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Autocrine Induction of Substance P mRNA and Peptide in Cultured Normal Human Keratinocytes
Biochemical and Biophysical Research Communications 263, 327–333 (1999) Article ID bbrc.1999.1285, available online at http://www.idealibrary.com on
Autocrine Induction of Substance P mRNA and Peptide in Cultured Normal Human Keratinocytes SangJae Bae, 1 Yoshitaka Matsunaga, Yoichi Tanaka, and Ichiro Katayama Department of Dermatology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, Japan 852-8501
Received August 5, 1999
In this study, we have demonstrated that normal cultured keratinocytes (KCs) could generate significant endogenous substance P (SP) in a dose- and timedependent response to exogenous SP by sensitive ELISA assay and express preprotachinin-a mRNA by RT-PCR and Southern blotting. We performed immunohistochemical analysis to confirm the presence of SP in cultured keratinocytes. In contrast, adrenaline, acetylcholine, histamine and CGRP induced only low amount of SP from cultured normal human KCs. This is the first report that SP can be induced by skin epithelial cells in response to exogenous SP and KC derived SP might play an important role in induction and acceleration of certain cutaneous diseases. © 1999 Academic Press
Key Words: substance P; itch-scratch cycle; stress; keratinocyte.
Itching is the predominant symptom among inflammatory cutaneous diseases, such as atopic dermatitis, urticaria and pruriginous diseases. However, its mechanism is ill-understood. It is well known that mediators causing itch in inflamed skin are histamine (1), prostaglandin (2) and substance P (3). Among these, SP plays central role to mediate peripheral itchy sensation because Jorizzo et al. reported that intradermal injection of SP causes itching due to histamine release from mast cells (4) and capsaicin depletes cutaneous nerve endings of substance P resulting in inhibition of itch-scratch cycle in certain pruritic skin disorders (5). Substance P has been recognized as a peptide of neuron-origin for a long period. However, it has been reported that SP gene expression can be identified in non-neural cells, such as human eosinophils (6), Leydig cells of testis (7), endothelial cells (8), or lymphocytes (9, 10). Recently, Ho et al. have shown that human monocytes and macrophages express mRNA encoding SP and its receptor and produce SP (11). It was re1
To whom correspondence should be addressed. Fax: 095-8497335.
ported that human keratinocytes express neurokinin receptor (12). Therefore, in this study, we addressed the question whether cultured human keratinocytes could express SP and possible presence of neuroendocrine circuit in human skin. MATERIALS AND METHODS Reagents. Substance P (lot: 58H 5800), CGRP and histamine were obtained from Sigma Chemical Co. (St. Louis, MO); acetylcholine and adrenaline were purchased from Wako Pure Chemicals (Tokyo, Japan). For immunostaining, polyclonal rabbit anti-human substance P antibody (Chemicon, International, Inc., Temecula, CA; dilution 1:1000) and FITC labeling anti-rabbit immunoglobulin (Dako, Denmark; dilution 1:30) were used. Keratinocyte culture. Primary normal human keratinocytes were purchased from cell systems (Sanko Junyaku Co., LTD. Japan). These were pooled cells derived from neonatal donors. Each batch of cells used for the experiments reported in Figs. 1, 2, 3, and 6. Cells were cultured in serum-free, modified KGM CC-3111 medium (Sanko Junyaku Co., Ltd., Japan) containing bovine pituitary extract 15 mg/500 ml, epidermal growth factor 0.5 mg/500 ml, insulin 2.5 mg/500 ml, antibiotics 0.5 ml/500 ml and hydrocortisone 0.25 mg/500 ml. Cultures were maintained in a humidified incubator controlled at 37°C and having an atmosphere of 5% CO 2. The culture medium was changed every 3 days, and the cells were subcultured at subconfluence at a split ratio of about 1:3. After the fourth passage, the cells seeded at 1 3 10 5 cells/cm 2 in 6 wells culture cluster (Corning, NY). At confluent state, the culture medium replaced with fresh medium supplemented with the stimulants described in the text, but without hydrocortisone. The culture supernatants were harvested and stored at 280°C until use. For mRNA detection, the cells were lysed with lysis buffer as described below. Substance P determination by ELISA. To investigate the peptide of SP level, culture supernatants were subjected to a competitiveenzyme immunoassay kit (Cayman, Ann Arbor, MI) as Ho et al. (11). Briefly, the assay is based on the competition between free SP and a SP tracer (SP linked to an acetylcholinesterase molecule) for a limited number of SP-specific rabbit antiserum binding sites. The amount of SP tracer that is able to bind to the rabbit antiserum will be inversely proportional to the concentration of free SP in the well. Immunohistochemical analysis of substance P expression in keratinocytes. The keratinocytes were cultured in chamber slide until confluent and then stimulated with 10 28 M of SP in fresh medium for 24 h and then washed with PBS. As positive control, serial 7 mm cryostat sections from atopic dermatitis were prepared. SP immunostaining were obtained using rabbit anti-serum (dilution 1:1000) and
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FIG. 1. Time course and dose-dependent production of SP by cultured KCs. Normal human keratinocytes were cultured in KGM CC-3111 medium (complete medium) supplemented with BPE, hEGF, insulin, antibiotics and hydrocortisone as described above. At semiconfluent stage, the cells were washed with ice-chilled DMEM and replaced with KGM medium without hydrocortisone, for 24 h. At the start of the experiment, the medium was replaced with fresh KGM medium containing variously diluted SP and then culture supernatants were collected at various times and donated for ELISA assay. (A) Dose response effect of SP production in normal keratinocytes. (B) Time course of SP-induced SP production in normal keratinocytes. Results are expressed as the means 6 SD.
then antibody-antigen complex were visualized with FITC labeling immunoglobulin (dilution 1:30). RT-PCR analysis of substance P mRNA expression. Total RNA was extracted from the cultured keratinocytes by RNeasy Mini Kit (QIAGEN, Japan). cDNA synthesis was performed by Superscript II (Gibco BRL, Rockville, MD) according to the method by the manufacturer. In short, the cycle condition is 94°C for 9 min, followed 94°C for 30 s, 50°C for 30 s and 70°C for 45 s: 40 cycles for substance P, 35 cycles for b-actin) by Takara Ex Taq (Takara Shuzo, Shiga, Japan) using 59 and 39 primer sets and b-actin (Clonetech, Palo Alto, CA). Southern blotting analysis of substance P mRNA analysis. The amplified products were blotted onto Hybond-N1 membrane (Amersham, Buckinghamshire, England) using standard methods (13). Oligonucleotide probe of human substance P was labeled with peroxidase complexes, hybridized and detected using ECL direct nucleic acid labeling and detection systems (Amersham, Buckinghamshire, England) according to the methods by the manufacture. Statistical analysis. tistical significance.
Paired t-test was used to determine the sta-
RESULTS Induction of Substance P by Cultured Normal Human Keratinocytes (KCs) by SP At the start of experiment, the medium was replaced with fresh KGM medium containing variously diluted SP and then culture supernatants were collected at various time for ELISA assay. As results are shown in Fig. 1A and Fig. 1B, SP induced SP in normal keratinocytes in a dose dependent and autocrine fashion (Fig. 1A). SP concentration without KC
was unchanged during the culture period possibly due to the absence of degradating enzyme in KGM medium. While endogenous SP is stable in plasma, synthetic SP added to human plasma is rapidly destroyed. The degradation is temperature-dependent with a much larger loss of activity at 37°C than at 0°C. SP is inactivated at 37°C the half-life in plasma being 12–15 min (14). It is suggesting that SP added into medium in incubator at 37°C might be rapidly destroyed. However, SP concentration of medium with KCs was augmented in a dose and time dependent fashion. The peak response of SP production by KCs is at 10 h after SP stimulation respectively (Fig. 1B). To clarify the de novo synthesis of SP by KCs, KCs were preincubated with SP for several times, washed in fresh medium SP 3 times, and then incubated for another 18 h in the presence of SP. As is shown in Fig. 2A, SP was newly generated by KCs in a incubation timedependent fashion. This was further confirmed by the blockade of SP production by cycloheximide (Fig. 2B). To exclude the possibility that cell associated SP is responsible for SP secretion into the medium during possible apoptotic process, cell associated SP was measured after SP stimulation. As shown, in Fig. 2C, cell associated SP in SP stimulated-KCs after several freeze and thaw treatment was unchanged at 3 and 18 h after stimulation.
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FIG. 2. Kinetics of de novo synthesis of SP by KCs. (A) To exclude the carry over of SP by exogenously added SP, KCs were stimulated with SP several times and then washed fresh medium without SP three times, incubated for another 18 h, and secreted SP was measured by ELISA. (B) Effect of cycloheximide on SP-induced SP production by KCs was studied by addition of various concentrations of cycloheximide to keratinocyte cultures and secreted SP was measured ELISA. (C) To exclude the possibility that cell associated SP is responsible for SP secretion into the medium during possible apoptotic process, cell associated SP was measured after SP stimulation. Results are expressed as the means 6 SD (B and C).
Effect of Substance P Antagonists on Substance P Production by KCs
dilution) clearly downregulated the SP production (Fig. 3B).
We then analyzed the effect of SP antagonists on SP induced-SP production by keratinocytes. As shown, NK1 antagonist, spantide did not inhibit SP production by KC rather augmented its autocrine production in a dose dependent fashion as reported by Ando et al. (15) (Fig. 3A). In contrast, anti-SP antibody (at 1:1000
Presence of Cytoplasic Substance P in Normal KCs To confirm the presence of SP in normal keratinocytes, we then performed immunohistochemical analysis. Normal human KCs were cultured on chamber slide glass (Nunc Inc., IL) and stimulated with SP for
FIG. 3. Effect of substance P antagonists on substance P production by KCs. (A) NK1 antagonist spantide was added to the culture and SP production by KCs was measured by ELISA to study the effect of SP antagonists on SP-induced SP production by keratinocytes. (B) Anti-SP antibody was added to the culture and SP production by KC was measured by ELISA. Results are expressed as the means 6 SD. 329
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FIG. 4. Presence of cytoplasic substance P in normal KCs. To confirm the presence of SP in normal keratinocytes, we then performed immunohistochemical analysis. Normal human KCs were cultured on chamber and at semiconfluent stage, the cells were fixed with 4% paraformaldehyde and then treated with polyclonal rabbit anti-P antibody followed by FITC-labeled anti-rabbit immunoglobulin. (A) Immunohistochemical staining of SP positive cultured KCs. (B) Negative control using SP-absorbed anti-SP Ab.
24 h, at semiconfluent stage, the cells were washed with PBS (2) for 3 times and fixed with 4% paraformaldehyde and then treated with polyclonal rabbit anti-SP antibody followed by FITC-labeled anti-rabbit immunoglobulin. Specific cytoplasmic staining of SP in normal KCs was observed in SP stimulated KCs (Fig. 4A). As negative control, we performed immunostaining using SP absorbed anti-SP antibody solution (Fig. 4B). In short, SP (5 mg) was dissolved with 1.5 ml PBS plus 1.5 ml anti-SP Ab, and shaken for 4 h at 4°C. Then, SP plus anti-SP Ab solution was poured into a visking tube and dialyzed 3 times with PBS. Substance-P Gene Expression in Substance P Stimulated Normal KCs
described in Materials and Methods (Fig. 5A and 5B). Figure 5C showed that the expressed ppt-a mRNA and b-actin bands are calculated by using NIH image. Effect of Various Agents on Substance P Production in Normal KCs In addition to SP, several biological active substances modulate KC functions in both in vitro and in vivo. We next screened several agents on SP production by normal KCs. As shown in Fig. 6A– 6D, relatively low compound to SP but significant SP was induced by high concentration of acetylcholine, adrenaline, histamine and CGRP (Fig. 6A– 6D). DISCUSSION
To clarify the SP gene expression in normal KCs, we performed RT-PCR and Southern blot analysis. As is described in Materials and Methods, first we performed RT-PCR using preprotachynin-A (ppt-a) specific primers. In non-stimulated condition, normal keratinocytes express moderate amount of ppt-a mRNA expression and addition of exogenous SP significantly augmented ppt-a mRNA expression a dose dependent fashion in normal KCs (Fig. 5A). This basal expression of ppt-a mRNA may be due to non-specific process such as effect of medium change or change of incubation temperature on mRNA induction. Figure 5B showed that time course of expression of ppt-a mRNA in normal KCs stimulated with exogenous SP. Peak time of expression of ppt-a mRNA is 10 h after stimulation with SP (Fig. 5B). To confirm expression of mRNA ppt-a, we then performed Southern-blot analysis as
A number of studies reported that substance P (SP) is a potent proinflammatory peptide and evokes an immunoinflammatory response involving proliferation of antigen-induced lymphocyte (16 –18). Matis et al. showed that endogenously released SP regulates the expression of an endothelial-leukocyte adhesion molecule (19). It was reported that SP induces eosinophil infiltration through degranulation of mast cells (20) and topically administrated SP elicits an itching sensation in mouse (15). In this study, we have demonstrated that normal cultured KCs generate endogenous SP in response to exogenous SP but NK-1 antagonist (spantide) did not suppress production of SP by keratinocytes as reported by Ando et al. (15). However, anti-SP Ab inhibited SP induction by keratinocytes and blockage of SP synthesis, cyclo-
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FIG. 5. Substance P gene expression in substance P-stimulated normal KCs. To clarify the SP gene expression in normal KC, we performed RT-PCR and Southern blot analysis. Under nonstimulated conditions, normal keratinocytes express ppt-a mRNA expression and addition of exogenous SP augmented ppt-a mRNA expression in normal KCs dose dependently (A). This basal expression of ppt-a mRNA may be due to nonspecific process such as effect of medium change or change of incubation temperature on mRNA induction. B showed that time course of expression of ppt-a mRNA in normal KCs stimulated with SP. Specificity of mRNA expression of ppt-a we then performed Southern blot analysis using as described in Materials and Methods (A and B). The values were determined by scanning densitometry and expressed in relation to b-actin scanning density (C and D).
heximide, suggest that SP might be synthesized by conventional ribosomal mechanism (21). This is the first report that SP is induced by epithelial cells in response to exogenous SP. These results provide very important clues for the understanding of physical stimuli-induced cutaneous diseases such as atopic dermatitis, chronic urticaria, psoriasis or autoimmune cutaneous diseases. Because numerous previous studies suggest that these stress-aggregated diseases are precipitated by stress-induced mediators such as SP (22), CGRP (23), adrenaline (24) or glucocorticoid (25) all of which have been believed to be released from cutaneous nerve endings except glucocorticoid under the control of neuroendocrine axis. Itching is the predominant symptom, among these cutaneous disease, i.e. atopic dermatitis, urticaria and pruriginous diseases. However, its mechanism is ill-understood. It was well known that mediators causing itch in inflamed skin is histamine (1), pros-
taglandin (2) and SP (3). Among these, SP plays central role to mediate peripheral itchy sensation because SP causes itching due to histamine release from mast cells (4). SP has been recognized as peptide of neuron origin for a long period; however, it has been reported that SP can be identified in nonneural cells, human eosinophils (6), Leydig cells of testis (7), endothelial cells (8), lymphocytes (9 –10) or macrophage lineage cells (11). In addition, nonneural cells, such as human keratinocytes (12), fibroblasts (26) and endothelial cells (12) express neurokinin receptor. It was reported that the distribution density of the cutaneous nerve fibers of atopic skin was much higher than in normal controls (27) and SP not only activates the keratinocytes to contribute to the initiation and perpetuation of the local immune reaction by expression of ICAM-1 and production of cytokine, IL-1a, TNFa and IL-8 (28) but also stimulates murine epidermal keratinocyte proliferation
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FIG. 6. Effect of various agents on substance P production in normal KCs. In addition to SP, several biological active substances modulate KCs functions in both in vitro and in vivo. We next screened several agents on SP production by normal KC. As shown in A–D, relatively low but significant SP was induced by high concentration of acetylcholine, adrenaline, CGRP and histamine. Results are expressed as the means 6 SE.
(29) suggesting that SP released by nerve terminals might act as first messenger of SP induction in epidermis. In summary, KC-derived SP might play an important role in induction and acceleration of certain cutaneous diseases precipitated by stress and physical stimuli and provide new clues for the understanding and treatment of these diseases. REFERENCES 1. Davies, M. G., and Greaves, M. W. (1980) J. Clin. Pharmacol. 9, 461– 465. 2. Greaves, M. W., and McDonald, G. W. (1973) BMJ ii, 608 – 609. 3. Hagemark, O., Hokfelt, T., and Pernow, B. (1978) J. Invest. Dermatol. 71, 233–235. 4. Jorrizo, J. L., Coutts, A. A., Eady, R. A., and Greaves, M. W. (1983) Eur. J. Pharmacol. 94, 67–76. 5. Lynn, B. (1992) Skin Pharmacol. 5, 9 –13.
6. Aliakbari, J., Sreedharan, S. P., Turck, C. W., and Goetzl, E. J. (1987) Biochem. Biophys. Res. Commun. 148, 1440 –1445. 7. Chiwakata, C., Blackmann, B., Hunt, N., Davidoff, M., and Schulze, W. (1991) Endocrinology 128, 2441–2448. 8. Linnik, M. D., and Moscowitz, M. A. (1989) Peptides 10, 957–962. 9. Pernow, B. (1983) Pharmacol. Rev. 35, 85–133. 10. Lai, J. P., Douglas, S. D., and Ho, W. Z. (1998) J. Neuroimmunol. 86, 80 – 86. 11. Ho, W. Z., Lai, J. P., Zhu, X. H., Uvaydova, M., Hu, B. F., and Douglas, S. D. (1997) J. Immunol. 159, 5654 –5660. 12. Bunnett, N. W., Song, I. S., and Grady, E. F. (1996) J. Invest. Dermatol. 106, 814. 13. Lichtenstein, A. V., Moiseev, V. L., and Zaboikin, M. M. (1990) Anal. Biochem. 191(1), 187–91. 14. Couture, R., and Regoli, D. (1981) Can. J. Physiol. Pharmacol. 59, 621– 625. 15. Andoh, T., Nagasawa, T., Satoh, M., and Kuraishi, Y. (1998) J. Pharmacol. Exp. Ther. 286(3), 1140 –1145. 16. Okamoto, Y., Shiratori, K., Kudo, K., Ishikawa, K., Ito, E., Togawa, K., and Saito, I. (1993) J. Immunol. 151, 4391– 4398.
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17. Pascual, D. W., Xu-Amano, J., Kiyono, H., McGhee, J. R., and Bost, K. L. (1991) J. Immunol. 146, 2130 –2136. 18. Yokote, R., Yagi, H., Furukawa, F., and Takigawa, M. (1998) Arch. Dermatol. Res. 290, 191–197. 19. Matis, W. L., Lavker, R. M., and Murphy, G. F. (1990) J. Invest. Dermatol. 94(4), 492– 495. 20. Matsuda, H., Kawakita, K., Kiso, Y., Nakano, T., and Kitamura, Y. (1989) J. Immunol. 142(3), 927–931. 21. Harmar, A., Schofield, J. G., and Keen, P. (1980) Nature 284, 267–269. 22. Fehder, W. P., Sachs, J., Uvaydova, M., and Douglas, S. D. (1997) Neuroimmunomodulation 4(1), 42– 48. 23. Tidgren, B., Theodorsson, E., and Hjemdahl, P. (1991) Clin. Physiol. 11(1), 9 –19.
24. Grant, P. J. (1990) Blood Coagul. Fibrinolysis 1(3), 299 –306. 25. Sternberg, E. M., Glowa, J. R., Smith, M. A., Calogero, A. E., Listwak, S. J., Aksentijevich, S., Chrousos, G. P., Wilder, R. L., and Gold, P. W. (1992) Brain Res. 570(1–2), 54 – 60. 26. Parenti, A., Amerini, S., Ledda, F., Maggi, C. A., and Ziche, M. (1996) Naunyn-Schmiedebergs Arch. Pharmacol. 353, 475– 481. 27. Urashima, R., and Mihara, M. (1998) Virchows Arch. 432(4), 363–370. 28. Viac, J., Gueniche, A., Doutremepuich, J. D., Reichert, U., Claudy, A., and Schmitt, D. (1996) Arch. Dermatol. Res. 288(2), 85–90. 29. Paus, R., Heinzelmann, T., Robicsek, S., Czarnetzki, B. M., and Maurer, M. (1995) Arch. Dermatol. Res. 287(5), 500 –502.
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Autocrine Induction of Substance P mRNA and Peptide in Cultured Normal Human Keratinocytes SangJae Bae, 1 Yoshitaka Matsunaga, Yoichi Tanaka, and Ichiro Katayama Department of Dermatology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki, Japan 852-8501
Received August 5, 1999
In this study, we have demonstrated that normal cultured keratinocytes (KCs) could generate significant endogenous substance P (SP) in a dose- and timedependent response to exogenous SP by sensitive ELISA assay and express preprotachinin-a mRNA by RT-PCR and Southern blotting. We performed immunohistochemical analysis to confirm the presence of SP in cultured keratinocytes. In contrast, adrenaline, acetylcholine, histamine and CGRP induced only low amount of SP from cultured normal human KCs. This is the first report that SP can be induced by skin epithelial cells in response to exogenous SP and KC derived SP might play an important role in induction and acceleration of certain cutaneous diseases. © 1999 Academic Press
Key Words: substance P; itch-scratch cycle; stress; keratinocyte.
Itching is the predominant symptom among inflammatory cutaneous diseases, such as atopic dermatitis, urticaria and pruriginous diseases. However, its mechanism is ill-understood. It is well known that mediators causing itch in inflamed skin are histamine (1), prostaglandin (2) and substance P (3). Among these, SP plays central role to mediate peripheral itchy sensation because Jorizzo et al. reported that intradermal injection of SP causes itching due to histamine release from mast cells (4) and capsaicin depletes cutaneous nerve endings of substance P resulting in inhibition of itch-scratch cycle in certain pruritic skin disorders (5). Substance P has been recognized as a peptide of neuron-origin for a long period. However, it has been reported that SP gene expression can be identified in non-neural cells, such as human eosinophils (6), Leydig cells of testis (7), endothelial cells (8), or lymphocytes (9, 10). Recently, Ho et al. have shown that human monocytes and macrophages express mRNA encoding SP and its receptor and produce SP (11). It was re1
To whom correspondence should be addressed. Fax: 095-8497335.
ported that human keratinocytes express neurokinin receptor (12). Therefore, in this study, we addressed the question whether cultured human keratinocytes could express SP and possible presence of neuroendocrine circuit in human skin. MATERIALS AND METHODS Reagents. Substance P (lot: 58H 5800), CGRP and histamine were obtained from Sigma Chemical Co. (St. Louis, MO); acetylcholine and adrenaline were purchased from Wako Pure Chemicals (Tokyo, Japan). For immunostaining, polyclonal rabbit anti-human substance P antibody (Chemicon, International, Inc., Temecula, CA; dilution 1:1000) and FITC labeling anti-rabbit immunoglobulin (Dako, Denmark; dilution 1:30) were used. Keratinocyte culture. Primary normal human keratinocytes were purchased from cell systems (Sanko Junyaku Co., LTD. Japan). These were pooled cells derived from neonatal donors. Each batch of cells used for the experiments reported in Figs. 1, 2, 3, and 6. Cells were cultured in serum-free, modified KGM CC-3111 medium (Sanko Junyaku Co., Ltd., Japan) containing bovine pituitary extract 15 mg/500 ml, epidermal growth factor 0.5 mg/500 ml, insulin 2.5 mg/500 ml, antibiotics 0.5 ml/500 ml and hydrocortisone 0.25 mg/500 ml. Cultures were maintained in a humidified incubator controlled at 37°C and having an atmosphere of 5% CO 2. The culture medium was changed every 3 days, and the cells were subcultured at subconfluence at a split ratio of about 1:3. After the fourth passage, the cells seeded at 1 3 10 5 cells/cm 2 in 6 wells culture cluster (Corning, NY). At confluent state, the culture medium replaced with fresh medium supplemented with the stimulants described in the text, but without hydrocortisone. The culture supernatants were harvested and stored at 280°C until use. For mRNA detection, the cells were lysed with lysis buffer as described below. Substance P determination by ELISA. To investigate the peptide of SP level, culture supernatants were subjected to a competitiveenzyme immunoassay kit (Cayman, Ann Arbor, MI) as Ho et al. (11). Briefly, the assay is based on the competition between free SP and a SP tracer (SP linked to an acetylcholinesterase molecule) for a limited number of SP-specific rabbit antiserum binding sites. The amount of SP tracer that is able to bind to the rabbit antiserum will be inversely proportional to the concentration of free SP in the well. Immunohistochemical analysis of substance P expression in keratinocytes. The keratinocytes were cultured in chamber slide until confluent and then stimulated with 10 28 M of SP in fresh medium for 24 h and then washed with PBS. As positive control, serial 7 mm cryostat sections from atopic dermatitis were prepared. SP immunostaining were obtained using rabbit anti-serum (dilution 1:1000) and
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FIG. 1. Time course and dose-dependent production of SP by cultured KCs. Normal human keratinocytes were cultured in KGM CC-3111 medium (complete medium) supplemented with BPE, hEGF, insulin, antibiotics and hydrocortisone as described above. At semiconfluent stage, the cells were washed with ice-chilled DMEM and replaced with KGM medium without hydrocortisone, for 24 h. At the start of the experiment, the medium was replaced with fresh KGM medium containing variously diluted SP and then culture supernatants were collected at various times and donated for ELISA assay. (A) Dose response effect of SP production in normal keratinocytes. (B) Time course of SP-induced SP production in normal keratinocytes. Results are expressed as the means 6 SD.
then antibody-antigen complex were visualized with FITC labeling immunoglobulin (dilution 1:30). RT-PCR analysis of substance P mRNA expression. Total RNA was extracted from the cultured keratinocytes by RNeasy Mini Kit (QIAGEN, Japan). cDNA synthesis was performed by Superscript II (Gibco BRL, Rockville, MD) according to the method by the manufacturer. In short, the cycle condition is 94°C for 9 min, followed 94°C for 30 s, 50°C for 30 s and 70°C for 45 s: 40 cycles for substance P, 35 cycles for b-actin) by Takara Ex Taq (Takara Shuzo, Shiga, Japan) using 59 and 39 primer sets and b-actin (Clonetech, Palo Alto, CA). Southern blotting analysis of substance P mRNA analysis. The amplified products were blotted onto Hybond-N1 membrane (Amersham, Buckinghamshire, England) using standard methods (13). Oligonucleotide probe of human substance P was labeled with peroxidase complexes, hybridized and detected using ECL direct nucleic acid labeling and detection systems (Amersham, Buckinghamshire, England) according to the methods by the manufacture. Statistical analysis. tistical significance.
Paired t-test was used to determine the sta-
RESULTS Induction of Substance P by Cultured Normal Human Keratinocytes (KCs) by SP At the start of experiment, the medium was replaced with fresh KGM medium containing variously diluted SP and then culture supernatants were collected at various time for ELISA assay. As results are shown in Fig. 1A and Fig. 1B, SP induced SP in normal keratinocytes in a dose dependent and autocrine fashion (Fig. 1A). SP concentration without KC
was unchanged during the culture period possibly due to the absence of degradating enzyme in KGM medium. While endogenous SP is stable in plasma, synthetic SP added to human plasma is rapidly destroyed. The degradation is temperature-dependent with a much larger loss of activity at 37°C than at 0°C. SP is inactivated at 37°C the half-life in plasma being 12–15 min (14). It is suggesting that SP added into medium in incubator at 37°C might be rapidly destroyed. However, SP concentration of medium with KCs was augmented in a dose and time dependent fashion. The peak response of SP production by KCs is at 10 h after SP stimulation respectively (Fig. 1B). To clarify the de novo synthesis of SP by KCs, KCs were preincubated with SP for several times, washed in fresh medium SP 3 times, and then incubated for another 18 h in the presence of SP. As is shown in Fig. 2A, SP was newly generated by KCs in a incubation timedependent fashion. This was further confirmed by the blockade of SP production by cycloheximide (Fig. 2B). To exclude the possibility that cell associated SP is responsible for SP secretion into the medium during possible apoptotic process, cell associated SP was measured after SP stimulation. As shown, in Fig. 2C, cell associated SP in SP stimulated-KCs after several freeze and thaw treatment was unchanged at 3 and 18 h after stimulation.
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FIG. 2. Kinetics of de novo synthesis of SP by KCs. (A) To exclude the carry over of SP by exogenously added SP, KCs were stimulated with SP several times and then washed fresh medium without SP three times, incubated for another 18 h, and secreted SP was measured by ELISA. (B) Effect of cycloheximide on SP-induced SP production by KCs was studied by addition of various concentrations of cycloheximide to keratinocyte cultures and secreted SP was measured ELISA. (C) To exclude the possibility that cell associated SP is responsible for SP secretion into the medium during possible apoptotic process, cell associated SP was measured after SP stimulation. Results are expressed as the means 6 SD (B and C).
Effect of Substance P Antagonists on Substance P Production by KCs
dilution) clearly downregulated the SP production (Fig. 3B).
We then analyzed the effect of SP antagonists on SP induced-SP production by keratinocytes. As shown, NK1 antagonist, spantide did not inhibit SP production by KC rather augmented its autocrine production in a dose dependent fashion as reported by Ando et al. (15) (Fig. 3A). In contrast, anti-SP antibody (at 1:1000
Presence of Cytoplasic Substance P in Normal KCs To confirm the presence of SP in normal keratinocytes, we then performed immunohistochemical analysis. Normal human KCs were cultured on chamber slide glass (Nunc Inc., IL) and stimulated with SP for
FIG. 3. Effect of substance P antagonists on substance P production by KCs. (A) NK1 antagonist spantide was added to the culture and SP production by KCs was measured by ELISA to study the effect of SP antagonists on SP-induced SP production by keratinocytes. (B) Anti-SP antibody was added to the culture and SP production by KC was measured by ELISA. Results are expressed as the means 6 SD. 329
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FIG. 4. Presence of cytoplasic substance P in normal KCs. To confirm the presence of SP in normal keratinocytes, we then performed immunohistochemical analysis. Normal human KCs were cultured on chamber and at semiconfluent stage, the cells were fixed with 4% paraformaldehyde and then treated with polyclonal rabbit anti-P antibody followed by FITC-labeled anti-rabbit immunoglobulin. (A) Immunohistochemical staining of SP positive cultured KCs. (B) Negative control using SP-absorbed anti-SP Ab.
24 h, at semiconfluent stage, the cells were washed with PBS (2) for 3 times and fixed with 4% paraformaldehyde and then treated with polyclonal rabbit anti-SP antibody followed by FITC-labeled anti-rabbit immunoglobulin. Specific cytoplasmic staining of SP in normal KCs was observed in SP stimulated KCs (Fig. 4A). As negative control, we performed immunostaining using SP absorbed anti-SP antibody solution (Fig. 4B). In short, SP (5 mg) was dissolved with 1.5 ml PBS plus 1.5 ml anti-SP Ab, and shaken for 4 h at 4°C. Then, SP plus anti-SP Ab solution was poured into a visking tube and dialyzed 3 times with PBS. Substance-P Gene Expression in Substance P Stimulated Normal KCs
described in Materials and Methods (Fig. 5A and 5B). Figure 5C showed that the expressed ppt-a mRNA and b-actin bands are calculated by using NIH image. Effect of Various Agents on Substance P Production in Normal KCs In addition to SP, several biological active substances modulate KC functions in both in vitro and in vivo. We next screened several agents on SP production by normal KCs. As shown in Fig. 6A– 6D, relatively low compound to SP but significant SP was induced by high concentration of acetylcholine, adrenaline, histamine and CGRP (Fig. 6A– 6D). DISCUSSION
To clarify the SP gene expression in normal KCs, we performed RT-PCR and Southern blot analysis. As is described in Materials and Methods, first we performed RT-PCR using preprotachynin-A (ppt-a) specific primers. In non-stimulated condition, normal keratinocytes express moderate amount of ppt-a mRNA expression and addition of exogenous SP significantly augmented ppt-a mRNA expression a dose dependent fashion in normal KCs (Fig. 5A). This basal expression of ppt-a mRNA may be due to non-specific process such as effect of medium change or change of incubation temperature on mRNA induction. Figure 5B showed that time course of expression of ppt-a mRNA in normal KCs stimulated with exogenous SP. Peak time of expression of ppt-a mRNA is 10 h after stimulation with SP (Fig. 5B). To confirm expression of mRNA ppt-a, we then performed Southern-blot analysis as
A number of studies reported that substance P (SP) is a potent proinflammatory peptide and evokes an immunoinflammatory response involving proliferation of antigen-induced lymphocyte (16 –18). Matis et al. showed that endogenously released SP regulates the expression of an endothelial-leukocyte adhesion molecule (19). It was reported that SP induces eosinophil infiltration through degranulation of mast cells (20) and topically administrated SP elicits an itching sensation in mouse (15). In this study, we have demonstrated that normal cultured KCs generate endogenous SP in response to exogenous SP but NK-1 antagonist (spantide) did not suppress production of SP by keratinocytes as reported by Ando et al. (15). However, anti-SP Ab inhibited SP induction by keratinocytes and blockage of SP synthesis, cyclo-
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FIG. 5. Substance P gene expression in substance P-stimulated normal KCs. To clarify the SP gene expression in normal KC, we performed RT-PCR and Southern blot analysis. Under nonstimulated conditions, normal keratinocytes express ppt-a mRNA expression and addition of exogenous SP augmented ppt-a mRNA expression in normal KCs dose dependently (A). This basal expression of ppt-a mRNA may be due to nonspecific process such as effect of medium change or change of incubation temperature on mRNA induction. B showed that time course of expression of ppt-a mRNA in normal KCs stimulated with SP. Specificity of mRNA expression of ppt-a we then performed Southern blot analysis using as described in Materials and Methods (A and B). The values were determined by scanning densitometry and expressed in relation to b-actin scanning density (C and D).
heximide, suggest that SP might be synthesized by conventional ribosomal mechanism (21). This is the first report that SP is induced by epithelial cells in response to exogenous SP. These results provide very important clues for the understanding of physical stimuli-induced cutaneous diseases such as atopic dermatitis, chronic urticaria, psoriasis or autoimmune cutaneous diseases. Because numerous previous studies suggest that these stress-aggregated diseases are precipitated by stress-induced mediators such as SP (22), CGRP (23), adrenaline (24) or glucocorticoid (25) all of which have been believed to be released from cutaneous nerve endings except glucocorticoid under the control of neuroendocrine axis. Itching is the predominant symptom, among these cutaneous disease, i.e. atopic dermatitis, urticaria and pruriginous diseases. However, its mechanism is ill-understood. It was well known that mediators causing itch in inflamed skin is histamine (1), pros-
taglandin (2) and SP (3). Among these, SP plays central role to mediate peripheral itchy sensation because SP causes itching due to histamine release from mast cells (4). SP has been recognized as peptide of neuron origin for a long period; however, it has been reported that SP can be identified in nonneural cells, human eosinophils (6), Leydig cells of testis (7), endothelial cells (8), lymphocytes (9 –10) or macrophage lineage cells (11). In addition, nonneural cells, such as human keratinocytes (12), fibroblasts (26) and endothelial cells (12) express neurokinin receptor. It was reported that the distribution density of the cutaneous nerve fibers of atopic skin was much higher than in normal controls (27) and SP not only activates the keratinocytes to contribute to the initiation and perpetuation of the local immune reaction by expression of ICAM-1 and production of cytokine, IL-1a, TNFa and IL-8 (28) but also stimulates murine epidermal keratinocyte proliferation
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FIG. 6. Effect of various agents on substance P production in normal KCs. In addition to SP, several biological active substances modulate KCs functions in both in vitro and in vivo. We next screened several agents on SP production by normal KC. As shown in A–D, relatively low but significant SP was induced by high concentration of acetylcholine, adrenaline, CGRP and histamine. Results are expressed as the means 6 SE.
(29) suggesting that SP released by nerve terminals might act as first messenger of SP induction in epidermis. In summary, KC-derived SP might play an important role in induction and acceleration of certain cutaneous diseases precipitated by stress and physical stimuli and provide new clues for the understanding and treatment of these diseases. REFERENCES 1. Davies, M. G., and Greaves, M. W. (1980) J. Clin. Pharmacol. 9, 461– 465. 2. Greaves, M. W., and McDonald, G. W. (1973) BMJ ii, 608 – 609. 3. Hagemark, O., Hokfelt, T., and Pernow, B. (1978) J. Invest. Dermatol. 71, 233–235. 4. Jorrizo, J. L., Coutts, A. A., Eady, R. A., and Greaves, M. W. (1983) Eur. J. Pharmacol. 94, 67–76. 5. Lynn, B. (1992) Skin Pharmacol. 5, 9 –13.
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