cutaneous vasodilatation was achieved after stimulation of cut dorsal nerve roots. As described above, the identification and characterization of polymodal and chemosensitive small afferent nerve fibers (C and A nociceptors) provided evidence that cutaneous nerves may participate in skin inflammation. Thus neurogenic inflammation was found to be predominantly or exclusively mediated by afferent chemosensitive C nociceptors. The role of A fibers in skin inflammation and pain is still not understood. According to the "classical" concept of neurogenic inflammation, the mediators of the antidromic axon reflex were released from different specialized afferent nerve terminals and not from the sensory nerves themselves (48, 119, 479) (Fig. 3).
This classical concept could be extensively completed by using a vanilloid compound, capsaicin, which directly stimulated the sensory nerve. Capsaicin, the pungent ingredient of "hot" chili peppers has become an important topic for understanding neurogenic inflammation, pain, and pruritus in various tissues including the skin. Capsaicin applied to the skin produces a burning sensation that is abolished by cold and intensified by heat. Jancso et al. (372), and later Szolcsanyi (839), initially observed the phenomenon of "capsaicin desensitization," a long-lasting chemoanalgesia and impairment in thermoregulation against heat. The pharmacological properties of capsaicin in sensory-innervated tissues like the skin led to the hypothesis of an existing "capsaicin receptor" on polymodal C fibers (reviewed in Refs. 372–374, 839, 841). Hence, this view fostered a new way of understanding sensory nerves as peptidergic regulators of inflammation. Thus the capsaicin-sensitive sensory nervous system serves as a "dual afferent-efferent" sensor whereby initiation of afferent signals and neuropeptide release are coupled at the same nerve endings. A new highlight was the discovery of the "capsaicin receptor," a six-transmembrane temperature-gated ion channel, now defined as "transient receptor potential vanilloid 1" (TRPV1).
Although electric stimulation of capsaicin-insensitive afferent nerve fibers may result in pain or pruritus, it does not lead to inflammatory responses in normal skin underlining the specific role of capsaicin-chemosensitive C fibers in this process (840). The cutaneous flare response can be inhibited by prior treatment of the skin with topical capsaicin over several days because sensory nerves are depleted of neuropeptides (58, 144, 252). Thus capsaicin-sensitive C fibers and to a lesser extent A fibers are not only capable of transporting impulses to the CNS (orthodromic signal) but also releasing neuropeptides (antidromic signal) that result in inflammatory activities within the skin.
Neuropeptides released from cutaneous nerves act on target cells via a paracrine, juxtacrine, or endocrine pathway. These target cells express specific neuropeptide receptors that are appropriately coupled to an intracellular signal transduction pathway or ion channels, which, when activated, may result in activation of biological responses such as erythema, edema, hyperthermia, and pruritus. Because of their anatomical association to cutaneous nerves, mast cells and their released products appear to play an important role in mediating neuronal antidromic responses in the skin, although their precise role in cutaneous inflammation is not known. Because afferent sensory neurons express specific receptors for neuropeptides, prostaglandins, histamine, neurotrophins, opioids, proteases, cytokines, and immunoglobulins (19), an interactive communication network between sensory nerves and immune cells likely exists during cutaneous inflammation (103, 787). Finally, cell-associated neuropeptide-degrading peptidases such as neutral endopeptidase (NEP), angiotensin converting enzyme (ACE), or endothelin converting enzyme (ECE)-1 have been shown to modulate neurogenic inflammation by limiting the effects of neuropeptides in the skin (739, 740, 804). Thus the interaction between sensory nerves releasing neuropeptides, target cells with functional receptors, and neuropeptide-degrading peptidases is critical for determining neurogenic inflammation (Fig. 1). The roles of NO (457, 833), purinergic receptors (127, 133, 342, 733, 748), prostaglandin (102, 163, 400, 879) and leukotriene receptors (102, 701, 971), and voltage-gated ion channels (302, 318, 531, 553, 900) in the interaction with the skin nerval system have been extensively reviewed.
B. Cutaneous Neuropeptides and Neuropeptide Receptor Biology
With a few exceptions, neuropeptides consist of a group of small peptides of 4 or more than 40 amino acids that exert their effects by interacting with members of a superfamily of G protein-coupled receptors with seven transmembrane domains (GPCRs). Immunohistochemistry studies in the skin have demonstrated the presence of multiple neuropeptides, neurotransmitters, and neurohormones in sensory nerves including substance P (SP), neurokinin A (NKA) (180), neurotensin, CGRP, VIP, pituitary adenylate cyclase activating polypeptide (PACAP) (549, 809), peptide histidine-isoleucinamide (PHI), NPY (380), somatostatin (SST) (76), -endorphin, enkephalin, galanin, dynorphin, secretoneurin, ACh, epinephrine, norepinephrine (NE), - or 
-melanocyte-stimulating hormone (MSH) (386, 497), and corticotropin-releasing hormone (CRH) (64, 240, 743, 768, 769, 845). Colocalization of distinct neuropeptides can be observed in different tissues including the skin. For instance, sensory nerve fibers are immunoreactive for SP and CGRP (761), SP and PACAP (549), or CGRP and SST (76). However, the factors that determine the relative concentration of these neuropeptides in different nerve fibers of the skin are not well understood, although distinct regulatory functions of neuropeptide-neuropeptide interactions have been observed (33, 418).
Various neuropeptides are produced and released by a subpopulation of unmyelinated afferent neurons (C fibers) defined as C-polymodal nociceptors, which, as mentioned above, represent 70% of all cutaneous C fibers in the skin. To a lesser extent, small myelinated A fibers and autonomic nerve fibers are also capable of releasing a number of neuropeptides that also act on neuronal and nonneuronal target cells. Despite similarities in structure, a large variety of neuropeptides have been identified; some of them have been generated by posttranslational modifications of a precursor molecule. In addition, recently, cutaneous cells themselves such as keratinocytes, microvascular endothelial cells, Merkel cells, fibroblasts, or leukocytes were found to be capable of releasing neuropeptides under physiological circumstances (477, 919).
Dermal blood vessels are not only highly innervated by sensory and autonomic nerve fibers, but they also synthesize certain neuropeptides after activation and express receptors for neuropeptides, which suggests that a complex autocrine and paracrine neuroendocrine system may exist in the skin. Arterial sections of arteriovenous anastomoses, precapillary sphincters of metarterioles, arteries, and capillaries appear to be the most intensely innervated regions. Although sensory nerves are important for vasodilatation, neuropeptides from sympathetic neurons such as NPY mediate vasoconstriction supporting an important role for neuropeptides in vascular regulation. Both endothelial cells and smooth muscle cells respond to neuronal modulation during processes such as inflammation, cellular immune responses, neovascularization, and wound healing (for catecholamines, see Ref. 4).
This section summarizes our current knowledge about the role of crucial neuromediators, neurotrophins, and neurotransmitters as well as their receptors in modulating skin physiology and pathophysiology. The role of certain neuromediators in the skin has been comprehensively reviewed and is thus only mentioned under certain aspects (39, 137, 176, 177, 211, 437, 440, 667, 769, 770, 884, 920).
1. Tachykinins and neurokinin receptors
Tachykinins are small peptides consisting of 10–13 amino acids with a conserved COOH-terminal sequence (FXGLM) and different ionic charges at the NH2 terminus, the latter of which is crucial for receptor binding and affinity. In mammals, SP, NKA, and NKB, and the NKA-variants neuropeptide K (NP-K) and neuropeptide (NP-) are encoded by two distinct genes. Specific mRNA splice variants of the preprotachykinin A gene encodes SP, NKA, substance K, and NP-, whereas the preprotachykinin B gene encodes NKB (reviewed in Refs. 130, 510, 734).
Only recently, the novel SP-like peptide hemokinin-1 (HK-1) that is encoded by the preprotachykinin C gene was identified in mouse B cells and shown to be a potentially important regulator of B-cell development (959, 960). In humans, a homologous preprotachykinin C polypeptide was found to be expressed in a variety of tissues with strong signals detected in the skin. Binding and functional analysis indicated that human HK-1 peptides were nearly identical to SP in their overall activity profile on the three NK receptors with the most potent affinity for the NK1 receptor. The results indicate that preprotachykinin C encodes another high-affinity ligand of the NK1 receptor which may play an important role in mediating some of the physiological roles previously assigned to the NK1 receptor (460).
The expression of preprotachykinin A mRNA, SP, and NKA in cells of neuronal and nonneuronal origin have been shown to be regulated by certain proinflammatory mediators [interleukin (IL)-1, lipopolysaccharide (LPS)] and neurotrophins [nerve growth factor (NGF)], respectively (91, 255, 893). In human skin, a dense innervation with tachykinin-immunoreactive nerves in the upper and lower dermis, as well as epithelium, supports the capacity for these neuropeptides to participate in sensory nerve transmission as well as interaction with epidermal and dermal target cells (228, 230).
In the skin, tachykinin-immunoreactive sensory nerves are often associated with dermal blood vessels, mast cells, hair follicles, or epidermal cells (671). Increased epidermal SP-immunoreactive nerve fibers have been observed in certain inflammatory human skin diseases such as psoriasis, atopic dermatitis, and contact dermatitis (reviewed in Refs. 540, 811). Moreover, several immune cells are capable of generating SP induced by stress, inflammation, or infection (569, 570, 596). For example, SP appears to be involved in keratinocyte/antigen-presenting cell interactions during chronic stress (569), T-cell regulation (607), natural killer cell activation (485), innate host defense (116), human immunodeficiency virus (HIV)-associated psoriasis (338, 570), wound healing (189), murine hair follicle apoptosis (636), genital herpes infection (836), and immunosurveillance during experimentally induced tumor growth (murine melanoma) (509). SP may be also involved in inflammation and host responses of the CNS (511) as well as transmitting sensory signals (neurogenic inflammation, pain, pruritus) to the CNS (reviewed in Refs. 622, 805). In addition, in a murine disease model, the NK1 receptor was recently shown to play an important role in the development of airway inflammation and hyperresponsiveness (889).
SP released by sensory neurons after noxious stimuli provokes erythema, edema, and pruritus. Tumor necrosis factor (TNF)- release from human skin may be induced by SP via activation of the mitogen-activated protein kinase (MAPK) pathway (599). SP is also capable of mediating secretion of histamine and TNF- from mast cells, which results in vasodilatation via activation of H1 receptors on vascular smooth muscle cells (23, 170). SP also directly induces the release of cytokines such as TNF-, IL-1, IL-2, and IL-6 from rat leukocyte subpopulations (190). SP may also induce the release of leukotriene B4, and prostaglandin D2 from skin mast cells, suggesting that granulocyte infiltration mediated by LTB4 may be generated in response to SP (257). Another study (762) recently showed that acute immobilization stress triggers skin mast cell degranulation via SP, CRH, and neurotensin. This finding agrees with observations from other studies that found in vitro activation of murine DRG neurons by CGRP-mediated mucosal mast cell degranulation during acute immobilization stress in experimental murine cutaneous leishmaniasis (689). This effect was reduced when animals were treated neonatally with capsaicin to deplete their sensory neurons of their neuropeptides. Thus stress via release of certain neuropeptides may trigger degranulation of skin mast cells and influence certain inflammatory skin responses and pruritus by release of SP, CGRP, and other neuropeptides (860).
Neuropeptides are capable of activating dermal microvascular endothelial cells by binding high-affinity receptors. For example, SP can directly modulate proinflammatory biological activities of human dermal microvascular endothelial cells (HDMEC) (936), such as upregulation of cell adhesion molecules such as intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 (656, 657). In addition, intracutaneous SP and CGRP rapidly induce cutaneous neutrophilic and eosinophilic infiltration that is accompanied by translocation of P-selectin to luminal endothelial cell membranes and expression of E-selectin (775). SP can also induce a concentration-dependent induction of IL-8 in HDMEC (452, 736). Taken together, these results suggest an important direct effect for neuropeptides in modulating proinflammatory activities of endothelial cells in the skin.
SP and NKA are also capable of activating keratinocytes resulting in a number of proinflammatory cytokines (527, 619). For example, production of the proinflammatory cytokines IL-1, IL-1, and IL-8 as well as the IL-1 receptor antagonist in murine and human keratinocytes is upregulated by SP (118, 780, 903). SP is capable of directly activating both murine and normal human keratinocytes to induce IL-1 in a dose-dependent manner (23, 780, 831), suggesting a regulatory role of sensory nerve fibers that extend directly into the epidermis where they come in direct contact with both keratinocytes and Langerhans cells (181, 348). Interestingly, this effect can be inhibited by NK receptor antagonists. Recent findings suggest that SP may induce NF
B activation and interferon-induced protein of 10-kDa production in synergy with interferon- via neurokinin-1 receptor on keratinocytes. SP induction of murine keratinocyte PAM 212 IL-1 production is mediated by the neurokinin 2 receptor (NK-2R) (129, 738).
These effects of SP may be mediated via phospholipase C activation, intracellular Ca2+ signal, and reactive oxygen intermediates (415). Furthermore, during wound healing, SP may promote the healing process by affecting the expression of both epidermal growth factor and epidermal growth factor receptor in the granulation tissues as demonstrated in a rat model (464). In addition, keratinocyte NGF is induced by sensory nerve-derived neuropeptides such as SP and NKA (129). This direct effect of the neurosensory system on keratinocyte NGF production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing. Keratinocytes themselves have been reported to express preprotachykinin A mRNA and SP, indicating an autocrine induction of SP in human keratinocytes (37). Finally, SP may modulate cutaneous inflammatory responses by upregulation of cell adhesion molecule expression on keratinocytes (903).
In cultured normal human fibroblasts a moderate amount of preprotachykinin A was found, which was significantly upregulated by exogenous SP. Also the expression of NEP was increased in fibroblasts stimulated with SP (38). Accordingly, SP was found to promote human fibroblast chemotaxis in a dose-dependent manner (406). Moreover, SP fragments (from endopeptidase degradation) [SP-(1–4) and SP-(3–11)] were used to find that the chemoattractant potency of these fragments was due to the COOH terminus of SP which is known to be active on neurokinin receptors (904, 906). The involvement of the NK1R in the chemotactic response to SP was also indicated by fibroblast migration toward optimal concentration of a selective NK1R agonist but not a NK2R agonist, suggesting a NK1R-mediated role of SP on human fibroblast chemotaxis (406). SP also augments fibrogenic cytokine-induced fibroblast proliferation (417) and works synergistically with IL-1 and platelet-derived growth factor to stimulate the proliferation of bone marrow fibroblasts (661). SP was also shown to enhance dose-dependently the proliferation of fibroblasts derived from human normal skin. After 48 h of culture with SP, fibroblasts expressed significantly more transforming growth factor (TGF)-1 mRNA than unstimulated fibroblasts. The effects of SP on both fibroblast proliferation and TGF-1 mRNA expression could be antagonized by a selective NK1R antagonist, suggesting that SP may play an important role in phenotype changes of fibroblast proliferation. In cultured rheumatoid fibroblast-like synoviocytes, SP enhances cytokine-induced VCAM-1 expression in a dose-dependent manner, probably via NK1R activation. This finding favors a role for SP in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (465) and is supported by observations in human skin fibroblasts (614, 969). Furthermore, SP may be linked to wound healing via fibroblast activity. The cell surface enzyme NEP degrades SP, thereby regulating its biologic actions. In fact, it was shown that elevated NEP activity in the skin and chronic ulcers of subjects with diabetes combined with peripheral neuropathy may contribute to deficient neuroinflammatory signaling and impaired wound healing (25). The selective nonpeptide antagonist for NK1R [(+/–)CP 96,345] diminished the effects elicited by the NK1 selective agonist [Sar9]-SP-sulfone ([Sar9]-SP) on cellular transduction mechanisms in stable, cultured, human skin fibroblasts. The exposure of the cells to the agonist [Sar9]-SP produced an early increase in inositol 1,4,5-trisphosphate (IP3) levels and a later rise in cellular inositol 1-phosphate (IP1) content, whereas cAMP level was not significantly modified. The [Ca2+]i mobilization in response to the NK1 agonist produced a rapid increase in the intracellular Ca2+ level, indicating a concentration-dependent increase in both the ratio and the number of cells responding to [Sar9]-SP. These results clearly demonstrate that NK1R stimulation results in cellular transduction mechanisms in human skin fibroblasts. In human lung fibroblasts neuropeptides were shown to modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. NKA and SP stimulated human lung fibroblast proliferation, whereas VIP and CGRP had no such effect. NKA alone stimulated fibroblast chemotaxis, and phosphoramidon, a NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, which is potentially regulated, at least in part, by NEP activity (320). In summary, tachykinins may modulate inflammation in the skin by a direct effect of neurokinins on several target cells in normal and inflamed skin.
SP, NKA, and NKB bind with different affinities to neurokinin receptors (NKRs) that belong to the G protein-coupled receptor family (251, 669). Mast cells, fibroblasts, keratinocytes, Merkel cells, endothelial cells, and Langerhans cells (23, 241, 283, 339, 614, 780, 794) express functional NKR, albeit G proteins of mast cells can be additionally activated by SP in a non-receptor-mediated fashion (125, 126, 559, 560). To date, three neurokinin receptors and one splice variant have been cloned and characterized, all of which differ in their binding affinity to SP, NKA, and NKB.
Binding of SP, NKA, and NKB by NK receptors is primarily determined by the NH2-terminal portion of the tachykinin peptide, whereas the COOH terminus is essential for receptor desensitization (339, 904, 906). Several studies have identified transcriptional gene regulators mediated by SP, including NFB, NFAT (482, 655), cAMP responsive elements (CRE), and activator protein-1 (AP-1) (158). However, there seem to be species-specific differences in neurokinin receptor expression. For example, NK2R expression is significantly higher in murine keratinocytes (780), whereas NK1R is preferentially expressed on human keratinocytes (654). In summary, tachykinins may modulate inflammation in the skin by a direct effect of neurokinins on several target cells in normal and inflamed skin.
Previous studies using the tachykinin NK1R antagonist SR140333 indicate that cutaneous edema can be mediated by NK1Rs and is independent of histamine effects (613). This finding is supported by experiments using NK1R knockout mice, which showed that intradermally injected SP, NK1R agonists (GR-73632), and the mast cell-degranulating agent compound 48/80 induced dose-dependent cutaneous edema in wild-type mice that was lacking in knockout mice. Capsaicin and exogenous tachykinins induced edema formation, which was reduced by a histamine (H1) receptor antagonist (mepyramine), indicating that tachykinins are capable of mediating cutaneous edema formation via NK1R activities. Additionally, edema induced by tachykinins may be partially affected by NK1Rs on mast cells, since capsaicin- and SP-induced edema formation was reduced by the histamine H(1) antagonist mepyramine (140). Moreover, in vivo studies using neurokinin-1 receptor knockout mice have demonstrated that NK1R agonists are involved in modulating neutrophil accumulation in the inflamed, but not normal cutaneous microvasculature (740). A similar result was observed in a tissue culture model of human skin in which SP induced a dose-dependent edema, vasodilation, and extravasation of lymphocytes and mast cells through the microvascular wall and the release of proinflammatory mediators IL-1 and TNF- in vitro (113). SP may directly cause vasodilatation on vascular endothelial cells via NK1R (100). Recent studies show that SP-induced vasodilatation is partly mediated by NO, whereas CGRP-induced vasodilatation appears to be NO independent (436).
In a rat model, it was shown that tachykinin receptor antagonists exerted inhibitory effects on thermally induced inflammatory reactions (487) through both NK1R and NK2R. Thus SP and probably NKA contribute to inflammatory reactions after thermal injury and increase both local edema as well as the nociceptive transmission at the spinal cord level. Studies using a specific NK1R antagonist and a specific CGRP receptor antagonist [CGRP-(8–37)] in rats support the role that SP, NKA, and CGRP play in mediating antidromic vasodilatation in the skin (309, 487).
Whether neutrophil accumulation occurs after neuropeptide-mediated inflammatory responses is not known. Some authors failed to detect a role for the NK1R in cutaneous neutrophil recruitment (645), but others have shown that the number of neutrophils is reduced in NK1R knockout mice during contact hypersensitivity (CHS) (735) or SP inhibition (634).
In summary, various important target cells in the skin that participate in cutaneous inflammation express appropriate tachykinin receptors for released neurokinins by sensory fibers or immunocompetent cells, respectively. These findings strongly indicate that SP- and NKA-mediated activation of tachykinin receptors contribute to inflammatory reactions and that the tachykinin receptor antagonists can reduce both the local inflammatory response and the nociceptive transmission at the spinal cord level.
VIP is a 28-amino acid peptide that derived from a precursor mRNA (preproVIP) that also encodes histidine-methionine (PHM) (187, 280, 699). In the skin, VIP-like immunoreactivity was detected in nerve fibers associated with dermal vessels; glands such as sweat, apocrine, and Meibominan glands; hair follicles; and Merkel cells. VIP immunoreactivity was less abundant than SP immunoreactivity in the epidermal layer (322, 743). VIP-staining fibers can be also found in close anatomical connection to mast cells (299, 323, 571). VIP immunoreactivity can be detected in various immunocompetent cells in different species, and VIP is an important molecule within the "neuroimmunological network" (186, 199, 269, 272, 652) (Table 2).
In the skin, functional studies with VIP found that this peptide may also mediate vasodilatation (52, 938) and proliferation (312, 943) as well as induce migration of keratinocytes that may be important in wound healing (943) and psoriasis (154, 379, 408, 572, 615, 641, 832).
Moreover, VIP reportedly regulates sweat production and accumulation of intracellular cAMP (229, 230, 703). However, VIP was not only identified as a physiologically active neuropeptide and neurotransmitter, but is further involved in neurogenic inflammation possibly through histamine release from mast cells and bradykinin-induced edema (17, 922). VIP may also play a role during infection. For example, antibodies against VIP were found in patients with HIV and were more prevalent in asymptomatic carriers, i.e., their titer correlated with disease progression (894).
VIP-induced stimulation of histamine release may lead to subsequent vasodilatation and increased plasma extravasation, suggesting a direct effect of VIP on the regulation of blood vessel function (52, 774). For example, VIP may cause direct vasodilatation by inducing NO synthesis, which results in vasorelaxation (270). The migration of monocytes from blood vessels into the inflammatory tissue is also increased by VIP. However, the molecular mechanisms and the receptors involved in this process are still unclear. Although VIP1-R mRNA was detected almost exclusively in endothelial cells with radioactive in situ hybridization, the VIP2-R could be seen in endothelial as well as smooth muscle cells (887). Immunohistochemical studies with affinity-purified polyclonal antibodies confirm this observation in human skin tissue (759).
It is well established that VIP is involved in neuroimmunomodulation (199). This cytokine-like peptide exerts a broad spectrum of anti-inflammatory effects in mammals including humans (272). In murine T cells, VIP has the capacity to modulate CD4+CD25+ Foxp3-expressing regulatory T cells in vivo. Application of VIP into T-cell receptor transgenic mice resulted in the expansion of T cells that inhibited the responder T-cell proliferation, increased the level of CD4+CD25+ Treg cells, inhibited delayed-type hypersensitivity in TCR-Tg hosts, and prevented graft-versus-host disease in vivo (192).
In human T cells and T-cell lines, VIP modulates IL-2 secretion (296) and induces Th2 responses by promoting Th2 differentiation and survival. Interestingly, VIP modulates the upregulation of granzyme B, FasL, and perforin in Th2 but not Th1 cells, thereby regulating Th2 cell survival by preventing apoptosis (749). VIP seems to be directly involved in regulating dendritic cell (DC)/T-cell interactions. VIP induced the generation of tolerogenic DCs, thereby producing CD4 and CD8 Treg cells (271). Moreover, VIP induced upregulation of IL-10 in human DCs in vitro. Thus VIP may be involved in regulating Th1 cell responses and may be an effective compound for the treatment of autoimmune diseases. Finally, VIP inhibits antigen-induced apoptosis of mature T lymphocytes by suppressing Fas ligand expression (193). Together, these results strongly support a regulatory immunosuppressive role of VIP on T cells and DCs by downregulating TNF-, IL-1, IL-6, and NO (115, 196), while stimulating the release of IL-10, for example.
In macrophages, VIP and PACAP protect mice from lethal endotoxemia through the inhibition of TNF- and IL-6, suggesting a protective role of both neuropeptides in innate immunity (197) by downregulating TNF- production (200). VIP and PACAP inhibited TNF- activation via regulating NFB and cAMP response element-binding protein (CREB)/c-jun, a cAMP-dependent pathway that increases CREB binding versus c-jun binding to the cAMP response element-binding site (CRE), and a cAMP-independent pathway that inhibits binding of NFB (194, 198, 473). VIP is also involved in modulating innate immunity by downregulating TLR4 expression and TLR4-mediated chemokine generation (CCL2, CXCL8) (306).
Animal studies clearly indicate a role for VIP in modulating inflammatory diseases in vivo. For example, VIP regulates CD4+CD25+ Treg cells during experimental autoimmune encephalomyelitis and T cells as well as DCs during contact hypersensitivity and arthritis (191, 441, 892). In neutrophils and macrophages, VIP regulates the outcome of animal survival during sepsis (195, 197, 515). Together, these findings suggest an important protective role of VIP in T cell-mediated diseases as well as innate immunity and host defense.
PACAP is a relatively new member of the VIP/secretin peptide family (542). Two forms can be distinguished, PACAP-38 and a truncated product PACAP-27, both of which are derived from a 176 precursor protein (19.5 kDa) by posttranslational cleavage (349). The mature peptide has a molecular mass of 5 kDa. PACAP has been localized in nerve fibers of different tissues, including skin (Table 2), from a number of species as well as in lymphoid tissues of the rat and lymphocytes from the peripheral blood (reviewed in Ref. 27).
PACAP is present in sensory and autonomic nerve fibers of dorsal root ganglia, the spinal cord, and the adrenal glands, suggesting involvement in sensory and nociceptive pathways (223, 549). Moreover, PACAP-immunoreactive fibers are sensitive to capsaicin (549). In various organs of rodents and humans, PACAP displays neuroprotective, regenerative, and immunomodulatory functions. In SCID mice, CD4+ T cells appear to induce PACAP gene expression, suggesting a regulatory role of immune cells on PACAP-induced immunomodulation and nerve regeneration (28).
In the skin, PACAP was detected in sensory nerve fibers (597, 809) coexisting with VIP, SP, or CGRP, respectively, all of which may play an important role in inflammatory skin diseases like psoriasis, urticaria, or atopic dermatitis (643) (reviewed in Ref. 24). In rat skin epithelium, PACAP has been detected especially within highly innervated structures like the tongue and the nose (549).
The distribution of PACAP-38 (597, 809) and the presence of the high-affinity PACAP1 receptor (PAC1R) (809) was described in normal and inflamed human skin. The concentration of PACAP-38 appears to be enhanced in lesional skin of psoriasis patients, indicating that this neuropeptide has a role in the pathophysiology of this skin disease (809). Moreover, the peptide level was significantly lower in nonlesional psoriatic skin than in lesional psoriatic skin, but was about twice as much as in normal human skin. Interestingly, immunoreactivity was significantly increased at the dermal-epidermal border in psoriasis (809). Further immunoreactivity was localized between connective tissue, around hair follicles, and close to sweat glands of normal skin. In contrast, no significant increase of positive nerve fibers was observed around blood vessels. PACAP appears to be involved in cutaneous inflammation, e.g., by releasing histamine from mast cells (597).
Several observations support the idea that PACAP modulates inflammatory responses in the skin: PACAP-27 produces a long-lasting depression of a C fiber-evoked flexion reflex in rats (961), indicating that PACAP plays an essential role in nociceptive transmission in the skin (394). Moreover, PACAP is a potent vasodilatator and edema potentiator in rabbit skin (921) and mediates plasma extravasation in rat skin (142, 730). From these data one may speculate that C fibers release PACAP in response to activation by a currently unknown stimulus that leads to vasodilatation and extravasation. Additionally, in a rodent model, VPAC1 and VPAC2 receptors play an important role in pressure-induced vasodilatation, suggesting a protective feature against applied pressure (247).
Recent findings suggest that PACAP stimulates histamine release from murine mast cells via direct stimulation of G proteins (730, 746). Using mast cell-deficient mice with or without transplantation of mast cells, Schmidt-Choudhury et al. (732) were able to show that intracutaneously injected PACAP produces a long-lasting, partially mast cell-dependent edema compared with mast cell-deficient mice, supporting a close interaction of PACAP-positive nerve fibers and mast cell regulation of murine skin. In humans, intravenous injection of PACAP leads to a long-lasting flush phenomenon. Thus PACAP may have a vasodilatory function in human skin that may also contribute to neurogenic inflammation.
Recent observations indicate that PACAP is also involved in immunomodulation. In murine T cells, PACAP can downregulate IL-2 and inhibit IL-10 expression (512) and IL-6 production; in murine peritoneal macrophages, PACAP can inhibit secretion (513, 514). These findings were recently confirmed in a study in which PACAP inhibited the induction of contact hypersensitivity by reducing murine LC antigen-presenting cell (primary and XS106 cell line) properties (441). Additionally, PACAP inhibits the LPS/granulocyte-macrophage colony stimulating factor (GM-CSF)-induced stimulation of IL-1 and augments IL-10, presumably by modulation of cytokine production (442). VIP and PACAP both inhibit the LPS-stimulated production of TNF- via VPAC1R and activation of the adenylate cyclase system in vitro and in vivo, suggesting a protective role for VIP and PACAP regulating the release of TNF- during inflammation (194, 200). Finally, PACAP and VIP via VPAC1R inhibit TNF- production at a transcriptional level in murine macrophages through two pathways, a cAMP-dependent pathway that increases CREB binding versus c-jun binding to the CRE, and a cAMP-independent pathway that inhibits binding of NFB (194, 198, 473). Finally, VIP and PACAP inhibit antigen-induced apoptosis of mature T lymphocytes by inhibiting Fas ligand expression (193). These results strongly support a regulatory role of PACAP and VIP for proinflammatory molecules such as TNF-, IL-1, IL-6, and NO (115, 196).
In this context, Delgado and co-workers (192, 193) recently showed that PACAP itself also regulates human T-cell function. Human DCs express receptors for PACAP and VIP, predominantly VPAC1R. Interestingly, PACAP exhibits a diverse role of action depending on the status of the inflammatory response. During an ongoing inflammation, PACAP drives T cells into an anti-inflammatory response (downregulation of proinflammatory cytokines), whereas in an ongoing immune response, PACAP upregulates CD86 expression on DCs thereby stimulating T-cell proliferation and differentiation into Th2 helper cells (201).
In summary, these results suggest an important role of PACAP during inflammation and neurotransmission within the neurocutaneous network.
So far, three different VIP/PACAP receptors (PVRs) with additional splicing products have been cloned (reviewed in Ref. 27) that were recently defined as PAC1-R (=PACAP1-R), VPAC1-R (=PACAP2-R = VIP1-R), and VPAC2-R (=PACAP3-R = VIP2-R). Since VIP and PACAP are capable of binding identical receptors in the same tissue but with different affinities (PAC1-R = high-affinity receptor for PACAP; VPAC1-R = low-affinity receptor for PACAP and high-affinity receptor for VIP; VPAC2-R = low-affinity receptor for VIP and PACAP), a differential fine-tuned interaction between these two peptides can be suggested. The PACAP/VIP receptor family can be found in several species. In humans, all three receptor subtypes can be detected in different peripheral organs, and the human high-affinity PAC1-R consists of at least five splice variants (648). PAC1-R, for example, has a significantly higher affinity for PACAP than VIP (207). Immunohistochemical and biochemical studies support a regulatory role of PVRs in skin inflammation. Binding sites for VIP have been identified on a variety of cells including sweat glands (329
