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Specific Binding and Lack of Growth-Promoting Activity of Substance P in Cultured Human Keratinocytes
LETTERS TO THE EDITOR
Specific Binding and Lack of Grow^th-Promoting Activity of Substance P in Cultured Human Keratinocytes T o the Editor: T h e neuropeptide substance P (SP) is presumed to be an important inflammatory mediator in the skin, and has been suggested to be involved in the pathogenesis of certain inflammatory skin diseases such as psoriasis [1]. However, the exact mode of action of SP in skin is still not fully clarified and data available on this issue remain contradictory. SP was found to stimulate the proliferation of a mouse transformed keratinocyte cell line [2]. In accordance with this, specific binding sites for SP has been detected on mouse keratinocytes and this neuropeptide was able to induce cytokine synthesis in these cells [3]. On the other hand, in a recent issue of the Journal Pincelli et al [4] reported that SP not only failed to stimulate the growth of cultured normal human keratinocytes as measured by counting cell number, but also blocked the vasoactive intestinal polypeptide (VIP)-induced stimulation of these cells. Recently, we were interested in whether normal human keratinocytes possess SP receptors, and the effect of SP on the proliferation of cultured normal human keratinocytes was also studied [5]. Keratinocytes were isolated from newborn foreskin and cultured as described earlier [6]. After second passage and reaching confluency, cells were transferred to 24-multiwell tissue-culture trays, and after adhering prepared for receptor studies. To characterize the SP binding sites, radioligand binding assays were performed. Cell monolayers were washed three times with ice-cold phosphate-buffered saline and incubated with increasing concentrations of ['^^I]-SP (specific activity 2200 Ci/mmol; New England Nuclear/DuPont, Dreieich, FRG) in the range of 0.05-10 nM in a final volume of 400 fll per well. The incubation was performed for 120 min at 4°C under continuous shaking. Nonspecific binding was determined in the presence of a thousandfold excess of unlabeled ligand. Saturation curves were analysed for Kd and Bmax using a computerized non-linear curve fitting program MxN-FIT [7]. Incubation of keratinocytes with increasing concentrations of [^^^I]-SP revealed increasing specific binding ofthe ligand to the cells (Fig 1). Batchanalysis of the binding data revealed a Kd of 8 nM and Bmax of 46,000 binding sites per cells. The Scatchard plot analysis demon-
a 3
r 0 Oourid [pM]
1 ? 1
Figure 1. Concentration dependence of specific and nonspecific [»25j].sp binding to cultured normal human keratinocytes. Values represent mean + SD offiveexperiments performed in duplicates. The inset shows Scatchard analysis of the data.
0022-202X/94/S07.00
IIBEGF
24 h
48 h
72 h
Incubation (h)
Figure 2. Effects of 10"' M SP and lO"' M EGF on the growth of cultured human keratinocytes. Measurements were made by counting cell number and 'H-thymidine incorporation. Values repersent mean ± SD of five experiments performed in duplicates. * p < 0.05 as compared with the 0.5% fetal bovine serum - treated control. strated that SP interacted with a single class of binding sites under the experimental conditions used. To study the effect of SP on the growth of human keratinocytes, cultured cells were plated in six-well plates. After 48 h the culture medium was removed and the cells were exposed to medium containing 0.5% fetal bovine serum with or without 10~' M SP and/or 10-" M epidermal growth factor (EGF). Cell proliferation was determined by counting cell number and ^H-thymidine incorporation as described earlier [8]. Our data demonstrate that SP had no effect on the growth of human keratinocytes as measured by counting cell number and 'H-thymidine incorporation (Fig 2). In addition, SP did not influence the EGF-induced increase in cell number and •'H-thymidine incorporation. This latter observation suggests that the reported inhibition of VIP-induced cell-proliferation by SP [4] is a rather specific effect. Our data apparently contradict the findings reported in the mouse system [2] and support the observations reported by Pincelli et al [4]. Furthermore, we could show for the first time that cultured normal human keratinocytes possess specific binding sites for SP. The exact biochemical characterization of the SP binding sites needs further investigations. This work was supported by Deutsche Forschungsgemeinsehaft (Ru 292/4-1).
Copyright © 1994 by The Society for Investigative Dermatology, Inc. 605
lEGF+SP
606
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
LETTERS TO THE EDITOR
Lajos Kem^ny Department of Dermatology Albert Szent-Gyorgyi Medical University Szeged, Hungary Birgitt von RestorfF Glinter Michel Thomas Ruzicka Department of Dermatology University of Munich Munich, FRG REFERENCES 1. 2. 3. 4.
5. 6. 7. 8.
Farber EM, Nickoloff BJ, Rccht B, Fraky JE: Stress, symmetry, atid psoriasis: possible role of neuropeptides.J/4m Acad Dermatol 14:305-311, 1986 Tanaka T, Danno K, Ikai K, Imamura S: Effects of substance P and substance K on the growtb of cultured keratinocytes.J Invest Dermatol 90:399-401, 1988 Ansel J, Perry P, Brown J, Damm D, Pban T, Hart C, Luger T, Hefeneider S: Cytokine modulation of keratinocyte cytokines. / Invest Dermatol 94:101S107S, 1990 Pincelli C, Fantini F, Romualdi P, Sevignani C, Lesa G, Benassi L, Giantietti A: Substance P is diminished and vasoactive intestinal peptide is augmented in psoriatic lesions and these peptides exert disparate effects on tbe proliferation of cultured buman keimnocyKs.] Invest Dermatol 98:421-427, 1992 Restorff B, Kemeny L, Michel G, Ruzicka T: Specific binding of substance P in normal bumati keratinocytes (abstr).y/iii'esf Dermatol 98:510, 1992 Aretiberger P, Kemeny L, Ruzicka T: Defect of epidermal 12(S)-hydroxyeicosatetraenoic acid receptors in psoriasis. EurJ Clin Invest 22:235-243, 1992 Burgisser EJ: MxN-FIT. An IBM-PC program for analysis of complex binding data according to the law of mass action. Rbeinfelden, BURECO AG, 1988 Gross E, Ruzicka T, von Restorff B, Stolz W, Lotz K-N: Higb-affinity binding and lack of growtb-promoting activity of 12(S)-bydroxyeicosatetraenoic acid (12(S)-HETE) in a human epidermal cell line. J Invest Dermatol 94:446-451, 1990
To the Editor: We read with interest the article by Garmyn et al regarding the effect of aging and sun exposure on the genetic response of cultured human keratinocytes [1]. The authors report that ultraviolet B (UVB) irradiation of human keratinocyte cultures with 16 mj/cm^ (160 J/m^) increases the mRNA level for interleukin-lyS (IL-1;8) fourfold over control levels 8 h post-irradiation but does not induce the mRNA for IL-la at the time points studied (4 and 8 h). Recently we studied the induction of IL-la in normal neonatal human keratinocytes (Clonetics), in the squamous cell carcinoma lines COLO-16 and SCC as well as the epidermal carcinoma cell line A431, Although previous reports using Sl-nuclease [2] and northern analysis [3] of IL-1 gene expression have purported to show that both IL-la and IL-1;9 mRNA levels increase markedly 6-24 h post-irradiation, we could find no reproducible evidence that IL-la mRNA levels were increased significantly over the unirradiated controls 1-48 h post-irradiation. We used UVB doses of 30-150 J/m^ in an irradiation protocol essentially the same as used by Garmyn et al but determined the expression of IL-la message using the sensitive reverse transcription-polymerase chain reaction (RT-PCR) protocol [4] with labeled primers. In none of the cell lines or normal human keratinocytes were we able to detect significant upregulation of IL-la message. The integrity of the extracted RNA and effectiveness of the reverse transcription and polymerase reactions was supported by the presence of strong signals for both IL-la and the housekeeping gene/?-actin in control cultures. We had also previously carried out experiments to ensure that our polymerase chain reaction (PCR) product yield was linear with respect to both template concentration and PCR cycle number. At 25 cycles, we were well within the exponential portion of the PCR, However, under similar PCR conditions in normal human keratinocytes we were able to demonstrate that IL-8 mRNA elevated twelvefold above control levels 12 h post-irradiation with 70 J/m^ UVB, Interleukin-la was actually suppressed to 25% of control level at the same time point. Similarly, in A431 cells, IL-la was not induced by 35-150J/m^ at 1-48 h post-irradiation. In
these same samples we were able to show that the melanoma growth factor activity (GRO) mRNA was ninefold greater than unirradiated control levels 24 h after irradiation with 100 J/m^ and that IL-8 mRNA was increased twelvefold in these cells 24 h post-irradiation with 70 J/m^- Other attempts including use of early passage cells, different batches of the cell lines from other laboratories, and changing the serum content of the growth media for the cell lines were to no avail. We therefore conclude that keratinocyte ILl a mRNA was not significantly increased by UVB. Several investigators have shown that UVB irradiation increases the release of IL-1 activity from keratinocyte cultures [5,6] and that UVB irradiation of normal human volunteers increases IL-1 activity in the skin [7,8] and in the serum [9], Keratinocytes are well known to contain pre-formed IL-1; we suggest that in the absence of UVB induction of IL-1 mRNA that the increased IL-la protein results from leakage from UVB-damaged cells or possibly from post-transcriptional regulation of IL-1 expression. Post-transcriptional regulation by UV has been demonstrated in a recent study on the UV induction of transforming growth factor-a (TGF-a) in melanoma cells showing that TGF-a protein is elevated in the absence of any increase in TGF-a mRNA [10], Roderick C. Mckenzie Division of Dermatology University of Toronto Kevin A, Galley Department of Surgical Research Hospital for Sick Children Thomas J, Venner Division of Dermatology Seiji Kondo Division of Dermatology Daniel N, Sauder Division of Dermatology University of Toronto Toronto, Ontario, Canada
REFERENCES 1. 2.
3. 4. 5.
6.
7. 8. 9. 10.
Garmyn M, Yaar M, Boileau N, Backendorfe C, Gilcbrest BA: Effect of aging and habitual sun exposure on the genetic response of cultured human keratinocytes to solar-simulated irradiation.y/nral Dermatol 99:743-748, 1992 KupperTS, Chua AO, Flood P, McGuireJ, Gubler U: Interleukin-1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation./ Clin Invest 88:430-436, 1980 Ansel JC, Luger TA, Quinn I: The effect of in vitro and in vivo UV irradiation on the productioti of ETAF activity by buman and murine keratinocytes.y/ni'a' Dcrmato/81:619-624, 1983 O'Garra A, Vieira P: Polymerase chain reaction for detection of cytokine gene expression. Curr Opin Immunol 4:211-215, 1992 Gahring L, Baltz M, Pepys MB, Daynes R: Effect of ultraviolet irradiation on production of epidermal cell tbymocytc-activating factor/interleukin-1 in vivo and in vitro. Proc Natl Acad Sci USA 81:1198- 1202, 1984 Nozaki S, Abrams JS, Pearce MK, Sauder DN: Augmentation of granulocyte/ tnacropbage colony-stitnulating factor expression by ultraviolet irradiation is mediated by interleukin-1 in Pam 212 keratinocytes.J Jni'esf Dermatol 97:1014, 1991 Oxholm A, Oxholtn P, Starberg B, Bendtzen K: Immunobistological detection of interleukin-1-like molecules and tumor necrosis factor in human epidermis before and after UVA-irradiation in vivo. BrJ Dermatol 118:369-377, 1988 Murphy GM, Dowd PN, Hudspith BN, Brostoff J, Greaves MW: Local increase in interleukin-1 -like activity following UVB irradiation of human skin in vivo. Photodermatol 6:26»-21A, 1989 Granstein R, Sauder DN: Whole body exposure to ultraviolet irradiation results in increased serum interleukin-1 activity in bumans. Lymphokine Res 6:187193, 1987 Chenevix-Trench G, Cullinan M, Ellem KAO, Hayward NK: UV induction of transforming growth factor a in melanoma cell lines is a posttranslational event.J Celt Physiol 152:328-336, 1992
Specific Binding and Lack of Grow^th-Promoting Activity of Substance P in Cultured Human Keratinocytes T o the Editor: T h e neuropeptide substance P (SP) is presumed to be an important inflammatory mediator in the skin, and has been suggested to be involved in the pathogenesis of certain inflammatory skin diseases such as psoriasis [1]. However, the exact mode of action of SP in skin is still not fully clarified and data available on this issue remain contradictory. SP was found to stimulate the proliferation of a mouse transformed keratinocyte cell line [2]. In accordance with this, specific binding sites for SP has been detected on mouse keratinocytes and this neuropeptide was able to induce cytokine synthesis in these cells [3]. On the other hand, in a recent issue of the Journal Pincelli et al [4] reported that SP not only failed to stimulate the growth of cultured normal human keratinocytes as measured by counting cell number, but also blocked the vasoactive intestinal polypeptide (VIP)-induced stimulation of these cells. Recently, we were interested in whether normal human keratinocytes possess SP receptors, and the effect of SP on the proliferation of cultured normal human keratinocytes was also studied [5]. Keratinocytes were isolated from newborn foreskin and cultured as described earlier [6]. After second passage and reaching confluency, cells were transferred to 24-multiwell tissue-culture trays, and after adhering prepared for receptor studies. To characterize the SP binding sites, radioligand binding assays were performed. Cell monolayers were washed three times with ice-cold phosphate-buffered saline and incubated with increasing concentrations of ['^^I]-SP (specific activity 2200 Ci/mmol; New England Nuclear/DuPont, Dreieich, FRG) in the range of 0.05-10 nM in a final volume of 400 fll per well. The incubation was performed for 120 min at 4°C under continuous shaking. Nonspecific binding was determined in the presence of a thousandfold excess of unlabeled ligand. Saturation curves were analysed for Kd and Bmax using a computerized non-linear curve fitting program MxN-FIT [7]. Incubation of keratinocytes with increasing concentrations of [^^^I]-SP revealed increasing specific binding ofthe ligand to the cells (Fig 1). Batchanalysis of the binding data revealed a Kd of 8 nM and Bmax of 46,000 binding sites per cells. The Scatchard plot analysis demon-
a 3
r 0 Oourid [pM]
1 ? 1
Figure 1. Concentration dependence of specific and nonspecific [»25j].sp binding to cultured normal human keratinocytes. Values represent mean + SD offiveexperiments performed in duplicates. The inset shows Scatchard analysis of the data.
0022-202X/94/S07.00
IIBEGF
24 h
48 h
72 h
Incubation (h)
Figure 2. Effects of 10"' M SP and lO"' M EGF on the growth of cultured human keratinocytes. Measurements were made by counting cell number and 'H-thymidine incorporation. Values repersent mean ± SD of five experiments performed in duplicates. * p < 0.05 as compared with the 0.5% fetal bovine serum - treated control. strated that SP interacted with a single class of binding sites under the experimental conditions used. To study the effect of SP on the growth of human keratinocytes, cultured cells were plated in six-well plates. After 48 h the culture medium was removed and the cells were exposed to medium containing 0.5% fetal bovine serum with or without 10~' M SP and/or 10-" M epidermal growth factor (EGF). Cell proliferation was determined by counting cell number and ^H-thymidine incorporation as described earlier [8]. Our data demonstrate that SP had no effect on the growth of human keratinocytes as measured by counting cell number and 'H-thymidine incorporation (Fig 2). In addition, SP did not influence the EGF-induced increase in cell number and •'H-thymidine incorporation. This latter observation suggests that the reported inhibition of VIP-induced cell-proliferation by SP [4] is a rather specific effect. Our data apparently contradict the findings reported in the mouse system [2] and support the observations reported by Pincelli et al [4]. Furthermore, we could show for the first time that cultured normal human keratinocytes possess specific binding sites for SP. The exact biochemical characterization of the SP binding sites needs further investigations. This work was supported by Deutsche Forschungsgemeinsehaft (Ru 292/4-1).
Copyright © 1994 by The Society for Investigative Dermatology, Inc. 605
lEGF+SP
606
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
LETTERS TO THE EDITOR
Lajos Kem^ny Department of Dermatology Albert Szent-Gyorgyi Medical University Szeged, Hungary Birgitt von RestorfF Glinter Michel Thomas Ruzicka Department of Dermatology University of Munich Munich, FRG REFERENCES 1. 2. 3. 4.
5. 6. 7. 8.
Farber EM, Nickoloff BJ, Rccht B, Fraky JE: Stress, symmetry, atid psoriasis: possible role of neuropeptides.J/4m Acad Dermatol 14:305-311, 1986 Tanaka T, Danno K, Ikai K, Imamura S: Effects of substance P and substance K on the growtb of cultured keratinocytes.J Invest Dermatol 90:399-401, 1988 Ansel J, Perry P, Brown J, Damm D, Pban T, Hart C, Luger T, Hefeneider S: Cytokine modulation of keratinocyte cytokines. / Invest Dermatol 94:101S107S, 1990 Pincelli C, Fantini F, Romualdi P, Sevignani C, Lesa G, Benassi L, Giantietti A: Substance P is diminished and vasoactive intestinal peptide is augmented in psoriatic lesions and these peptides exert disparate effects on tbe proliferation of cultured buman keimnocyKs.] Invest Dermatol 98:421-427, 1992 Restorff B, Kemeny L, Michel G, Ruzicka T: Specific binding of substance P in normal bumati keratinocytes (abstr).y/iii'esf Dermatol 98:510, 1992 Aretiberger P, Kemeny L, Ruzicka T: Defect of epidermal 12(S)-hydroxyeicosatetraenoic acid receptors in psoriasis. EurJ Clin Invest 22:235-243, 1992 Burgisser EJ: MxN-FIT. An IBM-PC program for analysis of complex binding data according to the law of mass action. Rbeinfelden, BURECO AG, 1988 Gross E, Ruzicka T, von Restorff B, Stolz W, Lotz K-N: Higb-affinity binding and lack of growtb-promoting activity of 12(S)-bydroxyeicosatetraenoic acid (12(S)-HETE) in a human epidermal cell line. J Invest Dermatol 94:446-451, 1990
To the Editor: We read with interest the article by Garmyn et al regarding the effect of aging and sun exposure on the genetic response of cultured human keratinocytes [1]. The authors report that ultraviolet B (UVB) irradiation of human keratinocyte cultures with 16 mj/cm^ (160 J/m^) increases the mRNA level for interleukin-lyS (IL-1;8) fourfold over control levels 8 h post-irradiation but does not induce the mRNA for IL-la at the time points studied (4 and 8 h). Recently we studied the induction of IL-la in normal neonatal human keratinocytes (Clonetics), in the squamous cell carcinoma lines COLO-16 and SCC as well as the epidermal carcinoma cell line A431, Although previous reports using Sl-nuclease [2] and northern analysis [3] of IL-1 gene expression have purported to show that both IL-la and IL-1;9 mRNA levels increase markedly 6-24 h post-irradiation, we could find no reproducible evidence that IL-la mRNA levels were increased significantly over the unirradiated controls 1-48 h post-irradiation. We used UVB doses of 30-150 J/m^ in an irradiation protocol essentially the same as used by Garmyn et al but determined the expression of IL-la message using the sensitive reverse transcription-polymerase chain reaction (RT-PCR) protocol [4] with labeled primers. In none of the cell lines or normal human keratinocytes were we able to detect significant upregulation of IL-la message. The integrity of the extracted RNA and effectiveness of the reverse transcription and polymerase reactions was supported by the presence of strong signals for both IL-la and the housekeeping gene/?-actin in control cultures. We had also previously carried out experiments to ensure that our polymerase chain reaction (PCR) product yield was linear with respect to both template concentration and PCR cycle number. At 25 cycles, we were well within the exponential portion of the PCR, However, under similar PCR conditions in normal human keratinocytes we were able to demonstrate that IL-8 mRNA elevated twelvefold above control levels 12 h post-irradiation with 70 J/m^ UVB, Interleukin-la was actually suppressed to 25% of control level at the same time point. Similarly, in A431 cells, IL-la was not induced by 35-150J/m^ at 1-48 h post-irradiation. In
these same samples we were able to show that the melanoma growth factor activity (GRO) mRNA was ninefold greater than unirradiated control levels 24 h after irradiation with 100 J/m^ and that IL-8 mRNA was increased twelvefold in these cells 24 h post-irradiation with 70 J/m^- Other attempts including use of early passage cells, different batches of the cell lines from other laboratories, and changing the serum content of the growth media for the cell lines were to no avail. We therefore conclude that keratinocyte ILl a mRNA was not significantly increased by UVB. Several investigators have shown that UVB irradiation increases the release of IL-1 activity from keratinocyte cultures [5,6] and that UVB irradiation of normal human volunteers increases IL-1 activity in the skin [7,8] and in the serum [9], Keratinocytes are well known to contain pre-formed IL-1; we suggest that in the absence of UVB induction of IL-1 mRNA that the increased IL-la protein results from leakage from UVB-damaged cells or possibly from post-transcriptional regulation of IL-1 expression. Post-transcriptional regulation by UV has been demonstrated in a recent study on the UV induction of transforming growth factor-a (TGF-a) in melanoma cells showing that TGF-a protein is elevated in the absence of any increase in TGF-a mRNA [10], Roderick C. Mckenzie Division of Dermatology University of Toronto Kevin A, Galley Department of Surgical Research Hospital for Sick Children Thomas J, Venner Division of Dermatology Seiji Kondo Division of Dermatology Daniel N, Sauder Division of Dermatology University of Toronto Toronto, Ontario, Canada
REFERENCES 1. 2.
3. 4. 5.
6.
7. 8. 9. 10.
Garmyn M, Yaar M, Boileau N, Backendorfe C, Gilcbrest BA: Effect of aging and habitual sun exposure on the genetic response of cultured human keratinocytes to solar-simulated irradiation.y/nral Dermatol 99:743-748, 1992 KupperTS, Chua AO, Flood P, McGuireJ, Gubler U: Interleukin-1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation./ Clin Invest 88:430-436, 1980 Ansel JC, Luger TA, Quinn I: The effect of in vitro and in vivo UV irradiation on the productioti of ETAF activity by buman and murine keratinocytes.y/ni'a' Dcrmato/81:619-624, 1983 O'Garra A, Vieira P: Polymerase chain reaction for detection of cytokine gene expression. Curr Opin Immunol 4:211-215, 1992 Gahring L, Baltz M, Pepys MB, Daynes R: Effect of ultraviolet irradiation on production of epidermal cell tbymocytc-activating factor/interleukin-1 in vivo and in vitro. Proc Natl Acad Sci USA 81:1198- 1202, 1984 Nozaki S, Abrams JS, Pearce MK, Sauder DN: Augmentation of granulocyte/ tnacropbage colony-stitnulating factor expression by ultraviolet irradiation is mediated by interleukin-1 in Pam 212 keratinocytes.J Jni'esf Dermatol 97:1014, 1991 Oxholm A, Oxholtn P, Starberg B, Bendtzen K: Immunobistological detection of interleukin-1-like molecules and tumor necrosis factor in human epidermis before and after UVA-irradiation in vivo. BrJ Dermatol 118:369-377, 1988 Murphy GM, Dowd PN, Hudspith BN, Brostoff J, Greaves MW: Local increase in interleukin-1 -like activity following UVB irradiation of human skin in vivo. Photodermatol 6:26»-21A, 1989 Granstein R, Sauder DN: Whole body exposure to ultraviolet irradiation results in increased serum interleukin-1 activity in bumans. Lymphokine Res 6:187193, 1987 Chenevix-Trench G, Cullinan M, Ellem KAO, Hayward NK: UV induction of transforming growth factor a in melanoma cell lines is a posttranslational event.J Celt Physiol 152:328-336, 1992