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Identification and occurrence of mRNAs for components of the kallikrein–kinin system in human skin and in skin diseases
Immunopharmacology 43 Ž1999. 287–291 www.elsevier.comrlocaterimmpharm
Identification and occurrence of mRNAs for components of the kallikrein–kinin system in human skin and in skin diseases Elisabeth Schremmer-Danninger a,b,) , Andrea Hermann Hans Fritz a , Adelbert A. Roscher b
a,1
, Edwin Fink a ,
a
b
Department of Clinical Chemistry and Clinical Biochemistry, Hospital of Surgery, Ludwig-Maximilians-UniÕersity, Munich, Germany Department of Clinical Biochemistry, Childrens Hospital, Ludwig-Maximilians-UniÕersity, Lindwurmstrabe 4, D-80337 Munich, Germany Accepted 3 May 1999
Abstract Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogen ŽHMW and LMW kininogen, respectively.. In the skin, kinins are involved, e.g., as co-mitogens in cellular proliferation or in processes propagating pain and inflammation. The aim of our study was to investigate the specific occurrence of mRNAs for components of the kallikrein–kinin system in normal human skin and in skin biopsies of patients with selected skin diseases Žpsoriasis, lichenificated atopic eczema, basalioma.. In normal skin, reverse transcription polymerase chain reaction ŽRT–PCR. with specific primer pairs followed by separation of products by polyacrylamide gel electrophoresis ŽPAGE. revealed the presence of mRNAs for tissue kallikrein, for the B2 and the B1 bradykinin receptors, but not for kininogen. In biopsies of lichenificated atopic eczema and basalioma, additionally, the mRNAs for HMW and LMW kininogen were detected, whereas in psoriatic skin mRNA for HMW kininogen was not expressed. These differences in mRNA expression may reflect the different contribution of kallikrein–kinin system components to the maintenance of chronic skin diseases like psoriasis. In acute dermal reactions occurring in lichenificated atopic eczema or in basalioma, tissue mRNA for HMW kininogen appears to be arisen from sources not pre-existing in normal skin. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Kallikrein–kinin system; mRNA; Human skin; Skin diseases
1. Introduction AbbreÕiations: PAGE, polyacrylamide gel electrophoresis; RT–PCR, reverse transcription polymerase chain reaction; HMW kininogen, high-molecular weight kininogen; LMW kininogen, low-molecular weight kininogen ) Corresponding author. Department of Clinical Biochemistry, Childrens Hospital, Ludwig-Maximilians-University, Lindwurmstrabe 4, D-80337 Munich, Germany. fax: q49-89-5160-4486; e-mail: [email protected] 1 Present address: November AG, Erlangen, Germany.
Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogens ŽHMW and LMW kininogens, respectively.. These kinins exert a broad spectrum of physiological and also pathophysiological effects including cell proliferation, pain and inflammation.
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 0 0 - 9
288
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
We have previously shown that B2 bradykinin receptors occur in the mitotically active keratinocytes of the basal layer ŽSchremmer-Danninger et al., 1995.. These receptors may mediate kinin effects related to cellular reactions such as keratinocyte proliferation following inflammatory conditions. The occurrence and localization of the other components of the kallikrein–kinin system Žkininogen, kallikrein. or of B1 bradykinin receptors in human skin has not yet been investigated. The specific aim of our study was to find out whether bradykinin can be generated directly from a local source of kininogen or from an external source originating, e.g., from immigrating leucocytes during inflammation. Therefore, we looked for the occurrence of specific mRNAs for components of the kallikrein–kinin system in normal human skin and in skin biopsies of patients with selected skin diseases Žpsoriasis, lichenificated atopic eczema, basalioma..
2. Material and methods 2.1. Tissue samples and RNA preparation Histologically normal adult skin was obtained from areas surrounding dermal naevi excised for suspicion of melanoma; biopsies from psoriatic skin, lichenificated atopic eczema and from basalioma tissue were recruited from patients giving their permission. Tissue samples were rinsed in ice-cold saline and frozen immediately in liquid nitrogen. According to the method described by Chomczynski and Sacchi Ž1987. total cellular RNA was isolated from skin biopsies with an acidic phenol–chloroform extraction. The isolated RNA was spectrophotometrically quantified at 260 nm. Negative control samples were prepared by performing the entire procedure in the absence of tissue. 2.2. ReÕerse transcription, first PCR and second, semi-nested PCR In order to control for potentially contaminating genomic DNA primers for reverse transcription polymerase chain reaction ŽRT–PCR. ŽTable 1. were selected in a way that the PCR products include at least one exon–exon transition. Thus, in case of
genomic DNA contaminating the RNA preparation PCR-products of different length would be obtained. cDNAs were synthezised from 1 mg of total RNA preparations and the negative control preparations following the instruction of the ‘‘First-Strand cDNA Synthesis Kit’’ ŽPharmacia Biotech. using the reverse primers C ŽTable 1.. PCR was performed with 1r10 of the cDNA synthesis reaction mixture or the control incubation and the respective primer pairs ŽTable 1A and C.. Each of the 20 cycles consisted of a denaturation step at 928C for 40 s, an annealing step at 528C for 40 s, and an elongation step at 728C for 40 s. The PCR with the semi-nested primers ŽTable 1A and B. was performed with 1 ml of a 1:10 dilution of the reaction mixture of the first PCR. Thirty-five cycles were run with the profile described above. 2.3. Identification of amplification products The PCR products were digested with restriction enzymes as indicated in Table 2. The PCR products, restriction fragments and standard length DNA were separated on 14.5% polyacrylamide gels and DNA bands were visualized by silver staining ŽRiesener et al., 1989.. 3. Results and discussion mRNA expression of components of the kallikrein–kinin system was investigated by RT of total cellular RNA using specific primers followed by two PCR reactions Žfirst PCR and second, seminested PCR.. In general, this method has the advantage to detect very low abundance mRNA species. The proper interpretation, however, requires that even sources of contaminating genomic DNA have to be excluded by control experiments. Therefore, primers ŽTable 1. were designed in a way that amplicons of intron-containing genomic DNA were distinguishable by electrophoretic length determination from PCR products of cDNA. Furthermore, all amplicons were identified by restriction fragment length analysis ŽTable 2.. The possibility that the amplicons might be derived from contaminating genomic DNA was also excluded for all depicted components of the kallikrein–kinin system: when PCRs were carried
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
289
Table 1 Sequence and location of primers Sequence
GenBank locus and position
(I) High-molecular weight kininogen X X A LHMW F 5 -CTA GAT TGC AAC GCT GAA G-3 X X B HMW-3 R 5 -CGC TCT TCA TCT TGT GAC G-3 X X C HK-O R 5 -GTC ATG TCT ACG AGT ATG C-3
ŽHUMKIN09 position 17–35. ŽHUMKIN10 position 135–117. ŽHUMKIN10 position 175–157.
(II) Low-molecular weight kininogen X X A LHMW F 5 -CTA GAT TGC AAC GCT GAA G-3 X X B LMW-4 R 5 -TCG ACC CTT GTA CTC GCA-3 X X C LK-O R 5 -CCC TCT CAG ATG CTG GCT C-3
ŽHUMKIN09 position 17–35. ŽHUMKIN10 position 2225–2208. ŽHUMKIN10 position 2262–2244.
(III) Tissue kallikrein A GK 124 F B Kal-3 R C GK 754 R
5 -CTG TAC CAT TTC AGC ACT TTC-3 X X 5 -CAT GAA CAA ACT GGG CTG TG-3 X X 5 -TCA CAT AAG ACA GCA CTC TGA-3
(IV) B1 bradykinin receptor A B1 32 F B B1 511 R C B1 972 R
5 -AGT GGC ACA ATC ATA GCT CG-3 X X 5 -CCA GGA AGA TGC TGA TGA AC-3 X X 5 -GAC TGC TTG CAC CTG GAA TA-3
(V) B2 bradykinin receptor A B2 9 F B B2 645 R C B2 680 R
5 -TGG CCT CAC TCA CAT CC-3 X X 5 -GCT GTA CTC CTT CAT GGT CC-3 X X 5 -CTG ATG ACA CAA GCG GTG AC-3
X
X
ŽHUMKALR2 position 127–147. ŽHUMKALR3 position 95–76. ŽHUMKALR5 position 148–128.
X
X
ŽHSU30271 position 111–130. ŽHSU12512 position 388–369. ŽHSU12512 position 849–830.
X
X
out with genomic DNA in all cases the products were either longer than products derived from cDNA or no product was amplified. This combination of control experiments assured that these RT–PCR procedures were specific for mRNA detection of the respective components of the kallikrein–kinin system.
ŽHSB2BRX2C position 82–98. ŽHSB2BRC14 position 606–587. ŽHSB2BRC14 position 641–622.
Biopsies of normal skin and of diseased skin were investigated for the expression of HMW and LMW kininogen, tissue kallikrein, and the B1 and B2 bradykinin receptors. The results are compiled in Table 3 and polyacrylamide gels are shown in Fig. 1. Since the B2 bradykinin receptor is expressed in human skin ŽSchremmer-Danninger et al., 1995., the
Table 2 Expected chain length of products of semi-nested PCR and fragments obtained by restriction enzyme cleavage HMW kininogen Žexon 9–10.
LMW kininogen Žexon 9–11.
Tissue kallikrein Žexon 2–5.
B2 BK receptor Žexon 2–3.
B1 BK receptor Žexon 2–3.
RT–PCR products Restriction enzyme
206 bp StyI HaeIII
192 bp DdeI HaeIII
151 bp Fok I BsT NI
Cleavage products Žbp.
27, 179 98, 108
166, 26 98, 94
33, 118 36, 115
637 bp DdeI Fok I HaeIII 22, 44, 244, 327 93, 191, 353 3, 21, 23, 25, 41, 86, 87, 92, 106, 132
480 bp DdeI Fok I XBaI 60, 420 106, 374 119, 361
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
290
Table 3 Occurrence mRNAs for components of the kallikrein–kinin system in normal and dieseased human skin biopsies
HMW kininogen LMW kininogen Tissue kallikrein B1 BK receptor B2 BK receptor
Lichenificated atopic eczema
Psoriatic skin
Basalioma tissue
Normal skin
q q q q q
y q q q q
q q q q q
y y q q q
detection of the respective mRNA was used as an internal control for the quality and suitability for RT–PCR analysis of the respective RNA preparation. Usually, B1 receptors are expressed only after stimulation due to pathological conditions. Our finding that the B1 bradykinin receptor gene is constitutively expressed in normal skin ŽTable 3. agrees with
results of studies of Chai et al. Ž1996. showing also B1 receptor gene expression in various normal human tissues. Therefore, human skin appears to belong to the few tissues where B1 receptors are transcribed under physiological conditions. Tissue kallikrein mRNA was detected in all biopsies investigated. mRNA for kininogen was not detected at all in normal skin. This implies that in
Fig. 1. Expression of mRNAs for components of the kallikrein–kinin system in biopsies of normal human skin and of diseased skin. Total cellular RNA was isolated from skin biopsies according to the method described by Chomczynski and Sacchi Ž1987., cDNA synthesis and PCR reactions were performed as described under Section 2. RT–PCR products were separated by polyacrylamide gel electrophoresis ŽPAGE. and visualized by silver staining. ŽA. Lichenificated atopic eczema, ŽB. psoriasis, ŽC. basalioma, and ŽD. normal skin.
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
human skin the kallikrein–kinin system is likely to be operative only under conditions where kininogen is transported to distinct locations within the skin. Such a hypothesis is supported by our finding that mRNAs for HMW and LMW kininogen are expressed in biopsies of lichenificated atopic eczema and basalioma tissue. In these skin lesions, immigrating leucocytes are known to contribute to an accompanying inflammatory reaction. Notably, kininogen has previously been described on the surface of polymorphonuclear leucocytes by immunological techniques ŽWilliams et al., 1997.. In biopsies from psoriatic skin mRNA for LMW kininogen but not HMW kininogen was expressed. These biopsies usually do not show the presence of leucocytes by histological criteria. Based on the findings of Hermann et al. Žthis issue., as depicted there in Table 3, cultured keratinocytes ŽHaCaT cell line. express mRNAs of all these components of the kallikrein–kinin system except of HMW kininogen. This could be a result of the artificial condition of cultivating and may represent a status of chronic inflammation like in psoriatic skin. In summary, normal human skin shows constitutive expression of tissue kallikrein as well as both types of bradykinin receptors. The full functionally active kallikrein–kinin system is, however, only observed under acute pathological conditions when kininogen is arising from other sources.
291
Acknowledgements This work was supported by Sonderforschungsbereich 469, Ludwig-Maximilians-University, Munich.
References Chai, K.X., Ni, A., Wang, D., Ward, D.C., Chao, J., Chao, L., 1996. Genomic DNA sequence, expression, and chromosomal localization of the human B1 bradykinin receptor gene BDKRB1. Genomics 31, 51–57. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Hermann, A., Arnhold, M., Kresse, H., Neth, P., Fink, E., this issue. Expression of components of the kallikrein–kinin system in human cell lines. Immunopharmacology. Riesener, D., Steger, G., Zimmat, R., Owens, R.A., Wagenhoefer, M., Hillen, W., Vollbach, S., Henco, K., 1989. Temperaturegradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein– nucleic acid interactions. Electrophoresis 10, 377–389. Schremmer-Danninger, E., Heinz-Erian, P., Toepfer-Petersen, E., Roscher, A.A., 1995. Autoradiographic localization and characterization of bradykinin receptors in human skin. Eur. J. Pharm. 283, 207–216. Williams, R.J., Henderson, L.M., Naidoo, Y., Cassim, B., Elson, C.J., Bhoola, K.D., 1997. Immunocytochemical analysis of tissue kallikrein and the kinin moiety on rheumatoid synovial fluid neutrophils. Br. J. Rheumatol. 36, 420–425.
Identification and occurrence of mRNAs for components of the kallikrein–kinin system in human skin and in skin diseases Elisabeth Schremmer-Danninger a,b,) , Andrea Hermann Hans Fritz a , Adelbert A. Roscher b
a,1
, Edwin Fink a ,
a
b
Department of Clinical Chemistry and Clinical Biochemistry, Hospital of Surgery, Ludwig-Maximilians-UniÕersity, Munich, Germany Department of Clinical Biochemistry, Childrens Hospital, Ludwig-Maximilians-UniÕersity, Lindwurmstrabe 4, D-80337 Munich, Germany Accepted 3 May 1999
Abstract Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogen ŽHMW and LMW kininogen, respectively.. In the skin, kinins are involved, e.g., as co-mitogens in cellular proliferation or in processes propagating pain and inflammation. The aim of our study was to investigate the specific occurrence of mRNAs for components of the kallikrein–kinin system in normal human skin and in skin biopsies of patients with selected skin diseases Žpsoriasis, lichenificated atopic eczema, basalioma.. In normal skin, reverse transcription polymerase chain reaction ŽRT–PCR. with specific primer pairs followed by separation of products by polyacrylamide gel electrophoresis ŽPAGE. revealed the presence of mRNAs for tissue kallikrein, for the B2 and the B1 bradykinin receptors, but not for kininogen. In biopsies of lichenificated atopic eczema and basalioma, additionally, the mRNAs for HMW and LMW kininogen were detected, whereas in psoriatic skin mRNA for HMW kininogen was not expressed. These differences in mRNA expression may reflect the different contribution of kallikrein–kinin system components to the maintenance of chronic skin diseases like psoriasis. In acute dermal reactions occurring in lichenificated atopic eczema or in basalioma, tissue mRNA for HMW kininogen appears to be arisen from sources not pre-existing in normal skin. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Kallikrein–kinin system; mRNA; Human skin; Skin diseases
1. Introduction AbbreÕiations: PAGE, polyacrylamide gel electrophoresis; RT–PCR, reverse transcription polymerase chain reaction; HMW kininogen, high-molecular weight kininogen; LMW kininogen, low-molecular weight kininogen ) Corresponding author. Department of Clinical Biochemistry, Childrens Hospital, Ludwig-Maximilians-University, Lindwurmstrabe 4, D-80337 Munich, Germany. fax: q49-89-5160-4486; e-mail: [email protected] 1 Present address: November AG, Erlangen, Germany.
Bradykinin and kallidin are released during dermal injury and inflammation as a result of activation of kallikreins which cleave high- and low-molecular weight kininogens ŽHMW and LMW kininogens, respectively.. These kinins exert a broad spectrum of physiological and also pathophysiological effects including cell proliferation, pain and inflammation.
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 0 0 - 9
288
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
We have previously shown that B2 bradykinin receptors occur in the mitotically active keratinocytes of the basal layer ŽSchremmer-Danninger et al., 1995.. These receptors may mediate kinin effects related to cellular reactions such as keratinocyte proliferation following inflammatory conditions. The occurrence and localization of the other components of the kallikrein–kinin system Žkininogen, kallikrein. or of B1 bradykinin receptors in human skin has not yet been investigated. The specific aim of our study was to find out whether bradykinin can be generated directly from a local source of kininogen or from an external source originating, e.g., from immigrating leucocytes during inflammation. Therefore, we looked for the occurrence of specific mRNAs for components of the kallikrein–kinin system in normal human skin and in skin biopsies of patients with selected skin diseases Žpsoriasis, lichenificated atopic eczema, basalioma..
2. Material and methods 2.1. Tissue samples and RNA preparation Histologically normal adult skin was obtained from areas surrounding dermal naevi excised for suspicion of melanoma; biopsies from psoriatic skin, lichenificated atopic eczema and from basalioma tissue were recruited from patients giving their permission. Tissue samples were rinsed in ice-cold saline and frozen immediately in liquid nitrogen. According to the method described by Chomczynski and Sacchi Ž1987. total cellular RNA was isolated from skin biopsies with an acidic phenol–chloroform extraction. The isolated RNA was spectrophotometrically quantified at 260 nm. Negative control samples were prepared by performing the entire procedure in the absence of tissue. 2.2. ReÕerse transcription, first PCR and second, semi-nested PCR In order to control for potentially contaminating genomic DNA primers for reverse transcription polymerase chain reaction ŽRT–PCR. ŽTable 1. were selected in a way that the PCR products include at least one exon–exon transition. Thus, in case of
genomic DNA contaminating the RNA preparation PCR-products of different length would be obtained. cDNAs were synthezised from 1 mg of total RNA preparations and the negative control preparations following the instruction of the ‘‘First-Strand cDNA Synthesis Kit’’ ŽPharmacia Biotech. using the reverse primers C ŽTable 1.. PCR was performed with 1r10 of the cDNA synthesis reaction mixture or the control incubation and the respective primer pairs ŽTable 1A and C.. Each of the 20 cycles consisted of a denaturation step at 928C for 40 s, an annealing step at 528C for 40 s, and an elongation step at 728C for 40 s. The PCR with the semi-nested primers ŽTable 1A and B. was performed with 1 ml of a 1:10 dilution of the reaction mixture of the first PCR. Thirty-five cycles were run with the profile described above. 2.3. Identification of amplification products The PCR products were digested with restriction enzymes as indicated in Table 2. The PCR products, restriction fragments and standard length DNA were separated on 14.5% polyacrylamide gels and DNA bands were visualized by silver staining ŽRiesener et al., 1989.. 3. Results and discussion mRNA expression of components of the kallikrein–kinin system was investigated by RT of total cellular RNA using specific primers followed by two PCR reactions Žfirst PCR and second, seminested PCR.. In general, this method has the advantage to detect very low abundance mRNA species. The proper interpretation, however, requires that even sources of contaminating genomic DNA have to be excluded by control experiments. Therefore, primers ŽTable 1. were designed in a way that amplicons of intron-containing genomic DNA were distinguishable by electrophoretic length determination from PCR products of cDNA. Furthermore, all amplicons were identified by restriction fragment length analysis ŽTable 2.. The possibility that the amplicons might be derived from contaminating genomic DNA was also excluded for all depicted components of the kallikrein–kinin system: when PCRs were carried
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
289
Table 1 Sequence and location of primers Sequence
GenBank locus and position
(I) High-molecular weight kininogen X X A LHMW F 5 -CTA GAT TGC AAC GCT GAA G-3 X X B HMW-3 R 5 -CGC TCT TCA TCT TGT GAC G-3 X X C HK-O R 5 -GTC ATG TCT ACG AGT ATG C-3
ŽHUMKIN09 position 17–35. ŽHUMKIN10 position 135–117. ŽHUMKIN10 position 175–157.
(II) Low-molecular weight kininogen X X A LHMW F 5 -CTA GAT TGC AAC GCT GAA G-3 X X B LMW-4 R 5 -TCG ACC CTT GTA CTC GCA-3 X X C LK-O R 5 -CCC TCT CAG ATG CTG GCT C-3
ŽHUMKIN09 position 17–35. ŽHUMKIN10 position 2225–2208. ŽHUMKIN10 position 2262–2244.
(III) Tissue kallikrein A GK 124 F B Kal-3 R C GK 754 R
5 -CTG TAC CAT TTC AGC ACT TTC-3 X X 5 -CAT GAA CAA ACT GGG CTG TG-3 X X 5 -TCA CAT AAG ACA GCA CTC TGA-3
(IV) B1 bradykinin receptor A B1 32 F B B1 511 R C B1 972 R
5 -AGT GGC ACA ATC ATA GCT CG-3 X X 5 -CCA GGA AGA TGC TGA TGA AC-3 X X 5 -GAC TGC TTG CAC CTG GAA TA-3
(V) B2 bradykinin receptor A B2 9 F B B2 645 R C B2 680 R
5 -TGG CCT CAC TCA CAT CC-3 X X 5 -GCT GTA CTC CTT CAT GGT CC-3 X X 5 -CTG ATG ACA CAA GCG GTG AC-3
X
X
ŽHUMKALR2 position 127–147. ŽHUMKALR3 position 95–76. ŽHUMKALR5 position 148–128.
X
X
ŽHSU30271 position 111–130. ŽHSU12512 position 388–369. ŽHSU12512 position 849–830.
X
X
out with genomic DNA in all cases the products were either longer than products derived from cDNA or no product was amplified. This combination of control experiments assured that these RT–PCR procedures were specific for mRNA detection of the respective components of the kallikrein–kinin system.
ŽHSB2BRX2C position 82–98. ŽHSB2BRC14 position 606–587. ŽHSB2BRC14 position 641–622.
Biopsies of normal skin and of diseased skin were investigated for the expression of HMW and LMW kininogen, tissue kallikrein, and the B1 and B2 bradykinin receptors. The results are compiled in Table 3 and polyacrylamide gels are shown in Fig. 1. Since the B2 bradykinin receptor is expressed in human skin ŽSchremmer-Danninger et al., 1995., the
Table 2 Expected chain length of products of semi-nested PCR and fragments obtained by restriction enzyme cleavage HMW kininogen Žexon 9–10.
LMW kininogen Žexon 9–11.
Tissue kallikrein Žexon 2–5.
B2 BK receptor Žexon 2–3.
B1 BK receptor Žexon 2–3.
RT–PCR products Restriction enzyme
206 bp StyI HaeIII
192 bp DdeI HaeIII
151 bp Fok I BsT NI
Cleavage products Žbp.
27, 179 98, 108
166, 26 98, 94
33, 118 36, 115
637 bp DdeI Fok I HaeIII 22, 44, 244, 327 93, 191, 353 3, 21, 23, 25, 41, 86, 87, 92, 106, 132
480 bp DdeI Fok I XBaI 60, 420 106, 374 119, 361
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
290
Table 3 Occurrence mRNAs for components of the kallikrein–kinin system in normal and dieseased human skin biopsies
HMW kininogen LMW kininogen Tissue kallikrein B1 BK receptor B2 BK receptor
Lichenificated atopic eczema
Psoriatic skin
Basalioma tissue
Normal skin
q q q q q
y q q q q
q q q q q
y y q q q
detection of the respective mRNA was used as an internal control for the quality and suitability for RT–PCR analysis of the respective RNA preparation. Usually, B1 receptors are expressed only after stimulation due to pathological conditions. Our finding that the B1 bradykinin receptor gene is constitutively expressed in normal skin ŽTable 3. agrees with
results of studies of Chai et al. Ž1996. showing also B1 receptor gene expression in various normal human tissues. Therefore, human skin appears to belong to the few tissues where B1 receptors are transcribed under physiological conditions. Tissue kallikrein mRNA was detected in all biopsies investigated. mRNA for kininogen was not detected at all in normal skin. This implies that in
Fig. 1. Expression of mRNAs for components of the kallikrein–kinin system in biopsies of normal human skin and of diseased skin. Total cellular RNA was isolated from skin biopsies according to the method described by Chomczynski and Sacchi Ž1987., cDNA synthesis and PCR reactions were performed as described under Section 2. RT–PCR products were separated by polyacrylamide gel electrophoresis ŽPAGE. and visualized by silver staining. ŽA. Lichenificated atopic eczema, ŽB. psoriasis, ŽC. basalioma, and ŽD. normal skin.
E. Schremmer-Danninger et al.r Immunopharmacology 43 (1999) 287–291
human skin the kallikrein–kinin system is likely to be operative only under conditions where kininogen is transported to distinct locations within the skin. Such a hypothesis is supported by our finding that mRNAs for HMW and LMW kininogen are expressed in biopsies of lichenificated atopic eczema and basalioma tissue. In these skin lesions, immigrating leucocytes are known to contribute to an accompanying inflammatory reaction. Notably, kininogen has previously been described on the surface of polymorphonuclear leucocytes by immunological techniques ŽWilliams et al., 1997.. In biopsies from psoriatic skin mRNA for LMW kininogen but not HMW kininogen was expressed. These biopsies usually do not show the presence of leucocytes by histological criteria. Based on the findings of Hermann et al. Žthis issue., as depicted there in Table 3, cultured keratinocytes ŽHaCaT cell line. express mRNAs of all these components of the kallikrein–kinin system except of HMW kininogen. This could be a result of the artificial condition of cultivating and may represent a status of chronic inflammation like in psoriatic skin. In summary, normal human skin shows constitutive expression of tissue kallikrein as well as both types of bradykinin receptors. The full functionally active kallikrein–kinin system is, however, only observed under acute pathological conditions when kininogen is arising from other sources.
291
Acknowledgements This work was supported by Sonderforschungsbereich 469, Ludwig-Maximilians-University, Munich.
References Chai, K.X., Ni, A., Wang, D., Ward, D.C., Chao, J., Chao, L., 1996. Genomic DNA sequence, expression, and chromosomal localization of the human B1 bradykinin receptor gene BDKRB1. Genomics 31, 51–57. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Hermann, A., Arnhold, M., Kresse, H., Neth, P., Fink, E., this issue. Expression of components of the kallikrein–kinin system in human cell lines. Immunopharmacology. Riesener, D., Steger, G., Zimmat, R., Owens, R.A., Wagenhoefer, M., Hillen, W., Vollbach, S., Henco, K., 1989. Temperaturegradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and protein– nucleic acid interactions. Electrophoresis 10, 377–389. Schremmer-Danninger, E., Heinz-Erian, P., Toepfer-Petersen, E., Roscher, A.A., 1995. Autoradiographic localization and characterization of bradykinin receptors in human skin. Eur. J. Pharm. 283, 207–216. Williams, R.J., Henderson, L.M., Naidoo, Y., Cassim, B., Elson, C.J., Bhoola, K.D., 1997. Immunocytochemical analysis of tissue kallikrein and the kinin moiety on rheumatoid synovial fluid neutrophils. Br. J. Rheumatol. 36, 420–425.