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Cutaneous allergic contact dermatitis responses are diminished in mice deficient in neurokinin 1 receptors and augmented by neurokinin 2 receptor blockage ¹

THOMAS E. SCHOLZEN^*,2, MARTIN STEINHOFF^*, ANCA SINDRILARU^#, AGATHA SCHWARZ^*, NIGEL W. BUNNETT, THOMAS A. LUGER^*, CHERYL A. ARMSTRONG and JOHN C. ANSEL

^* Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin and
^# Institute for Experimental Dermatology, Department of Dermatology, University of Münster, Germany;
Department of Dermatology, Northwestern University, Chicago, Illinois, USA; and
Departments of Surgery and Physiology, University of California San Francisco, San Francisco, California, USA

² Correspondence: University of Münster, Department of Dermatology, Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, Von-Esmarch-Strasse 58, 48149 Münster, Germany. E-mail: thoscho{at}uni-muenster.de

SPECIFIC AIMS

Sensory nerves respond to noxious stimuli by releasing neuropeptides such as the tachykinins substance P (SP) and neurokinin A (NKA). In previous studies, a primary role for SP and its principal neurokinin 1 receptor (NK-1R) was suggested for the initiation and propagation of neurogenic inflammation as part of the inflammatory process in general and also as a component of certain skin disorders such as psoriasis or allergic contact dermatitis (ACD), but the exact role of NKA and the NK-2R in these events is less determined. The availability of mice lacking the NK-1R (NK-1R^–/–) allowed us to explore whether alterations in the SP/NK-1R system may dysregulate inflammatory skin responses and to determine the relative contribution of SP and NKA and their respective receptors, NK-1R and NK-2R, on the outcome of an inflammatory response in the skin.

PRINCIPAL FINDINGS

1. Diminished allergic contact dermatitis (ACD) responses in NK-1R^–/– mice and modulation of ACD sensitization by blocking of NK-1R
In a murine model for ACD as a model for T cell-mediated skin inflammation, inflammatory responses were significantly diminished in NK-1R-deficient mice sensitized to the antigen dinitrofluorobenzene (DNFB) compared with NK-1R^+/+ animals, as determined by measurement of ear swelling, which resulted from challenge of the animals with the same antigen on one ear after 5 days to elicit the efferent phase of ACD (Fig. 1 a). Histologically, NK-1R^–/– mice had less dermal edema and epidermal hyperplasia and displayed fewer infiltrating leukocytes in the affected skin area (51.3±18.6% vs. NK-1R^+/+, P<0.05). In NK-1R^+/+ mice, transient systemic administration of an NK-1R antagonist (NK-1RA) before ACD antigen sensitization significantly diminished the ACD inflammatory response after antigen challenge (Fig. 1b ). We hypothesized that this transient NK-1R inhibition may functionally impair APC such as epidermal Langerhans cells and dermal dendritic cells (DC), which are fundamentally important for ACD sensitization. Bone marrow-derived dendritic cells (BMDC) generated from wild-type (wt) mice matured with GM-CSF/IL-4 in the presence of a NK-1RA for 7 days expressed fewer DC maturation markers and costimulatory molecules than did BMDC not exposed to the antagonist (data not shown). Adoptive transfer of BMDC in vitro haptenized with the soluble DNFB analog dinitrobenzene sulfonic acid (DNBS) into naive wt mice profoundly increased ACD ear swelling after DNFB challenge compared with mice injected with unpulsed BMDC (Fig. 1b ). Mice injected with NK-1RA-treated, haptenized BMDC demonstrated a markedly reduced ACD inflammatory response after antigen challenge compared with recipient mice injected with haptenized BMDC not treated with the antagonist (Fig. 1b ). In addition, the presence of a NK-1RA significantly reduced the mixed lymphocyte reaction between hapten-specific proliferation of T cells isolated from DNFB-sensitized NK-1R^+/+ mice exposed to Ag-laden BMDC (data not shown). Thus, the presence of NK-1R and activation by SP is mandatory for ACD sensitization, interaction of DC and T cells, and a full inflammatory response to cutaneous allergens.

View larger version (22K): [in this window] [in a new window]   Figure 1. Diminished ACD response in NK-1R–/– mice and modulation of ACD sensitization by blocking of NK-1Rs. NK-1R–/–, and NK-1R+/+ mice were sensitized with 0.5% DNFB on the shaved abdomen and challenged with 0.2% DNFB on the right ear on day 5 (a). The ACD response was determined by the degree of ear swelling of the hapten-exposed ear compared with that of the vehicle-treated contralateral ear before and after DNFB challenge (s+c). Some mice were challenged only with DNFB without prior sensitization (c only). b) NK-1R+/+ mice were sensitized and challenged as above (positive control; s+c). 1 x 106 DNBS-haptenized BMDC generated from NK-1R+/+ mice were matured in the presence or absence of a NK-1RA (SR140333 1 µM) and injected i.v. into the tail vein of naive wt mice ["+DC (D)" or "+DC (N, D)"]. Some mice were injected with untreated or NK-1RA-treated BMDC that were not haptenized with DNBS ["DC (control)"] or ["DC (N)"]. Some mice were treated with 1 µmol/kg SR140333 30 min before sensitization ["N, s+c"]. All mice were challenged ("c") after 5 days and ear swelling was determined as above. Ear swelling values in µm as mean ± SE, n = 5–8. a) ***P < 0.001 for NK-1R–/– vs. NK-1R+/+ mice. b) **P < 0.01 for "DC (N, D)" vs. "DC (D)"; #P < 0.05 and ##P < 0.01 for "DC (N, D)" and "N, s + c" vs. PoCo "s + c"; +++P < 0.001 for "DC (D), c" and "DC (D, N), C" vs. "DC (control), c" and "DC (N), c."

2. Modulation of ACD responses in NK-1R^+/+ and NK-1R^–/– mice by agonists and antagonist of the NK-2R
We next examined the relative contribution of NKA and NK-2R in the murine ACD response. Local epicutaneous or systemic application of a potent and highly specific NK-2R antagonist (GR 94800) during the first 6 h of the efferent phase of ACD significantly augmented ACD inflammatory responses in NK-1R^+/+ mice (Fig. 2 a) and in animals lacking NK-1Rs (Fig. 2b ). By contrast, epicutaneous NK-2R agonists such NKA or ßAla⁸NKA(4-10) after hapten challenge significantly diminished ACD inflammatory responses (Fig. 2a ). Immunohistochemical examination revealed increased dermal edema and epidermal hyperplasia, a higher density of CD4⁺, CD8⁺, and MAC-1⁺ infiltrating cells, as well as increased expression of CD54/ICAM-1 in the vascular endothelium and in some nonendothelial cells in ears from sensitized and challenged NK-2R antagonist-treated NK-1R^+/+ mice 72 h after DNFB challenge compared with mice not treated with the drug. By contrast, treatment with the NK-2R agonist ßAla⁸NKA(4-10) reduced cutaneous edema, the total number of infiltrating leucocytes (47.1±5.2%; P<0.01 vs. untreated NK-1R^+/+), the density of CD4⁺, CD8⁺, and MAC⁺ cells infiltrating into the skin, and expression of CD54 compared with wt mice not treated with this mediator. Thus, NK-2R activation during the efferent phase of ACD may decrease ICAM-1 expression and recruitment of inflammatory cells, including CD4⁺ and CD8⁺ T cells, which could be important for the outcome of a T cell-mediated immune response.

View larger version (22K): [in this window] [in a new window]   Figure 2. Allergic contact dermatitis in NK-1R–/– and NK-1R+/+ mice and the effects of topically delivered NK-2R antagonist and agonists on ACD inflammation. ACD in NK-1R–/– or NK-1R+/+ mice was induced and determined as described in the legend to Fig. 1 (s+c). The NK-2R antagonist GR 94800 (a, b) or NK-2R agonists ßAla8NKA(4-10) and NKA (a) were delivered topically (20 µg/ear) to the right ear 10 min before and 2 and 6 h after challenge (GR 98400 s+c, ßAla8NKA(4-10) s+c, or NKA s+c, respectively). Some mice were only challenged with DNFB without prior sensitization (c only) or sensitized with DNFB, followed by GR 98400 treatment without DNFB challenge (GR98400 s only), to study irritant effects of the DNFB or NK-2R antagonist, respectively. Ear swelling values are given in µm as mean ± SE, n = 5. a) *P < 0.05, **P < 0.01, ***P < 0.001 for GR 94800-treated NK-1R+/+ mice vs. untreated NK-1R+/+ mice, #P < 0.05 for ßAla8NKA(4-10) -treated NK-1R+/+ mice vs. untreated NK-1R+/+ mice, and +P < 0.05 for NKA-treated NK-1R+/+ mice vs. untreated NK-1R+/+ mice. b) *P < 0.05, **P < 0.01, for GR 94800-treated NK-1R–/– mice vs. untreated NK-1R–/– mice.

CONCLUSIONS AND SIGNIFICANCE

Cutaneous inflammation is a complex interdependent response of a host immune system to an exogenous or endogenous noxious stimulus that involves cellular and noncellular components. As important modulators of innate and adaptive immune responses, cutaneous neuropeptides (including SP, NKA, and CGRP) are released from unmyelinated sensory neurons in response to epicutaneously applied allergens. In this study, we have examined the relative contribution of SP, NKA, and their corresponding receptors NK-1R and NK-2R in experimental ACD as a common type of cutaneous inflammation. Our results demonstrate for the first time that sensitized mice lacking NK-1Rs, in contrast to NK-1R^+/+ animals, exhibit only limited ACD inflammatory responses after allergen challenge. In agreement with earlier studies in other models of inflammation, our ACD model reflects the important role of SP/NK-1R for cutaneous allergic inflammatory responses. By activating NK-1Rs, SP either directly or indirectly via the induction of IL-1 is capable of exerting proinflammatory effects on a variety of cells. Thus, transient inhibition or deletion of NK-1R disrupts a major proinflammatory circuit that would normally amplify the inflammatory response. In the present study, transient inhibition of NK-1R impaired ACD sensitization, which can be mimicked by the transfer of NK-1RA-treated, antigen-laden bone marrow-derived dendritic cells into naive wt mice. These data suggest that diminished ACD responses after NK-1R inhibition and before antigen sensitization are due at least in part to an intrinsic, NK-1R-dependent functional impairment of DC. The lack of functional NK-1Rs interferes with the DC-induced proliferative response of antigen-specific T cells. Therefore, NK-1Rs are important for DC function and the activation of T cells in vitro and in vivo, which is particularly relevant for ACD as a T cell-mediated immune response. Moreover, the present study revealed the novel and unexpected finding that blocking of the NK-2R augmented rather than further reduced the allergic inflammatory response in mice when applied in the early efferent phase of ACD. The antagonist used augmented the ACD response even in NK-1R^–/– mice, thus excluding that the observed ACD modulation by this drug involves NK-1Rs. This is further supported by the observation that specific NK-2R agonists decreased ACD inflammation in our mouse model. Therefore, our study for the first time demonstrates that NKA and NK-2Rs may have a novel regulatory role in ACD independent of NK-1R and may serve to control and dampen a neurogenic inflammatory response via a yet unknown cellular target (Fig. 3 ). In higher organisms, a variety of mechanisms have evolved that control the persistent inflammatory process, including the rapid desensitization of target cells responding to the inflammatory mediators and the presence of neuropeptide–proteolytic enzymes. Anti-inflammatory factors such as IL-10 or -melanocyte-stimulating hormone are often released in response to specific inflammatory molecules and so do not prevent the initial host inflammatory response, but rather limit this response and prevent it from becoming chronic, which would have fatal consequences for the host organism. NKA could be one such factor that exhibits a dual modulatory function for the outcome of an inflammatory response. Although our results were generated using a model for skin inflammation, it seems highly likely that NKA, by opposing SP/NK-1R-mediated proinflammatory effects via NK-2Rs, may have a similar function in other tissues. There is evidence that by activating NK-1Rs and NK-2Rs, respectively, SP and NKA have contrasting effects on hematopoiesis, with SP acting as a stimulatory and NKA, by inducing negative hematopoietic growth factors, acting as an inhibitory mediator. Our observations support the importance of tachykinins and their receptors in inflammatory skin disease and confirm that knowledge of the targeted modulation of the neurocutaneous system, but also of neurogenic inflammation in other tissues, may be essential for the development of novel therapeutic agents for treating inflammatory diseases.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0658fje;

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