Modern Aspects of Cutaneous Neurogenic Inflammation FREE

Afferent somatic nerves with fine unmyelinated (C-) or myelinated (Aδ-) fibers derive from dorsal root ganglia and innervate the skin. Both types of fibers respond to a range of physiologic stimuli such as physical trauma (heat, cold, nociception, mechanical distension, and UV light) and even low-intensity mechanical stimulation. Furthermore, specialized nociceptor fibers can be activated by a wide range of chemicals, which indicates that not only physical but also chemical stimuli are capable of activating the cutaneous nervous system. Moreover, biological factors such as microbiological agents and proteases from plants (eg, papain and trypsins) are capable of stimulating sensory nerves in the skin. On stimulation, the nerves rapidly release active neuropeptides into the microenvironment. A short graphic summary of these factors appears at the end of this article. In addition to these exogenous trigger factors, endogenous stimuli such as like protons (pH changes), hormones, cytokines, proteinases, kinins, and probably other undefined mediators released by various cells involved in inflammatory processes induce activation of primary afferent neurons in many organs, including the skin (Figure 1).

Autonomic nerve fibers represent only a minority of cutaneous fibers that generate predominantly neurotransmitters such as acetylcholine and catecholamines. Autonomic nerves may also generate neuropeptides such as neuropeptide Y, calcitonin gene-related peptide (CGRP), atrial natriuretic peptide, and vasoactive intestinal peptide (VIP). Neuropeptides may be involved in the regulation of sweat gland function and vasoregulation.^1- 2 Recent findings suggest that classic autonomic neurotransmitters can also be generated by nonneuronal cells such as keratinocytes. In addition, muscarinic and nicotinergic acetylcholine receptor expression has been described on keratinocytes and Langerhans cells,^3- 4 which indicates a role of autonomic neuromediators in inflammation, epidermal function, and hair growth.^5- 8

A century ago, cutaneous vasodilatation was achieved after stimulation of cut dorsal nerve roots. Later, the identification and characterization of polymodal and chemosensitive small nerve fibers (C- and Aδ-nociceptors) explained how nerves may participate in cutaneous inflammation.⁹ The cutaneous flare response can be inhibited by prior treatment of the skin with topical capsaicin over several days. After such treatment, sensory nerves will be depleted of neuropeptides. After their release, neuropeptides act on target cells via a paracrine, juxtacrine, or endocrine pathway resulting in erythema, edema, hyperthermia, and pruritus. Because of their anatomic association with cutaneous nerves, mast cells and their released products appear to play an important role in mediating neuronal antidromic responses in the skin, although the precise role of these cells in cutaneous inflammation remains to be determined.

In contrast, afferent nerves express specific receptors for neuropeptides, prostaglandins, histamine, neurotrophins, proteases, vanilloids, and cytokines. This would indicate that an interactive communication network between sensory nerves and immune cells likely exists during cutaneous inflammation.

Finally, most cells expressing receptors for neuropeptides also generate neuropeptide-degrading peptidases such as neutral endopeptidase (NEP) or angiotensin-converting enzyme (ACE), thereby terminating the inflammatory stimulus by neuropeptides. Similarly, cells synthesizing receptors for the neurotransmitters acetylcholine and noradrenaline also generate enzymes that control the effects of these molecules. Thus, a close interaction between neuromediators, target cell receptors, and neuropeptide-degrading enzymes is critical for controlling cutaneous neurogenic inflammation.

Neuropeptides are a group of small peptides with 4 to more than 40 amino acids. More than 20 neuropeptides have been identified and more or less characterized in the skin of various species. However, regarding the function of a neuropeptide, organ- and species-specific effects must be considered in the interpretation of the observed effects. The most recognized neuropeptides in the skin are substance P (SP), neurokinin (NK) A, neurotensin, CGRP, VIP, pituitary adenylate cyclase activating polypeptide (PACAP), neuropeptide Y, somatostatin (SOM), β-endorphin, enkephalin, galanin, dynorphin, atrial natriuretic peptide, α- or γ-melanocyte-stimulating hormone (MSH), parathyroid hormone–related protein, corticotropin-releasing hormone, and urocortin (Table 1). Under physiologic circumstances, cutaneous cells such as keratinocytes, microvascular endothelial cells, Merkel cells, fibroblasts, and leukocytes are also capable of releasing neuropeptides. Herein, we report our findings on some of these neuropeptides, while others are elucidated elsewhere.^{7- 8,10- 15}

Tachykinins are small peptides consisting of 10 to 13 amino acids. In the skin, SP effects have been described on keratinocytes including hair follicles, mast cells, fibroblasts, and endothelial cells. Increased epidermal SP-immunoreactive nerve fibers have been observed in certain inflammatory human skin diseases.¹⁶ The expression of tachykinins can be regulated by proinflammatory mediators (interleukin [IL] 1 and lipopolysaccharide) and neurotrophins (nerve growth factor [NGF]).^17- 18

Release of SP induces vascular responses and pruritus. For example, SP stimulates release of tumor necrosis factor α, histamine, prostaglandin D₂, and leukotriene B₄ from skin mast cells.¹⁹ Acute immobilization stress induces skin mast cell degranulation via SP, corticotropin-releasing hormone, and neurotensin release, which suggests that stress by activation of certain neuropeptides may trigger mast cell degranulation and thereby modulate cutaneous neurogenic inflammation and pruritus.²⁰

Neuropeptides are also capable of activating human dermal microvascular endothelial cells in vivo and in vitro.²¹ Substance P induces up-regulation of cell adhesion molecules such as P-selectin, intercellular adhesion molecule (ICAM) 1, and vascular cell adhesion molecule (VCAM) 1²²; recruitment of neutrophils and eosinophils; and the release of chemokines such as IL-8. Together, these results suggest an important direct effect of SP for leukocyte-endothelial interactions in the skin.²³

In keratinocytes, SP and NK-A stimulate production of the proinflammatory cytokines IL-1α, IL-1β, and IL-8 as well as the IL-1 receptor antagonist.^24- 25 Up-regulation of keratinocyte cell adhesion molecule expression (ICAM-1) can be also modulated by SP.²⁶

In fibroblasts, SP promotes chemotaxis²⁷ via activation of the neurokinin-1 receptor (NK-1R). By augmenting cytokine-induced fibroblast proliferation and VCAM-1 expression, SP may be involved in the pathophysiology of autoimmune diseases and fibrosis.²⁸ Transcription factors such as nuclear transcription factor κB (NF-κB), nuclear factor of activated T cells, and AP-1,²⁹ which are crucially involved in inflammation, can be regulated via tachykinins.

Substance P, NK-A, and NK-B bind with different affinities to 3 potential neurokinin receptors. Keratinocytes, Langerhans cells, Merkel cells, fibroblasts, mast cells, and endothelial cells express functional neurokinin receptors, while G proteins of mast cells can be additionally activated by SP in a nonreceptor-mediated fashion.³⁰ To date, 3 neurokinin receptors have been cloned and characterized that differ in their binding affinity to SP, NK-A, and NK-B. Expression of NK-2R is significantly higher in murine keratinocytes,²⁵ while NK-1R is preferentially expressed on human keratinocytes.²⁹ Whether dysregulation of receptor activation or termination (polymorphisms and/or signal transduction) actively contributes to cutaneous diseases is currently unknown. However, NK-1R gene–deficient mice do not show a specific skin-related phenotype under normal conditions but instead show characteristic signs of edema and plasma extravasation.³¹ No NK-1R–mediated effects were observed in an experimentally induced wound-healing model.³² Additionally, previous studies have demonstrated that neurokinin receptor antagonists may be beneficial substrates for the treatment of inflammatory skin diseases.^33- 35

Vasoactive intestinal peptide is a 28–amino acid peptide detected in nerve fibers associated with dermal vessels, sweat, apocrine, and meibomian glands, hair follicles, and Merkel cells. Fibers staining for VIP can also be found in close anatomic connection to mast cells and sweat glands.³⁶ In lesional skin of patients with psoriasis VIP was increased, whereas the concentration of this neuropeptide was diminished in atopic dermatitis.³⁷ Accordingly, VIP directly inhibited experimentally induced contact dermatitis in humans.^38- 40

Vasoactive intestinal peptide mediates vasodilatation by inducing nitric oxide synthesis⁴¹ and keratinocyte proliferation and migration⁴²; induces sweat production⁴³; and may be an important transmitter during neurogenic inflammation possibly by inducing histamine release from mast cells.⁴⁴ Similar to tachykinins, the distribution of the neuropeptide receptor subfamilies varies within the epidermal and dermal compartment. While VPAC-1R (vasoactive intestinal polypeptide/pituitary adenylate cyclase activating polypeptide 1 receptor) appears to be the predominant receptor on human dermal microvascular endothelial cells, keratinocytes predominantly express VPAC-2R.⁴⁵

Pituitary adenylate cyclase activating polypeptide is a novel member belonging to the VIP family. Two forms can be distinguished: PACAP-38 and a truncated product, PACAP-27.⁴⁶ While PACAP has been localized in cutanous nerve fibers of rodents and humans where it is involved in sensory, nociceptive, and probably autonomic pathways,⁴⁷ the highest immunoreactivity was found around blood vessels, hair follicles, and close to sweat glands.

Pituitary adenylate cyclase activating polypeptide modulates inflammatory responses in the skin: PACAP-27 produces a long-lasting depression of a C-fiber–evoked flexion reflex in rats, potentiates vasodilatation and edema, and mediates plasma extravasation in the skin.^48- 49 These effects may be indirectly mediated via mast cells.⁵⁰ Recently, the distribution of PACAP-38 was described in human skin, although its role is still uncertain.^51- 52 Additionally, enhanced concentrations of PACAP-38 were measured in lesional skin of patients with psoriasis.⁵²

Three different VIP/PACAP receptors (PAC-Rs) with additional splicing products have been cloned⁵³ and defined as PAC-1R, VPAC-1R (or VIP-1R), and VPAC-2R (or VIP-2R). Since VIP and PACAP are capable of binding identical receptors in the same tissue but with different affinities, a differential fine-tuned interaction between these 2 peptides is suggested. The PACAP/VIP receptor family can be found in several species. In humans, all 3 receptor subtypes can be detected in different peripheral organs. Receptor VPAC-1R was detected in endothelial cells, VPAC-1R and VPAC-2R in smooth muscle cells, and VPAC-1R in keratinocytes.^54- 55

Recent observations indicate that PACAP and VPAC-Rs are involved in the modulation of immune cells. In T cells or macrophages, PACAP down-regulates IL-2 and IL-6 production and inhibits IL-10 expression.⁵⁶ Vasoactive intestinal peptide and PACAP both inhibit the lipopolysaccharide-stimulated production of tumor necrosis factor α via VPAC-1R.^57- 58 Recently, an anti-inflammatory role of both VIP and PACAP was demonstrated in the chronic inflammatory disease rheumatoid arthritis.^59- 60 Thus, PACAP may have a proinflammatory effect on endothelial cells during acute inflammation, yet it exerts anti-inflammatory effects under chronic inflammatoy conditions.

Calcitonin gene-related peptide is one of the most prominent neuropeptides of the skin and is often associated with mast cells, Merkel cells, melanocytes, keratinocytes, and Langerhans cells. It is capable of increasing keratinocyte proliferation⁶¹ and regulating cytokine production in human keratinocytes, and it exerts its effects predominantly on arterioles, resulting in vasodilatation.⁶² In general, CGRP predominantly mediates anti-inflammatory and neurotrophic effects.⁶³ Under certain circumstances, CGRP enhanced the accumulation of neutrophils and edema formation induced by IL-1.^64- 65

Calcitonin gene-related peptide receptors can be subdivided into 2 classes: CGRP-1 and CGRP-2. Both receptor subtypes can be differentiated by their pharmacologic characteristics. Recently, the calcitonin gene–like receptor was localized in human skin.⁶⁶ Additionally, a family of CGRP receptor activity–modifying proteins have been identified in humans.⁶⁷ The fact that these proteins are differently regulated during different disease states indicates a regulatory role for them and calcitonin gene-related–like receptors in tissue pathophysiology.⁶⁸

Pro-opiomelanocortin (POMC) peptides are expressed by melanocytes, keratinocytes, microvascular endothelial cells, Langerhans cells, mast cells, and fibroblasts as well as by immune cells such as monocytes and macrophages. The POMC gene includes several bioactive peptides such as adrenocorticotrophic hormone, β-lipotropin, α-, β-, and γ-MSH, and β-endorphin. After cleavage by prohormone convertase 1 and 2, a POMC prohormone generates up to 8 different POMC peptides. These enzymes appear to be crucial for the tissue specificity of POMC-derived peptides.

Several studies have demonstrated a direct immunoregulatory and anti-inflammatory role of α-MSH in the skin in vivo and cutaneous cells in vitro. For example, α-MSH antagonizes the effects of proinflammatory cytokines such as IL-1α, IL-1β, IL-6, and tumor necrosis factor α or endotoxins^69- 70 and down-regulates the production of proinflammatory cytokines and accessory molecules on antigen-presenting cells, while production of suppressor factors such as IL-10 is up-regulated by α-MSH.⁷¹

α-Melanocyte-stimulating hormone up-regulates the expression of matrix metalloproteinase 1⁷² and induces IL-8 release in cultured fibroblasts,⁷³ which indicates a role of this neuropetide during dermal inflammatory processes. Moreover, fibroblast-derived β-endorphin induces histamine release, which demonstrates a possible interaction of fibroblasts and mast cells via POMC peptides.⁷⁴

Inflammatory mediators such as UV light or IL-1β up-regulate POMC messenger RNA in dermal endothelial cells.⁷⁵ Melanocortin 1 receptor (MC-1R) expression was also increased in these cells on stimulation with IL-1β. α-Melanocyte-stimulating hormone induces increased levels of the chemokines IL-8 and Gro-α (growth-related oncogene-α) in human dermal microvascular endothelial cells via MC-1R.⁶⁹ Some of the anti-inflammatory properties of α-MSH appear to be mediated by down-regulating NF-κB.^76- 78

Proopiomelanocortin peptides exert their effects via 5 subtypes of heterodimeric G protein–coupled melanocortin receptors (MC-1R–MC-5R) with 7 transmembrane domains: endothelial cells, fibroblasts, keratinocytes, monocytes, and melanocytes express MC-1R, while MC-2R and MC-5R are synthesized in muscle cells and adipocytes.⁷⁹

Acetylcholine, epinephrine, and norepinephrine are small nonpeptide messengers produced by neurons and non-neuronal cells of various tissues including the skin.⁷ Originally, these molecules were described as important mediators released from autonomic nerve fibers. Recent studies suggest that adrenergic and cholinergic transmitters play important roles in cutaneous homeostasis and inflammation.^3,6 For example, catecholamines down-regulate the capability of antigen presentation in murine Langerhans cells via activation of β₂ adrenergic receptors on these cells.⁴ Epinephrine also inhibits the induction of contact hypersensitivity to epicutaneously administered haptens in vivo, which dindicates a direct role of autonomic nerves in cutaneous immunomodulation.

The mammalian skin expresses a variety of neurotrophic growth factors that are essential for growth, proliferation, and maintenance of nerves such as NGF, brain-derived NGF, neurotrophin-3, and neurotrophin-4/5. Cutaneous neurotrophins are expressed by sensory and sympathetic neurons and non-neuronal cells thereby regulating various biological modalities such as nociception, proprioception, mechanoreception, nerve growth and development, apoptosis, epidermal homeostasis, inflammation, hair growth, and melanogenesis.⁸⁰ Several observations suggest that neurotrophins participate in the neuroimmunologic network, eg, expression of NGF and neurotrophins 3, 4, and 5 can be induced by cytokines such as IL-6 and soluble IL-6 receptor.⁸¹

During inflammation, NGF is markedly up-regulated in nerves associated with the inflamed area,⁸² and NGF levels are increased in inflammatory skin diseases such as psoriasis.⁸³ Nerve growth factor also directly stimulates degranulation of mast cells, increases the number of mast cells, modulates B-cell function, and enhances histamine release from basophils. It is also capable of suppressing leukotriene C₄ production in human eosinophils.⁸⁴

Clinically, topically applied capsaicin, a vanillyl-alkaloid found in red hot chili peppers, elicits a rapid sensation of burning pain by selectively activating small-diameter sensory neurons and triggering neurogenic inflammation.¹⁵ Long-term application of capsaicin leads to neurotoxic effects in sensory nerves and causes the termination of the inflammatory response, an effect that is used in the treatment of chronic inflammatory diseases.

Transient receptor potentials (TRPs) are a novel family of temperature-sensitive receptors with 6 transmembrane domains. These are nonselective cation channels that are structurally related to other members of store-operated calcium channels. Caterina et al⁸⁵ recently described the successful molecular cloning of the rat capsaicin receptor (TRP vanilloid receptor 1 [TRP-V1]). Low pH levels that accompany inflammatory responses can increase the response of TRP-V1 to noxious stimuli, which suggests that the response of sensory nerve fibers during neurogenic inflammation results, at least in part, from activation of vanilloid receptors through an excess of protons, endogenous cannabinoids, and probably bradykinin.⁸⁶ Recent data support a role of TRP-V1 in neurogenic inflammation.⁸⁷ However, the role of TRP-Vs in cutaneous neurogenic inflammation is still unclear.

Recent data strongly indicate that serine proteinases such as thrombin, cathepsin G, tryptase, and trypsin are not merely enzymes that degrade proteins and peptides in the extracellular space; they are also capable of mediating important effects during inflammation and immune response such as cytokine release, cell migration, recruitment of leukocytes, and endothelial cell activation.^88- 89 These processes are at least in part mediated by cleavage and activation of proteinase-activated receptors (PARs), which are G protein–coupled receptors with 7 transmembrane domains activated by proteolytic cleavage. So far, 4 PARs have been cloned and characterized.⁹⁰ Activation of PARs results in the activation of signal transduction pathways ultimately involved in inflammatory signalling such as NF-κB, mitogen-activated protein kinases, and protein kinase C.

Recent data suggest that PAR-1 and PAR-2 are involved in neurogenic inflammation,^91- 92 indicating that proteinases may activate PARs on sensory neurons to stimulate release of CGRP and SP, which mediate the inflammatory response. This is supported by several findings that natural and synthetic PAR agonists often mimic inflammatory effects of neuropeptides.⁹³ For example, tryptase also induces plasma extravasation⁹⁴ and neutrophil infiltration^89,91 and stimulates cytokine secretion.⁹⁵ Tryptase-releasing mast cells can be found in close proximity to PAR-2–expressing cells such as keratinocytes and dermal endothelial cells⁹⁶ or C-fibers during inflammation^97- 98 (Figure 1). Recent studies using PAR-2–deficient mice demonstrate that PAR-2 plays an important role in cutaneous inflammation in vivo, probably by stimulating nitric oxide release.^91,99 This is supported by findings in human skin tissues showing that PAR-2 agonists modulate plasma extravasation, edema, leukocyte recruitment, up-regulation of cell adhesion molecules on dermal microvascular endothelial cells, and pruritus.^91,99- 100 Addtionally, PAR-2 is an inducer of the transcription factor NF-κB, a factor that has been implicated in the pathophysiology of atopic dermatitis.^101- 102

The physiologic control of cell responses to various inflammatory stimuli requires regulation at several levels. Stimulation of mast cells, for example, by SP results in modification of cell function at the transcriptional and post-transcriptional level such as cytokine synthesis or secretion. Receptor regulation is another point in the process at which to control cell function. On the receptor level, a variety of factors regulate receptor-ligand interactions such as receptor number, receptor affinity, receptor desensitization, uncoupling of receptors from its ligands, endocytosis, receptor recycling, and lysosomal trafficking. Moreover, signal amplification may occur through activation of specific second-messenger pathways. Thus, dysregulation of these processes may result in disease or uncontrolled inflammation. For example, degradation or abnormalities in the release of neuropeptides in receptor regulation or during signal transduction may result in neurogenic inflammation, vascular permeability, hyperalgesia, analgesia, or pruritus.

Recent studies indicate that NEP and ACE play an important role in the control of neurogenic inflammation. While ACE is capable of degrading the tachykinin SP, bradykinin, and angiotensin, NEP additionally cleaves NK-A, NK-B, VIP, PACAP, atrial natriuretic peptide, and endothelins. Both NEP and ACE have been identified in vascular endothelial cells,¹⁰³ skin fibroblasts¹⁰⁴ and keratinocytes.^105- 106 Additionally, ACE is predominantly found on the luminal side of vascular endothelium, which limits its range of actions predominantly to vascular responses (vasodilatation, plasma extravasation, and leukocyte-endothelium adhesion¹⁰⁷).

In vivo studies using NEP^−/−mice demonstrated a significant increase of plasma extravasation and cutaneous inflammation. The maximum was reached after 6 hours in a model of experimentally induced contact dermatitis,¹⁰³ which indicates an anti-inflammatory role of NEP during cutaneous inflammation.

Neurokinin 1 receptor and NEP are often coexpressed by the same target cell. Thus, NEP can efficiently inhibit NK-1R signaling if expressed in high concentrations and in the vicinity of NK-1R at the cell surface.¹⁰⁸ Neutral endopeptidase activity was found to be increased after stimulation with proinflammatory cytokines¹⁰⁹ and glucocorticoids that also down-regulated NK-1R expression.^110- 111 Moreover, NEP is up-regulated during wound healing. Similar effects on cutaneous inflammation (contact hypersensitivity) were observed for ACE.^112- 113 These findings imply that up-regulation of NEP is a potential therapeutic approach to limiting the proinflammatory effects of neurogenic inflammation. Down-regulation of NEP and ACE may result in an uncontrolled stimulation of neuropeptides and lead to chronic inflammation.

There is evidence that peripheral nerves contribute to the pathophysiology of urticaria, psoriasis, atopic dermatitis, hypersensitivity reactions, rosacea, and wound healing. Moreover, several of the neuromediators discussed herein are involved in the pathophysiology of pruritus as well as immunmodulation. Certain neuropeptides have been found to be enhanced in urticaria (SP, CGRP) or psoriasis (SP, PACAP). In addition, anti-inflammatory neuropeptides such as α-MSH, VIP, and PACAP may be released to terminate inflammation under physiologic conditions. Thus, neuromediators are not necessarily proinflammatory but participate in all phases of the inflammatory response (acute, subacute, and chronic). For example, deficiency of PACAP and VIP had deleterious effects on the development of arthritis in a mouse model in vivo.

In patients with psoriasis, a significant reduction in the number of SOM- and factor XIIIa–positive dendritic cells were observed during topical treatment with clobetasol propionate or calcipotriol. Moreover, the efficiency of SOM or SOM analogues for the treatment of psoriasis and the observed reduction of SOM-positive cells during treatment support the idea that SOM might play a role during the clearing process of psoriasis.¹¹⁴

Recent evidence also suggests a role of NGF as a mediator of inflammatory responses during psoriasis.¹¹⁵ Also, protease inhibitors and PAR-modulating agents have been discussed as potential therapeutic molecules for the treatment of allegic, atopic, and inflammatory diseases.^91,116

Pharmacologic targets for the development of new agents will include the neuropeptides released in the skin, neuropeptide receptors expressed on target cells in the skin, proteases that degrade neuropeptides, agents that modify the function of vanilloid receptors, and growth factors that influence cutaneous innervation. Similar to capsaicin, topical calcineurin inhibitors such as pimecrolimus significantly inhibited histamine release from skin mast cells, which indicates a beneficial role of novel topical immunosuppressants also during neurogenic inflammation.¹¹⁷ Whether these drugs also modulate the signalling of cytokines to nerves remains to be clarified.

In summary, the interaction of the skin with the peripheral as well as central nervous system plays a crucial role in skin homeostasis and disease states (Table 1). Recent discoveries about molecular mechanisms of neuropeptide and neuropeptide receptor functions along with the development of modern techniques offer exciting insights into a complex network of the skin, the nerves, and the immune system during inflammation. Using novel technical approaches, physicians may define new ways to treat inflammatory skin diseases involving the neuro-immuno-endocrine axis.

Corresponding author and reprints: Martin Steinhoff, MD, PhD, Department of Dermatology, University of Münster, von-Esmarch-Str 58, 48129 Münster, Germany (e-mail: msteinho@uni-muenster.de).

Accepted for publication August 28, 2003.

This study was supported by grants IZKF, Fö.01KS9604/0, DFG STE 1014, and SFB 293 from the Federal Ministry of Education and Research, Münster, Germany (Dr Steinhoff); Centre de Recherches et d' Investigations Epidermiques et Sensorielles, Paris, France; and Boltzmann Institute, Münster, Germany (Drs Luger and Steinhoff).