Interactions of the Skin and Nervous System

CUTANEOUS NEUROBIOLOGY

Interactions of the Skin and Nervous System John C. Ansel*t, Cheryl A. Armstrong*t, InSung Song*, Kimberly L. Quinlan*,]ohn E. Olerud,:j: s. Wright Caughman,* and Nigel W. Bunnett§ 'Department of Dermatology, Emory University, Adanta, Georgia, U.S. A. ; tDermatology Service, Veterans Affairs Medical Center; Atlanta, Georgia, U.S.A.; :j:Departrnent of Dermatology, University of Washington, Seatde, Washington, U.S.A.; and

§ Departments

of Physiology and Surgery, UCSF, San Francisco, California, U.S.A.

There is increasing experimental evidence that the

lial cell (HDMEC) activities. Our studies indicate that

neurologic system can directly participate in cutane­

the c-fiber-derived calcitonin gene-related peptide

ous in1lammation and wound healing. Recent studies

(CGRP) is capable of stimulating HDMEC to secrete

indicate that neuropeptides released by cutaneous

the neutrophil chemotactic factor interleukin 8 (IL-

nerves such as c-fibers can activate a number of tar­

8). In addition, SP is able to directly activate HDMEC

get cells including keratinocytes, Langerhans cells,

to express high levels of the important cellular adhe­

mast cells, and endothelial cells. One such neuropep­

sion molecule vascular cellular adhesion molecule 1

tide, substance P (SP), is able to specifically bind to

(VCAM-l). Thus, these studies support the role that

murine and human keratinocytes and induce the

the neurologic system may play in mediating the

release of cytokines such as interleukin

(IL-l).

biologic processes that occur during inflammation

Other studies demonstrate that SP can also activate

and wound healing in the skin. Key words: neuropep­

1

tides/substance P/keratinocytes/wound healing. Journal of

mast cells to produce the potent pro-inflammatory cytokine tumor necrosis factor

a (TNFa).

Investigative Dermatology Symposium Proceedings 2:23-26,

More re­

cently, we examined the effect of cutaneous neu­

1997

ropeptides on human dermal microvascular endothe-

NEUROGENIC INFLAMMATION

inflammatory process in the skin soon focused on neuropeptides released by nerves such as c-fibers. Although these small peptides principally act as neurotransmitters, suggestive evidence supported their role as mediators of inflammation. Investigators detected increased levels of neuropeptides and their respective receptors in inflamed tissues (Levine et ai, 1984; Holzer, 1988). In addition, certain neuropeptide antagonists had anti-inflammatory properties in experimental animal models of inflammation (payan, 1992). The precise mechanism by which the activated nerves mediated inflam­ matory responses, however, was still unclear. To explain the pro-inflammatory interaction between nerves and tissue target cells, the axon-reflex model of neurogenic inflamma­ tion was proposed. This model proposes that when any tissue innervated by the sensory nervous system is injured, an immediate orthodromic signal is elicited by the sensory nerves from this tissue to the dorsal root ganglia and to the central nervous system, allowing the host to quickly sense and respond to the injury. In addition to this orthodromic response, the sensory nerves are capable of a second impulse in the reverse direction back to the innervated tissue--the so called antidromic response (Holzer, 1988). With this antidromic response, neuropeptides are released into the injured tissue and may interact with potential target cells located in close proximity to the activated nerve fibers (Holzer, 1988; Payan, 1992). This hypothesis presented another potential problem for investigators because it was believed that only nerves had specific receptors for these neurotransmitters.

It has been appreciated by dennatologists for many years that the skin is a complex tissue with a sophisticated cutaneous neurologic system and specialized afferent sensory structures capable of elicit­ ing the sensation of pain, itch, light touch, vibration, heat, and cold. It is less well known that this sensory system also is capable of efferent activities in the skin. Recent studies indicate that neuropep­ tides released by sensory nerves in the skin are capable of activating specific cutaneous target cells to induce a range of inflammatory activities (Baraniuk et ai, 1990; Bernstein, 1991; Payan, 1992; Eedy, 1993; Hosoi et ai, 1993; Pincelli et ai, 1993; Ansel et ai, 1996). In the early part of this century, Bayliss and Bruce noted that patients with defective cutaneous sensory systems were not capable of mounting a nonnal inflammatory responses in the skin to topically applied irritants (Bayliss, 1901). Likewise, clinicians noted that patients with congenital analgesia syndromes, post-herpes analgesia, and diabetic neuropathy have defective inflammatory responses in the skin (Lewis and Marvin, 1927; Lewis, 1927). These types of clinical observations led investigators to examine the role of the neurologic system in modulating infl ammatory responses in the skin. It remained to be determined, however, how the neuro­ sensory system was able to participate in cutaneous inflammation. The search for a link between the neurologic system and the Reprint requests to: Dr. John C. Ansel, Department of Dermatology, Emory University School of Medicine, 53 13 Woodrolf Memorial Building,

NEUROPEPTIDE RECEPTORS AND DEGRATORY ENZYMES IN NONNEURONAL TISSUE

Atlanta, GA 30322. Abbreviations: HDMEC, human dermal microvascular endothelial cells;

NK, neurokinin; NK1-R: neurokinin 1 receptor; NK2-R: neurokinin 2

A further breakthrough in this field occurred when it was discov­ ered that a number of nonneuronal-derived cells had receptors for

receptor; NK3-R, neurokinin 3 receptor; NEP: neutral endopeptidase; SP, substance P; VCAM-1, vascular cellular adhesion molecule

1087-0024/97/$10.50

•

1.

Copyright

© 1997 by The Society for Investigative Dermatology, Inc. 23

JID SYMPOSIUM PROCEEDII'i(;1

ANSEL ET AL

24

by some these secreted neuropeptides and could be activated such as T cells ly�phoid that reported ors Investigat tides. neuropep for these cells B cells and monocytic cells have specific receptors

:

nrltters

(Helke et ai, 1990; Tsuchida et ai, 1990). In addition. it was noted that immunologic tissues such as lymph nodes, spleen, and thymus were innervated by small nerves capable

neu otrans

of

secreting

a

wide range of neuropeptides ( Hadden

et

aI, 1977;

Payan, 1992). Therefore, the principal lymphoid organs are hard­ wired to the neurologic system, which is capable of activating resident leukocytes via the release of cutaneous neurotransmitters. Recent research also indicates that when these immune cells are exposed to specific neuropeptides, they become activated effector cells and demonstrate increased cytotoxic activities, antibody se­ cretion, and cytokine production (Hartung and Toyka, 1983; Payan

et ai, 1983; Hartung et ai, 1986; Payan and Goetzl, 1988; Wieder­ man et ai, 1989; McGillis et ai, 1991; Pascual et ai, 1991; Calvo et ai, 1992). Thus, in a very direct way the neurologic system can mod­

ulate immune responses. In addition to lymphoid cells, a wide range of nonimmune cells also express receptors for neurotransmitters.

In the skin both

epidermal and dermal cells express receptors for neuropeptides (Brown et ai, 1990; Matis et ai, 1990; Greeno et ai, 1992; Ansel et ai, 1993). It has been demonstrated that cutaneous sensory fibers are in

close contact not only with dermal blood vessels, mast cells, and fibroblasts but extend up into the epidermis where they are in intimate contact with both keratinocytes and Langerhans cells (Dalsgaard et ai, 1989; Hosoi et ai, 1993). Thus, in the skin there is also a direct link between the sensory nervous system and a range of potential cutaneous target cells. To date, three principal neurokinin receptors have been de­ scribed, NK-1R, NK-2R, and NK-3R, which bind with high affinity to SP, substance K, and neurokinin B, respectively, al­ though there is considerable cross-reactivity between these ligands and receptors (Sasai and Nakanishi, 1989; Tsuchida et ai, 1990; Hershey et ai, 1991). Recent immunohistochemistry studies in our laboratory indicate that keratinocytes in the epidermis predomi­ nantly express the NK-2R, whereas dermal endothelial cells and mast cells express the NK-1R.l Thus, three potential target cells in the skin for neuroinflammation express appropriate receptors for

THE ROLE OF NEUROGENIC INFLAMMATION IN CLINICAL DISEASE

r

There are a number of examples of clinical diseases that appe a 10 have a significant neurogenic component. These in lude ophthal­ � mic herpes virus infection s , inflammatory bowel dIsease, asthma.

and inflammatory arthritis (Jancso rt ai, 1985; Borson ft aI, 198� Hwang et ai, 1993). There is evidence of a neurogenic componem in the pathogenesis of urticaria, psoriasis, and atopic dermatitis. In the skin there is a close anatomic association of cutaneoU! nerves and mast cells, thus providing the physical link for these two systems to interact

(Naukkarinen

et ai, 1993). The ability of

neuropeptides to activate mast cells and induce urticaria has been appreciated for a number of years (Church et ai, 1989). Investigaton have demonstrated that neurokinins such as SP are capable of binding to and directly activating mast cells and triggering the release of immediate hypersensitivity mediators (Baxter and Ad­ amik, 1978; Hartung and Toyka, 1983; Payan et ai, 1984). As will be discussed below, neurokinins may also be responsible for the release of mast cell cytokines that may mediate late phase inflam­ matory responses (Ansel et ai, 1993). A neurogenic component for the pathophysiology of psoriasil i! suggested by both clinical and experimental findings (Bernstein (f

ai, 1986; Farber et ai, 1986; Giannetti and Girolomoni, 1989; Naukkarinen et ai, 1989; Pincelli et ai, 1990). Clinically, lesions often have a symmetrical distribution in regions that are trauma­ tized. The so-called Koebner phenomenon in psoriasis may be initiated by the release of pro-inflammatory neuropeptides in the traumatized skin

(Farber et ai,

1986). Investigators have also

reported increased levels of neuropeptides and sensory nerves in psoriatic skin lesions and capsaicin, a chemical that depletes neu­ ropeptides from nerve endings, has been reported to have some therapeutic value in clearing lesions (Bernstein et ai, 1986; Nauk­ karinen et ai, 1989). Likewise, in atopic dermatitis increased lesional staining of cutaneous nerves and abnormal cutaneous responses to injected neuropeptides have been reported (Giannetti and Girolo­ moni, 1989; Pincelli et ai, 1990). These types of prelintinary studies support,

but do not prove,

the potential involvement of the

neurologic system in inflammatory skin diseases.

released neuropeptides by cutaneous sensory fibers. In addition to released neuropeptides and appropriate target cell receptors, another key determinant of the neurogenic inflammatory response is the expression of neutral endopeptidase (NEP) (Kerr and Kenny, 1974; Bunnett, 1987). NEP is a principal protease for neurokinins such as SP and is believed to be an important regulator in terminating neurogenic inflammation (Borson et ai, 1989; Um­ eno et ai, 1989; Hwang et ai, 1993). When this cell membrane­ associated protolytic enzyme is located on the target cell that expresses the neurokinin receptors, it effectively competes for the released neuropeptides. Recent work in our laboratory indicates that in the skin significant NEP is detected in the epidermis and microvascular endothelial cells of the dermis. 1 Other studies dem­ onstrate that spontaneous vascular leakage and cutaneous edema occur in the dermis of NEP knockout mice, thereby supporting a role of this enzyme in preventing constitutive neurogenic inflam­ mation in the skin (Lu et ai, 1997). Thus, like all inflammatory processes, neurogenic inflammation is complicated and dependent on a number of factors including the types and quantities of released neuropeptides, the types of receptors on the target cells, NEP localization, activation state of the target cell, and the presence of other mediators of inflammation such as prostaglandins and cyto­ kines.

THE ROLE OF THE NEUROLOGICAL SYSTEM IN WOUND HEALING Increasing data suggest that the neurologic system also may play an important role in mediating normal wound healing.

Released

neuropeptides may modulate a number of important aspects of normal wound healing such as cell proliferation, cytokine and growth factor production, and neovascularization (Nilsson et ai, 1985; Payan, 1985; Nilsson et aI, 1986; Tanaka et ai, 1988; Brown

et ai, 1990; Ziche et ai, 1990). Surgical resection of cutaneous nerves results in delayed wound healing in animal models (Kjartansson et ai, 1987; Lusthaus et aI, 1993). In addition, patients with cutaneous sensory defects due to lepromatous leprosy, spinal cord injury, and diabetic neuropathy develop ulcers that fail to heal in spite of aggressive wound care and wound protection (Olivari ef ai, 1972; Basson and Burney, 1982; Johnson and Doll, 1984). Experimental evidence indicates that the neurologic system also appears to have an important trophic effect for tissue integrity and function. For example, the destruction of the ophthalmic branch of the trigeminal nerve results in atrophy, scarring, and ulceration of the cornea (Duke-Elder and Leigh, 1965). Currently, a number of animal models exist that will allow us to test the role of the neurologic system in normal wound healing. Murine gene knockout models are currently available in which various components of the neuro­ genic inflammatory system have been deleted including cutaneous sensory nerves, neurokinins, neurokinin receptors, and neuropep­

1 Bunnett

NW,

Song IS, Grady EF, Harten]B, Olerud ]E, Armstrong

CA, Payan DG, Ansel]C: Localization of neurokinin receptors on trans­ fected cell lines, keratinocytes, and dermal microvascular endothelial cells.

] Invest Dennatol106: 814, 1996

(abstr).

tidases (Lee et ai, 1992; Lu et aI, 1995). These animals will allow us to examine the role of the neurologic system on cutaneous inflammation and wound healing in a well controlled experimental system.

VOL. 2, NO.

1

SKIN-NERVOUS SYSTEM INTERACTIONS

AUGUST 1997

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Figure 1. SP induces expression of VCAM-1 by HDMEC in a dose-dependent fashion. Cultured HDMEC were treated with increas­

and

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"'" tj - '" "'" ... '" :::! � 0 '" '" '" N

Dose (nM)

ing concentrations of SP (0.1, 1.0, 10, and 100

"t:

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± SD of quadruplicate analyses.

NEUROPEPTIDE EFFECTS ON SPECIFIC TARGET CELLS Recent work in our laboratories has examined the effect of cutaneous neuropeptides on specific target cells in the skin. As previously mentioned, it has been established by a number of investigators that neuropeptides are capable of activating mast cells to release mediators of immediate hypersensitivity (Baxter and Adamik, 1978; Hartung and Toyka, 1983; Payan et aI, 1984). We have demonstrated that mast cells are also capable of secreting mediators of late phase inflammatory responses such as tumor necrosis factor a (TNFa) (Ansel et aI, 1993). SP is capable of inducing mast cell TNFa mRNA levels and increased secreted TNFa activity. This effect was not generalized inasmuch as SP did not a1fect mast cell interleukin-3 and interleukin-6 production. Thus, these studies support the role of the neurologic system in directly activating mast cells to produce cytokines such as TNFa, which may mediate delayed inflammatory responses in the skin. It has been noted that cutaneous sensory fibers extend directly into the epidermis, where they come in contact with both kerati­ nocytes and Langerhans cells (Dalsgaard et aI, 1989; Hosoi et aI, 1993). We examined the possibility that released neurokinins may modulate cutaneous inflammation in part by the induction of keratinocyte cytokine production: We have reported that SP is capable of directly activating both murine and normal human keratinocytes to induce interleukin 1 (IL-1) production in a dose­ dependent manner (Brown et aI, 1990). This effect can be inhibited by specific neurokinin receptor antagonists. In a related in vivo study, normal human volunteers were treated topically with the neuropeptide-releasing agent, capsaicin, and IL-1 production in the epidermis was measured 12 h later by immunohistochemistry.2 As in the in vitro studies, we found significant IL-1 produced in the epidermis after the capsaicin induction of cutaneous neuropeptides. Thus, the release of neurokinins by cutaneous sensory nerves may mediate cutaneous inflammation in part by the induction of kera­ tinocyte pro-inflammatory cytokines. Dermal microvascular endothelial cells are another potentially . unportant target cell in cutaneous neurogenic inflammation. Cuta­ neous sensory fibers are found in close contact with dermal microvascular endothelial cells. We have examined the effect of cutaneous neuropeptides on two important inflammatory activities of endothelial cells: cytokine production and cellular adhesion molecule expression. Recent studies in our laboratory indicate that certain neuropeptides released by cutaneous c-fibers are capable of inducing interleukin 8 (IL-8) production in normal human dermal

Figure 2. The kinetics of SP induction of HDMEC VCAM-1 ex­ pression. Cultured HDMEC were treated with SP (10 nM) for various amounts of time ranging from 1 to 24 h, or with TNFa (300 units per

rul)

for 16 h and then VCAM-l expression was measured by cell enzyme-linked

immunosorbent assay. Values are expressed as OD 450 the mean

oM and represent

± SD of quadruplicate analyses.

microvascular endothelial cells (HDMEC).3 IL-8 is a principal chemotactic factor for neutrophils and facilitates their vascular transmigration. Increased IL-8 secretion and IL-8 immunoreactivity were detected in neuropeptide-activated HDMEC and in endothe­ lial cells in an ex vivo cultured skin explant system.4 The effect of cutaneous neuropeptides on HDMEC cellular adhesion molecules was also examined. Our studies indicate that HDMEC can be directly activated to express increased cellular adhesion molecules in a dose-dependent fashion in vitro and in vivo by neuropeptides such as SP. As shown in Fig 1, vascular cellular adhesion molecule 1 (VCAM-1) is induced on the surface of cultured HDMEC by SP with maximum induction following treatment with 10 nM SP. Kinetics studies demonstrate that SP induction of VCAM-1 surface expression on HDMEC increases over time with maximal induction at 16 h (Fig 2). Thus, released cutaneous neurokinins can directly modulate dermal microvascular endothelial cell inflammatory ac­ tivities. CONCLUSION As our understanding of the interactions of the skin and nervous system continues to expand, it is likely that exciting, novel therapies will be developed to treat the inflammatory skin diseases that are mediated through neuroinflammation. Specific pharmacologic tar­ gets for the development of new agents will include the neuropep­ tides released in the skin, neuropeptide receptors expressed on target cells in the skin, proteases that degrade cutaneous neuropep­ tides, and growth factors that influence innervation in the skin. Thus, these approaches generate promise of more specific therapies for a wide range of chronic, debilitating infI.ammatory skin diseases. REFERENCES Ansel], Brown], Payan D, Brown M: Substance P selectively activates TNF-o< gene expression in murine mast cells.] Immu.wl 150:1-8, 1993 Ansel]C, Kaynard

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