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The Neurosensory Tachykinins Substance P and Neurokinin A Directly Induce Keratinocyte Nerve Growth Factor

2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês

10.1046/j.0022-202x.2001.01498.x

ISSN

1523-1747

Autores

Guido J. Burbach, Kyu Han Kim, Adam Zivony, Amy Kim, Jennifer Aranda, Stacey Wright, Shubhada M. Naik, S. Wright Caughman, John C. Ansel, Cheryl A. Armstrong,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

Nerve growth factor is an essential neurotrophic factor required for the growth and maintenance of cutaneous sensory nerves. In the skin, keratinocytes are a significant source of nerve growth factor; however, the regulation of cutaneous nerve growth factor production still remains to be fully understood. In this study we tested the hypothesis that neuropeptides released by cutaneous sensory nerves have the capacity to modulate directly the expression of keratinocyte nerve growth factor, which would have important implications for the maintenance and repair of nerves in the skin. In order to address this question experimentally we examined the effect of the neuropeptides, substance P and neurokinin A, on nerve growth factor expression in human keratinocytes and the murine keratinocyte PAM 212 cell line by quantitative reverse transcriptase–polymerase chain reaction, enzyme-linked immunosorbent assay, and the PC-12 nerve growth factor bioassay. The results of these studies indicated that substance P and neurokinin A can directly induce nerve growth factor mRNA expression and the secretion of bioactive nerve growth factor protein in both human and murine keratinocytes. The specificity of these responses was demonstrated using neuropeptide receptor antagonists and nerve growth factor blocking antibodies. Additional studies also demonstrated a significant in vivo upregulation of keratinocyte nerve growth factor expression in murine epidermis after the topical application of the neuropeptide releasing agent capsaicin. This is the first report demonstrating the induction of cutaneous nerve growth factor by sensory nerve-derived neuropeptides such as substance P and neurokinin A. This direct effect of the neurosensory system on keratinocyte nerve growth factor production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing. Nerve growth factor is an essential neurotrophic factor required for the growth and maintenance of cutaneous sensory nerves. In the skin, keratinocytes are a significant source of nerve growth factor; however, the regulation of cutaneous nerve growth factor production still remains to be fully understood. In this study we tested the hypothesis that neuropeptides released by cutaneous sensory nerves have the capacity to modulate directly the expression of keratinocyte nerve growth factor, which would have important implications for the maintenance and repair of nerves in the skin. In order to address this question experimentally we examined the effect of the neuropeptides, substance P and neurokinin A, on nerve growth factor expression in human keratinocytes and the murine keratinocyte PAM 212 cell line by quantitative reverse transcriptase–polymerase chain reaction, enzyme-linked immunosorbent assay, and the PC-12 nerve growth factor bioassay. The results of these studies indicated that substance P and neurokinin A can directly induce nerve growth factor mRNA expression and the secretion of bioactive nerve growth factor protein in both human and murine keratinocytes. The specificity of these responses was demonstrated using neuropeptide receptor antagonists and nerve growth factor blocking antibodies. Additional studies also demonstrated a significant in vivo upregulation of keratinocyte nerve growth factor expression in murine epidermis after the topical application of the neuropeptide releasing agent capsaicin. This is the first report demonstrating the induction of cutaneous nerve growth factor by sensory nerve-derived neuropeptides such as substance P and neurokinin A. This direct effect of the neurosensory system on keratinocyte nerve growth factor production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing. nerve growth factor substance P neurokinin A brain-derived neurotrophic factor neurotrophin-3 neurotrophin-4 neurotrophin-5 neurokinin-1 receptor neurokinin-2 receptor Nerve growth factor (NGF) is the first isolated and best characterized member of the neurotrophin (NT) family of growth factors (Cohen et al., 1954Cohen S. Levi-Montalcini R. Hamburger V. A nerve growth stimulating factor isolated from sarcoma 37 and 180.Proc Natl Acad Sci USA. 1954; 40: 1014-1018Crossref PubMed Google Scholar;Levi-Montalcini, 1987Levi-Montalcini R. The nerve growth factor: thirty-five years later. 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Neurotrophin-5. a novel neurotrophic factor that activates trk and trkB.Neuron. 1991; 7: 857-866Abstract Full Text PDF PubMed Scopus (702) Google Scholar). NGF is detected in neurons of the central and peripheral nervous system (Korsching and Thoenen, 1983Korsching S. Thoenen H. Nerve growth factor in sympathetic ganglia and corresponding target organs of the rat: correlation with density of sympathetic innervation.Proc Natl Acad Sci USA. 1983; 80: 3513-3516Crossref PubMed Scopus (484) Google Scholar;Korsching et al., 1985Korsching S. Auburger G. Heumann R. Scott J. Thoenen H. Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.EMBO J. 1985; 4: 1389-1393Crossref PubMed Scopus (743) Google Scholar;Heumann et al., 1987bHeumann R. Lindholm D. 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A significant source of NGF in normal skin appears to be keratinocytes (Tron et al., 1990Tron V.A. Coughlin M.D. Jang D.E. Stanisz J. Sauder D.N. Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212).J Clin Invest. 1990; 85: 1085-1089Crossref PubMed Scopus (114) Google Scholar;Di Marco et al., 1991Di Marco E. Marchisio P.C. Bondanza S. Franzi A.T. Cancedda R. De Luca M. Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.J Biol Chem. 1991; 266: 21718-21722Abstract Full Text PDF PubMed Google Scholar;Pincelli et al., 1994Pincelli C. Sevignani C. Manfredini R. et al.Expression and function of nerve growth factor and nerve growth factor receptor on cultured keratinocytes.J Invest Dermatol. 1994; 103: 13-18Abstract Full Text PDF PubMed Google Scholar). In addition, Langerhans cells (Torii et al., 1997Torii H. Yan Z. Hosoi J. Granstein R.D. Expression of neurotrophic factors and neuropeptide receptors by Langerhans cells and the Langerhans cell-like cell line XS52: further support for a functional relationship between Langerhans cells and epidermal nerves.J Invest Dermatol. 1997; 109: 586-591Crossref PubMed Scopus (66) Google Scholar) and dermal microvascular endothelial cells (Gibran NS, 2000, personal communication) have also been reported to express NGF in the skin. There is increasing evidence that the development and maintenance of the neurosensory system in the skin depends on the localized expression of NGF (Otten et al., 1980Otten U. Goedert M. Mayer N. Lembeck F. Requirement of nerve growth factor for development of substance P-containing sensory neurones.Nature. 1980; 287: 158-159Crossref PubMed Scopus (157) Google Scholar;Ross et al., 1981Ross M. Lofstrandh S. Gorin P.D. Johnson E.M. Schwartz J.P. Use of an experimental autoimmune model to define nerve growth factor dependency of peripheral and central substance P-containing neurons in the rat.J Neurosci. 1981; 1: 1304-1311PubMed Google Scholar;Albers et al., 1994Albers K.M. Wright D.E. Davis B.M. Overexpression of nerve growth factor in epidermis of transgenic mice causes hypertrophy of the peripheral nervous system.J Neurosci. 1994; 14: 1422-1432PubMed Google Scholar). Cutaneous NGF production may therefore serve as a key factor in neuronal maintenance, survival, and repair in the skin, especially during cutaneous inflammatory responses and wound healing (reviewed inPincelli and Yaar, 1997Pincelli C. Yaar M. Nerve growth factor: its significance in cutaneous biology. [Review].J Invest Dermatol Symp Proc. 1997; 2: 31-36Abstract Full Text PDF PubMed Scopus (68) Google Scholar;Ansel et al., 1996Ansel J.C. Kaynard A.H. Armstrong C.A. Olerud J. Bunnett N. Payan D. Skin–nervous system interactions.J Invest Dermatol. 1996; 106: 198-204Crossref PubMed Scopus (184) Google Scholar). It is now appreciated that the neurosensory system can modulate a wide range of inflammatory and proliferative processes in various tissues by the release of bioactive tachykinins such as substance P (SP) and neurokinin A (NKA) (reviewed inAnsel et al., 1996Ansel J.C. Kaynard A.H. Armstrong C.A. Olerud J. Bunnett N. Payan D. Skin–nervous system interactions.J Invest Dermatol. 1996; 106: 198-204Crossref PubMed Scopus (184) Google Scholar,Ansel et al., 1997Ansel J.C. Armstrong C.A. Song I. Quinlan K.L. Olerud J.E. Caughman S.W. Bunnett N.W. Interactions of the skin and nervous system.J Invest Dermatol Symp Proc. 1997; 2: 23-26Abstract Full Text PDF PubMed Scopus (140) Google Scholar;Scholzen et al., 1998Scholzen T. Armstrong C.A. Bunnett N.W. Luger T.A. Olerud J.E. Ansel J.C. Neuropeptides in the skin. interactions between the neuroendocrine and the skin immune systems. [Review].Exp Dermatol. 1998; 7: 81-96Crossref PubMed Scopus (353) Google Scholar). Tachykinin releasing sensory C and Aδ fibers extend from the dorsal root ganglia into the epidermis where they are in direct contact with keratinocytes (Kennedy et al., 1994Kennedy W.R. Wendelschafer-Crabb G. Brelje T.C. Innervation and vasculature of human sweat glands: an immunohistochemistry-laser scanning confocal fluorescence microscopy study.J Neurosci. 1994; 14: 6825-6833PubMed Google Scholar;Lawson, 1996Lawson S.N. Peptides and cutaneous polymodal nociceptor neurones.Prog Brain Res. 1996; 113: 369-385Crossref PubMed Scopus (39) Google Scholar;Reilly et al., 1997Reilly D.M. Ferdinando D. Johnston C. Shaw C. Buchanan K.D. Green M.R. The epidermal nerve fibre network. characterization of nerve fibres in human skin by confocal microscopy and assessment of racial variations.Br J Dermatol. 1997; 137: 163-170Crossref PubMed Scopus (85) Google Scholar;Schulze et al., 1997Schulze E. Witt M. Fink T. Hofer A. Funk R.H. Immunohistochemical detection of human skin nerve fibers.Acta Histochem. 1997; 99: 301-309Crossref PubMed Scopus (79) Google Scholar). Recent evidence indicates that SP and NKA released into the skin after injury can activate specific receptors on cutaneous target cells such as dermal endothelial cells, Langerhans cells, and keratinocytes thereby modulating cutaneous inflammation, wound healing, and tissue repair (reviewed inScholzen et al., 1998Scholzen T. Armstrong C.A. Bunnett N.W. Luger T.A. Olerud J.E. Ansel J.C. Neuropeptides in the skin. interactions between the neuroendocrine and the skin immune systems. [Review].Exp Dermatol. 1998; 7: 81-96Crossref PubMed Scopus (353) Google Scholar). In experimental models of inflammation, upregulation of neuropeptide levels in the primary sensory neurons innervating the inflamed tissue was detected as well as elevated NGF levels in skin (Donnerer et al., 1992Donnerer J. Schuligoi R. Stein C. Increased content and transport of substance P and calcitonin gene-related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo.Neuroscience. 1992; 49: 693-698Crossref PubMed Scopus (531) Google Scholar;Woolf et al., 1994Woolf C.J. Safieh-Garabedian B. Ma Q.P. Crilly P. Winter J. Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity.Neuroscience. 1994; 62: 327-331Crossref PubMed Scopus (583) Google Scholar;Safieh-Garabedian et al., 1995Safieh-Garabedian B. Poole S. Allchorne A. Winter J. Woolf C.J. Contribution of interleukin-1 beta to the inflammation-induced increase in nerve growth factor levels and inflammatory hyperalgesia.Br J Pharmacol. 1995; 115: 1265-1275Crossref PubMed Scopus (495) Google Scholar). Although it has been reported that NGF upregulates neuropeptide levels in nerves (Goedert et al., 1981Goedert M. Stoeckel K. Otten U. Biological importance of the retrograde axonal transport of nerve growth factor in sensory neurons.Proc Natl Acad Sci USA. 1981; 78: 5895-5898Crossref PubMed Google Scholar), the ability of neuropeptides to induce NGF production in skin tissue has not been studied. In this study we test the hypothesis that neuropeptides released by cutaneous sensory nerves may directly promote the local production of NGF, which may be important during cutaneous injury and wound healing. The effects of SP and NKA are mediated by specific neurokinin receptors that have been identified on a number of cell types in the skin, including human and murine keratinocytes. The functionality of these keratinocyte neuropeptide receptors has been demonstrated by neuropeptide induction of neurokinin receptor-mediated specific Ca2+ mobilization and cytokine production in these cells (Brown et al., 1990Brown J.R. Perry P. Hefeneider S. Ansel J.C. Neuropeptide modulation of keratinocyte cytokine production.in: Oppenheim J.J. Powanda M.C. Kluger M. Dinarello C.A. Somerset N.J. Molecular and Cellular Biology of Cytokines. Wiley-Liss, Inc., 1990: 451-456Google Scholar;Song et al, 1999). SP is known to preferentially bind to the neurokinin 1 receptor (NK1R) and NKA to the neurokinin 2 receptor (NK2R). As there is significant cross-reactivity of these ligand-receptor pairs, SP and NKA can bind to both neurokinin receptors but with diminished affinities (Regoli et al., 1997Regoli D. Nguyen K. Calo G. Neurokinin receptors. Comparison of data from classical pharmacology, binding, and molecular biology.Ann N Y Acad Sci. 1997; 812: 144-146Crossref PubMed Scopus (12) Google Scholar). The murine keratinocyte cell line PAM 212 expresses predominantly NK2R rather than NK1R and therefore both SP and NKA appear to activate murine keratinocytes primarily by activation of NK2R (Song et al, 1999). In contrast, human keratinocytes express both NK1R and NK2R (Brown et al., 1990Brown J.R. Perry P. Hefeneider S. Ansel J.C. Neuropeptide modulation of keratinocyte cytokine production.in: Oppenheim J.J. Powanda M.C. Kluger M. Dinarello C.A. Somerset N.J. Molecular and Cellular Biology of Cytokines. Wiley-Liss, Inc., 1990: 451-456Google Scholar), so SP and NKA stimulate specific responses through preferential binding to these neurokinin receptors, respectively. In this study we examined the ability of the sensory nerve-derived neuropeptides SP and NKA to modulate keratinocyte NGF expression in both human keratinocytes and the murine keratinocyte cell line PAM 212. Receptor blocking studies were conducted to investigate the specificity of our observations. Furthermore, the effect of in vivo cutaneous neuropeptide release on keratinocyte NGF production was determined. The spontaneously transformed keratinocyte cell line (PAM 212), originally derived from a newborn BALB/C mouse, was generously provided by Dr Stuart Yespa (National Cancer Institute, Bethesda, MD). Cells were cultured in RPMI-1640 media (Mediatech Inc., Herndon, VA) containing 10% fetal bovine serum (Gibco BRL/Life Technologies, Grand Island, NY) and 1% penicillin–streptomycin amphotericin B (PSF, Gibco). Human keratinocytes were isolated from neonatal foreskins at the Emory Skin Diseases Research Core Center as previously described (Boyce and Ham, 1985Boyce S.T. Ham R.G. Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum free media.J Tissue Culture Methods. 1985; 9: 83Crossref Scopus (251) Google Scholar) and cultured in keratinocyte growth media (Clonetics, Walkersville, MD) supplemented with bovine pituitary extract (Clonetics). PC-12 cells (American Type Culture Collection, ATCC, Rockville, MD), originally derived from rat pheochromocytoma tissue, were cultured in a medium of RPMI 1640, 10% donor horse serum (Sigma, St Louis, MO), 5% fetal bovine serum, and 1% PSF. Cell cultures were maintained at 37°C in humidified incubators with 5% CO2. PAM 212 cells and PC-12 cells were passaged at 60–70% confluency to avoid differentiation. Human keratinocytes were cultured until passages 3–5 for immediate use. Before stimulating cells with various concentrations of neuropeptides, proinflammatory stimuli, or conditioned media, human keratinocytes were starved in keratinocyte growth media without bovine pituitary extract; PAM 212 cells in RPMI-1640 media containing 1% fetal bovine serum and 1% PSF; and PC-12 cells in RPMI-1640 media containing 1% donor horse serum, 0.5% fetal bovine serum, and 1% PSF for 24 h, respectively. For quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) studies cells were either left untreated or were treated with NKA or SP (Peninsula Laboratories, Belmont, CA) in concentrations ranging from 0.1 to 100 nM. In some studies, treatment with 50 ng per ml phorbol 12-myristate 13-acetate (PMA, Sigma) served as a positive control. All mediators were diluted in fresh starving media and added directly to the cells. Before stimulating cells with 10 nM SP or NKA cells in selected studies were preincubated for 30 min or 2 h either with a 10 µM solution of the specific NK1R antagonist Spantide I (Peninsula;Yanagisawa and Otsuka, 1990Yanagisawa M. Otsuka M. Pharmacological profile of a tachykinin antagonist, spantide, as examined on rat spinal motoneurons.Br J Pharmacol. 1990; 100 (706): 711Crossref PubMed Scopus (45) Google Scholar) or a 1 µM solution of the specific NK2R antagonist GR-94800 (Peninsula;McElroy et al., 1992McElroy A.B. Clegg S.P. Deal M.J. et al.Highly potent and selective heptapeptide antagonists of the neurokinin NK-2 receptor.J Med Chem. 1992; 35: 2582-2591Crossref PubMed Scopus (71) Google Scholar), respectively. Spantide was dissolved in 1% acetic acid, GR-94800 in 100% acetone and both were diluted to their final concentration in fresh starving media. Normal human keratinocytes and murine PAM 212 cells were left untreated or treated with neuropeptide concentrations ranging from 0.1 to 100 nm and harvested after 3 h for total RNA isolation. This time-point was chosen as preliminary experiments had demonstrated that NGF mRNA expression after neuropeptide treatment was highest after 3 h (data not shown). RNA was then subjected to DNAse treatment (Ambion, Austin, TX) for 1 h at 37°C followed by a DNAse inactivation step at 70°C for 10 min and reverse transcribed using AMV reverse transcriptase (Promega, Madison, WI) and random primers (Promega). cDNAs were subjected to PCR utilizing the Perkin Elmer PCR 9600 sequence detection system (Perkin Elmer Applied Biosystems, Foster City, CA) and a SYBR green binding dye (Sybr Green PCR Core Reagents, Perkin Elmer) (Morrison et al., 1998Morrison T.B. Weis J.J. Wittwer C.T. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification.in: Biotechniques. 24. 1998: 954-958Google Scholar,Morrison et al., 1999Morrison T.B. Ma Y. Weis J.H. Weis J.J. Rapid and sensitive quantification of Borrelia burgdorferi-infected mouse tissues by continuous fluorescent monitoring of PCR.J Clin Microbiol. 1999; 37: 987-992Crossref PubMed Google Scholar;Steuerwald et al., 2000Steuerwald N. Cohen J. Herrera R.J. Brenner C.A. Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR.Mol Hum Reprod. 2000; 6: 448-453Crossref PubMed Scopus (74) Google Scholar). The following specific NGF primers were selected using Primer Express software (Perkin Elmer): human NGF sense primer 5′-AAGTGCCGG GACCCAAAT-3′; human NGF anti-sense primer 5′-TGAGTTCCA GTGCTTTGAGTCAA-3′; mouse NGF sense primer 5′-ACAGTGTA TTCAGACAGTACTTTTTTGAGA-3′; mouse NGF anti-sense primer 5′-GAGTTCCAGTGTTTGGAGTCGAT-3′; human and mouse 18 S ribosomal RNA sense primer 5′-CGGCTACCACATCCAAGGAA-3′; human and mouse 18 S ribosomal RNA anti-sense primer 5′-GCTGGAATTACCGCGGCT-3′. For the amplification of human NGF cDNA a standard amplification program was used (1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min), for amplifying PAM 212 NGF cDNA the following modifications were made: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, 35 cycles of 95°C for 15 s and 60°C for 1 min. Direct detection of the PCR product was monitored by measuring the increase in fluorescence caused by the binding of SYBR green to double-stranded DNA and the specificity of the PCR product was verified by melting curve analysis (Ririe et al., 1997Ririe K.M. Rasmussen R.P. Wittwer C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.Anal Biochem. 1997; 245: 154-160Crossref PubMed Scopus (1222) Google Scholar). The PCR product was furthermore sequenced and compared with the published nucleotide sequences verifying that the amplified PCR product indeed corresponds with the published NGF mRNA sequence (Ullrich et al., 1983Ullrich A. Gray A. Berman C. Dull T.J. Human beta-nerve growth factor gene sequence highly homologous to that of mouse.Nature. 1983; 303: 821-825Crossref PubMed Scopus (375) Google Scholar;Borsani et al., 1990Borsani G. Pizzuti A. Rugarli E.I. et al.cDNA sequence of human beta-NGF.Nucleic Acids Res. 1990; 18: 4020Crossref PubMed Scopus (18) Google Scholar). Using a standard curve of serial dilutions of an untreated cDNA sample, a relative quantitation of the respective target cDNA expressed in x-fold differences was performed. All quantitations were normalized to an endogenous control (18S ribosomal RNA (Schmittgen and Zakrajsek, 2000Schmittgen T.D. Zakrajsek B.A. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR [In Process Citation].J Biochem Biophys Methods. 2000; 46: 69-81Crossref PubMed Scopus (967) Google Scholar) to account for variability in the initial concentration and quality of total RNA and in the conversion efficiency of the reverse transcription reaction. Results are representative of three independent assays. To detect the presence of bioactive NGF in untreated and neuropeptide-treated 48 h keratinocyte supernatants, PC-12 bioassays were performed as previously described (Scott et al., 1981Scott S.M. Tarris R. Eveleth D. Mansfield H. Weichsel M.E.J. Fisher D.A. Bioassay detection of mouse nerve growth factor (mNGF) in the brain of adult mice.J Neurosci Res. 1981; 6: 653-658Crossref PubMed Scopus (22) Google Scholar;Varilek et al., 1991Varilek G.W. Weinstock J.V. Pantazis N.J. Isolated hepatic granulomas from mice infected with Schistosoma mansoni contain nerve growth factor.Infect Immun. 1991; 59: 4443-4449PubMed Google Scholar). Specifically, PC-12 cells were plated in 24-well dishes at defined concentrations and stimulated with conditioned 48 h supernatants from neuropeptide-treated keratinocytes after cell attachment. In order to prove the specificity of the bioassay, neuropeptide-treated keratinocyte supernatants were preincubated with specific anti-mouse and human NGF neutralizing antibodies (Roche Diagnostics Corp., Indianapolis, IN) before stimulating PC-12 cells. Untreated PC-12 cells served as a negative control and recombinant NGF (50 µg per ml, Roche) treated PC-12 cells as a positive control. After 48 h bioassays were quantified by counting a minimum of 200 cells in random fields per test well followed by determining the percentage of cells bearing neurites. Cells were considered positive for neurite outgrowth if they had one or more processes extending greater than one cell diameter. Counts were done twice, results were calculated as mean of counts and are representative of two independent assays. To quantitate secreted NGF by ELISA, 48 h supernatants were collected from untreated and neuropeptide-treated keratinocytes and utilized in a standard sandwich ELISA (Promega). The 48 h time-point was chosen as preliminary experiments had demonstrated that NGF protein secretion after neuropeptide treatment was maximal after 48 h (data not shown). Specifically, flat-bottom 96 well plates were coated using polyclonal anti-human and mouse NGF antibodies. Captured NGF was detected by a second specific monoclonal anti-NGF antibody and the amount of specifically bound monoclonal antibody was then detected using a species-specific antibody conjugated to horseradish peroxidase as a tertiary reactant. The unbound conjugate was removed by washing followed by an incubation with the chromogenic substrate 3,3′,5,5′-tetramethyl-benzidine. The colorimetric reaction was stopped by addition of 50 µl 1 M H2SO4 and plates were read on an ELISA reader (Spektramax 250, Molecular Devices Corp., Sunnyvale, CA) at OD 450 nm. A standard protein assay (Bio-Rad Laboratories, Hercules, CA) was used for normalizing the NGF protein content of the supernatants to the total cell lysate protein content. Data were analyzed utilizing Softmax Pro (Molecular Devices Corp.) and Microsoft Excel (Microsoft Corp., Redmond, WA) software. Results were calculated as the mean of three values ± SD and are representative of three independent assays. Statistical differences between NGF levels of untreated and treated keratinocyte supernatants were calculated using unpaired Student's t test and p ≤ 0.05 was considered significant. Normal female C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in the Emory Animal Facility. To cause the local release of neuropeptides from sensory nerves in the skin, 8 wk old mice were treated on the right ear with a single topical application of capsaicin (Zostrix HP 0.075%); the left ear was not treated. Cutaneous treatment sites and untreated control ears were collected from killed mice after 6, 12, 24, and 48 h, immediately frozen in OCT (Tissue-Tek, Sakura Finetechnical, Torrance, CA) and stored at -70°C. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University (Atlanta, GA). Cryostat sections of untreated or capsaicin-treated mouse skin (thickness: 8 µm) were fixed in ice-cold acetone, quenched in a 0.3% H2O2 solution and subjected to peroxidase immunostaining using VectaStain elite ABC kit (Vector Laboratories Inc., Burlingame, CA) according to the manufacturer's recommendations. A polyclonal rabbit-anti-mouse-NGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used as a primary antibody (1:100) and normal rabbit IgG (Santa Cruz Biotechnology) as a first step negative control at the same dilution. The primary antibody was detected using a biotinylated goat-anti-rabbit antibody and a preformed streptavidin horseradish peroxidase complex from the VectaStain elite ABC kit. To prevent unspecific binding of the secondary antibody, sections were blocked with 10% goat serum in Tris-buffered saline. Sections were stained with a 3,3′-diaminobenzidine solution (0.35 mg per ml; Sigma FAST, Sigma) and counterstained with Mayer's hematoxylin (Sigma). They were photographed with an Olympus C35 AD-4 camera, mounted on an Olympus BH2 microscope. Photographs of immunohistochemistry sections were scanned on a flatbed scanner (HP Scan Jet 6100 C, Hewlett Packard, Palo Alto, CA) utilizing Adobe Photoshop softw

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