Expression of Bcl-2 and Bcl-xL in Cutaneous and Bone Marrow Lesions of Mastocytosis
2003; Elsevier BV; Volume: 163; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63442-6
ISSN1525-2191
AutoresKarin Hartmann, Metin Artuc, Stephan Baldus, Thomas K. Zirbes, Bárbara Hermes, Jüergen Thiele, Yoseph A. Mekori, Beate M. Henz,
Tópico(s)Asthma and respiratory diseases
ResumoMastocytosis is a rare disease characterized by accumulation of mast cells in tissues. To investigate whether an altered regulation of mast cell apoptosis might be involved in the pathogenesis of mastocytosis, expression of the apoptosis-preventing molecules bcl-2 and bcl-xL was studied by immunohistochemistry in skin and bone marrow lesions of mastocytosis patients. In addition, reverse transcription-polymerase chain reaction was used to investigate levels of bcl-2 and bcl-xL mRNA in cutaneous mastocytosis lesions. Since activating mutations of c-kit are known to be associated with some forms of mastocytosis, human mast cell cultures were also stimulated via c-kit and the expression of bcl-2 and bcl-xL was assessed by immunoblotting. In patients with mastocytosis, the expression of bcl-2 protein but not bcl-xL in cutaneous mast cells was significantly enhanced, compared to healthy controls. Evaluating different subgroups of adult and pediatric mastocytosis patients, all groups were found to express significantly increased levels of bcl-2 protein, and none of the patient groups was found to overexpress bcl-xL, with the exception of solitary mastocytomas that showed a tendency for up-regulated bcl-xL protein. Furthermore, the expression of bcl-2 mRNA was significantly enhanced in cutaneous lesions of adult and pediatric patients, while bcl-xL mRNA levels were only slightly increased in pediatric, but not in adult patients with mastocytosis. In contrast to the skin lesions, bone marrow infiltrates of patients with systemic mastocytosis showed only low or absent immunoreactivity for bcl-2, but marked expression of bcl-xL. In vitro, stimulation of two different mast cell culture systems by activation of c-kit resulted in up-regulation of bcl-2 and also in an increase of bcl-xL, although less pronounced. Thus, overexpression of bcl-2 and bcl-xL leading to prolonged survival of mast cells may contribute to the pathogenesis of mastocytosis. Our findings may help to develop new strategies for the treatment of this disease. Mastocytosis is a rare disease characterized by accumulation of mast cells in tissues. To investigate whether an altered regulation of mast cell apoptosis might be involved in the pathogenesis of mastocytosis, expression of the apoptosis-preventing molecules bcl-2 and bcl-xL was studied by immunohistochemistry in skin and bone marrow lesions of mastocytosis patients. In addition, reverse transcription-polymerase chain reaction was used to investigate levels of bcl-2 and bcl-xL mRNA in cutaneous mastocytosis lesions. Since activating mutations of c-kit are known to be associated with some forms of mastocytosis, human mast cell cultures were also stimulated via c-kit and the expression of bcl-2 and bcl-xL was assessed by immunoblotting. In patients with mastocytosis, the expression of bcl-2 protein but not bcl-xL in cutaneous mast cells was significantly enhanced, compared to healthy controls. Evaluating different subgroups of adult and pediatric mastocytosis patients, all groups were found to express significantly increased levels of bcl-2 protein, and none of the patient groups was found to overexpress bcl-xL, with the exception of solitary mastocytomas that showed a tendency for up-regulated bcl-xL protein. Furthermore, the expression of bcl-2 mRNA was significantly enhanced in cutaneous lesions of adult and pediatric patients, while bcl-xL mRNA levels were only slightly increased in pediatric, but not in adult patients with mastocytosis. In contrast to the skin lesions, bone marrow infiltrates of patients with systemic mastocytosis showed only low or absent immunoreactivity for bcl-2, but marked expression of bcl-xL. In vitro, stimulation of two different mast cell culture systems by activation of c-kit resulted in up-regulation of bcl-2 and also in an increase of bcl-xL, although less pronounced. Thus, overexpression of bcl-2 and bcl-xL leading to prolonged survival of mast cells may contribute to the pathogenesis of mastocytosis. Our findings may help to develop new strategies for the treatment of this disease. Mastocytosis represents a heterogeneous group of diseases characterized by an increase of mast cells in different organs.1Metcalfe DD Akin C Mastocytosis: molecular mechanisms and clinical disease heterogeneity.Leuk Res. 2001; 25: 577-582Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 2Hartmann K Henz BM Mastocytosis: recent advances in defining the disease.Br J Dermatol. 2001; 144: 682-695Crossref PubMed Scopus (151) Google Scholar Cutaneous mastocytosis is limited to the skin, preferentially develops in infancy, often shows spontaneous regression after several years, and can be categorized into a maculopapular (traditionally named urticaria pigmentosa), nodular (also named mastocytoma and presents as solitary or multiple lesions), diffuse, and telangiectatic form.3Valent P Horny HP Escribano L Longley BJ Li CY Schwartz LB Marone G Nunez R Akin C Sotlar K Sperr WR Wolff K Brunning RD Parwaresch RM Austen KF Lennert K Metcalfe DD Vardimann JW Bennett JM Diagnostic criteria and classification of mastocytosis: a consensus proposal.Leuk Res. 2001; 25: 603-625Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar, 4Wolff K Komar M Petzelbauer P Clinical and histopathological aspects of cutaneous mastocytosis.Leuk Res. 2001; 25: 519-528Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 5Hartmann K Henz BM Classification of cutaneous mastocytosis: a modified consensus proposal.Leuk Res. 2002; 26: 483-484Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Systemic mastocytosis is defined by infiltrates of mast cells in the bone marrow or other extracutaneous organs, presents with or without skin lesions, usually affects adults, and shows an indolent or aggressive clinical course.3Valent P Horny HP Escribano L Longley BJ Li CY Schwartz LB Marone G Nunez R Akin C Sotlar K Sperr WR Wolff K Brunning RD Parwaresch RM Austen KF Lennert K Metcalfe DD Vardimann JW Bennett JM Diagnostic criteria and classification of mastocytosis: a consensus proposal.Leuk Res. 2001; 25: 603-625Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar Recent studies have demonstrated that activating mutations of c-kit, the receptor of the mast cell growth factor stem cell factor (SCF), play a key role in the pathogenesis of sporadic adult onset mastocytosis.6Nagata H Worobec AS Oh CK Chowdhury BA Tannenbaum S Suzuki Y Metcalfe DD Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematological disorder.Proc Natl Acad Sci USA. 1995; 92: 10560-10564Crossref PubMed Scopus (804) Google Scholar However, these mutations alone do not explain the heterogeneity of adult disease and are usually not present in pediatric and familial mastocytosis.1Metcalfe DD Akin C Mastocytosis: molecular mechanisms and clinical disease heterogeneity.Leuk Res. 2001; 25: 577-582Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 7Buettner C Henz BM Welker R Sepp NT Grabbe J Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behaviour.J Invest Dermatol. 1998; 111: 1227-1231Crossref PubMed Scopus (193) Google Scholar, 8Longley BJ Metcalfe DD Tharp M Wang X Tyrrell L Lu S Heitjan D Ma Y Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.Proc Natl Acad Sci USA. 1999; 96: 1609-1614Crossref PubMed Scopus (498) Google Scholar, 9Rosbotham JL Malik NM Syrris P Jeffery S Bedlow A Gharraie S Murday VA Holden CA Carter ND Lack of c-kit mutation in familial urticaria pigmentosa.Br J Dermatol. 1999; 140: 849-852Crossref PubMed Scopus (57) Google Scholar, 10Sato-Matsumura KC Matsumura T Koizumi H Sato H Nagashima K Ohkawara A Analysis of c-kit exon 11 and exon 17 of urticaria pigmentosa that occurred in monozygotic twin sisters.Br J Dermatol. 1999; 140: 1130-1132Crossref PubMed Scopus (23) Google Scholar To investigate additional mechanisms by which mast cell numbers are regulated in mastocytosis, we hypothesized that an altered control of mast cell apoptosis might also be involved in the pathogenesis of the disease. 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In the same patients, bone marrow infiltrates fail to express bcl-2, but strongly express bcl-xL. These results further support the concept that alterations in the control of apoptosis may contribute to accumulation of mast cells in mastocytosis and also suggest that survival of cutaneous mast cells in this disease may be differentially regulated compared to bone marrow mast cells. Cutaneous biopsies were obtained from a total of 39 patients with mastocytosis for diagnostic purposes. The diagnosis of mastocytosis with cutaneous involvement was made on the basis of clinical appearance and an increase of mast cells on histology, according to published criteria.3Valent P Horny HP Escribano L Longley BJ Li CY Schwartz LB Marone G Nunez R Akin C Sotlar K Sperr WR Wolff K Brunning RD Parwaresch RM Austen KF Lennert K Metcalfe DD Vardimann JW Bennett JM Diagnostic criteria and classification of mastocytosis: a consensus proposal.Leuk Res. 2001; 25: 603-625Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar, 4Wolff K Komar M Petzelbauer P Clinical and histopathological aspects of cutaneous mastocytosis.Leuk Res. 2001; 25: 519-528Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar Paraffin-embedded sections of 32 mastocytosis patients (12 adult and 20 pediatric patients) were analyzed by immunohistochemistry and compared with 7 healthy controls. Out of the 32 patients, 12 adults and 6 children had maculopapular cutaneous mastocytosis (urticaria pigmentosa), 7 children had solitary mastocytomas, and 7 children had multiple mastocytomas.4Wolff K Komar M Petzelbauer P Clinical and histopathological aspects of cutaneous mastocytosis.Leuk Res. 2001; 25: 519-528Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 5Hartmann K Henz BM Classification of cutaneous mastocytosis: a modified consensus proposal.Leuk Res. 2002; 26: 483-484Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar Lesional mast cell counts of skin biopsies were 6.58 ± 1.75 mast cells/mm2 (mean ± SEM) in 12 adult mastocytosis patients and 39.68 ± 5.31 mast cells/mm2 in 20 pediatric patients (29.23 ± 6.42 mast cells/mm2 in 6 maculopapular mastocytosis, 38.81 ± 5.90 mast cells/mm2 in 7 solitary mastocytoma, and 49.50 ± 11.73 mast cells/mm2 in 7 multiple mastocytoma patients), compared with 1.01 ± 0.07 mast cells/mm2 in 7 healthy controls. In addition, small snap-frozen pieces of 9 mastocytosis biopsies (2 biopsies that had also been used for immunohistochemical staining and 7 additional biopsies of different patients with mastocytosis) from 3 adult patients with maculopapular mastocytosis and 6 pediatric patients with maculopapular mastocytosis or mastocytomas as well as frozen tissue of 6 healthy controls were used for RNA isolation. Out of the12 adult patients analyzed by immunohistochemistry, 5 patients with systemic mastocytosis3Valent P Horny HP Escribano L Longley BJ Li CY Schwartz LB Marone G Nunez R Akin C Sotlar K Sperr WR Wolff K Brunning RD Parwaresch RM Austen KF Lennert K Metcalfe DD Vardimann JW Bennett JM Diagnostic criteria and classification of mastocytosis: a consensus proposal.Leuk Res. 2001; 25: 603-625Abstract Full Text Full Text PDF PubMed Scopus (954) Google Scholar associated with maculopapular cutaneous mastocytosis were chosen who had both a skin and bone marrow biopsy within the same year and their paraffin-embedded bone marrow biopsies were also investigated by immunohistochemistry. All patients displayed focal dense infiltrates of mast cells in the bone marrow. In 4 of these patients, tryptase levels were available and showed comparable systemic involvement (range of tryptase levels, 60.7 to 72.3 μg/L; mean, 67.3 μg/L; n = 4; measured by UniCAP Tryptase Fluoroenzyme Immunoassay, Pharmacia Diagnostics, Uppsala, Sweden). Most of the adult patients analyzed in this study were tested in the past for the presence of a codon 816 c-kit mutation and were all found to express this mutation.7Buettner C Henz BM Welker R Sepp NT Grabbe J Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behaviour.J Invest Dermatol. 1998; 111: 1227-1231Crossref PubMed Scopus (193) Google Scholar Written, informed consent was obtained from all patients and controls before biopsies were performed. The study was approved by the ethical committee of the Humboldt University, Berlin, Germany. For analysis of mast cells in cutaneous biopsies, all sections were first stained with toluidine blue at pH 0.5 for 24 hours, and costaining of mast cells with antibodies against bcl-2 and bcl-xL was then assessed in serial sections, as previously described.36Haas N Toppe E Henz BM Microscopic morphology of different types of urticaria.Arch Dermatol. 1998; 134: 41-46Crossref PubMed Scopus (83) Google Scholar, 37Hermes B Prochazka AK Haas N Jurgovsky K Sticherling M Henz BM Up-regulation of TNF-α and IL-3 expression in lesional and uninvolved skin in different types of urticaria.J Allergy Clin Immunol. 1991; 103: 307-314Abstract Full Text Full Text PDF Google Scholar Before exposure to antibodies, antigen unmasking was performed by incubating the skin sections in a commercial antigen retrieval buffer (pH 9.9) (Target Retrieval Solution; DakoCytomation, Hamburg, Germany) for 30 minutes at 95°C. Immunoreactivity for bcl-2 and bcl-xL was then investigated using a mouse anti-human bcl-2 mAb (clone 100/D5; dilution 1:100; Zymed Laboratories, South San Francisco, CA), a mouse anti-human bcl-xL mAb (clone 7B2.5; dilution 1:500; DPC Biermann, Bad Nauheim, Germany), and the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique.37Hermes B Prochazka AK Haas N Jurgovsky K Sticherling M Henz BM Up-regulation of TNF-α and IL-3 expression in lesional and uninvolved skin in different types of urticaria.J Allergy Clin Immunol. 1991; 103: 307-314Abstract Full Text Full Text PDF Google Scholar Optimal dilutions of the antibodies were tested with appropriate positive and negative control specimens such as tonsils or tumors. Evaluation of skin sections was performed by recording the distribution of positive cells and by counting the numbers of toluidine blue-positive and immunoreactive mast cells in at least five microscopic fields at high-power magnification (×400). Data are expressed as percentage (mean ± SEM) of bcl-2- or bcl-xL-positive mast cells. To compare the expression of bcl-2 and bcl-xL in skin and bone marrow sections and to exclude differences due to methodology, 5 adult patients with systemic mastocytosis who had both a skin and bone marrow biopsy within the same year were chosen and their skin sections, which had previously been stained by the APAAP technique, as well as their bone marrow sections, were also pretreated by incubation in 10 mmol/L citrate buffer at pH 6.0 in a microwave oven at 750 W three times for 4 minutes each and stained using a mouse anti-human bcl-2 mAb (clone 124; dilution 1:40; DakoCytomation), a mouse anti-human bcl-xL mAb (clone H-5; dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), and a commercial immunohistochemistry kit (EnVision AP; DakoCytomation). Again, optimal dilutions of the antibodies were tested with appropriate positive and negative controls. In the cutaneous sections, the APAAP and the EnVision AP technique showed the same results. For detection of mast cell infiltrates in the bone marrow sections, staining with chloroacetate esterase was performed on serial sections and compared to staining reactions obtained with the antibodies against bcl-2 and bcl-xL. In these 5 patients, the intensity of the EnVision AP-stained cutaneous and bone marrow sections was evaluated semiquantitatively and graded as 0 (bone marrow: reactivity in <5% of dense focal infiltrates containing mast cells; skin: reactivity in 65% of infiltrates; skin: reactivity in >65% of mast cells). Total RNA was isolated from frozen skin biopsies using the RNeasy Kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase I (Roche Molecular Biochemicals, Mannheim, Germany). After isopropanol precipitation, 1 μg of total RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Life Technologies, Karlsruhe, Germany) using random hexamers. Polymerase chain reaction (PCR) was performed using the following primers for bcl-2: 5′- CAGCTGCACCTGACGCCCTT-3′ and reverse 5′- CCCAGCCTCCGTTATCCTGGA-3′, the following primers for bcl-xL: 5′- AGGCAGGCGACGAGTTTGAACTG-3′ and reverse 5′-CAGGAACCAGCGGTTGAAGCGT-3′, the following primers for tryptase: 5′- GGAGCTGGAGGAGCCCGTGA-3′ and reverse 5′- ACCTGGGTAGGAAGCAGTGGTG-3′, and the following primers for GAPDH: 5′-GCTGTAGCCAAATTCGTTGTC-3′ and reverse 5′- GATGACATCAGAAGGTGGTG-3′. In all experiments, PCR conditions were standardized using a master mixture containing Taq polymerase, MgCl2, and 2 μl cDNA/50 μl PCR volume. PCR products were purified using the QIAquick Nucleotide Removal Kit (Qiagen) and spectrofluorometrically quantified using the double-stranded DNA-binding dye PicoGreen (Molecular Probes, Leiden, Netherlands). Results are given in ng bcl-2, bcl-xL or tryptase mRNA/ng GAPDH mRNA. The human basophilic cell line KU-812 (kindly provided by the Institute for Allergy Research, Borstel, Germany),38Kishi K A new leukemia cell line with Philadelphia chromosome characterized as basophil precursors.Leuk Res. 1985; 9: 381-390Abstract Full Text PDF PubMed Scopus (226) Google Scholar known to express non-mutated c-kit,7Buettner C Henz BM Welker R Sepp NT Grabbe J Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behaviour.J Invest Dermatol. 1998; 111: 1227-1231Crossref PubMed Scopus (193) Google Scholar was maintained in RPMI 1640 supplemented with 20% fetal calf serum, 2 mmol/L L-glutamine, antibiotics, and 0.04% 2-mercaptoethanol. Primary cord blood-derived mast cells were obtained from mononuclear cells of heparinized umbilical cord blood after incubation with a selective medium containing 100 ng/ml SCF (Peprotech, Rocky Hill, NJ) for 8 to 12 weeks, as described.39Hartmann K Henz BM Krueger-Krasagakes S Koehl J Burger R Guhl S Haase I Lippert U Zuberbier T C3a and C5a stimulate chemotaxis of human mast cells.Blood. 1997; 89: 2863-2870Crossref PubMed Google Scholar Mast cell development was confirmed by toluidine blue staining and fluorescence-activated cell sorter analysis of tryptase and KIT expression. KU-812 cells and cord blood-derived mast cells were cultured with or without 100 ng/ml SCF (Peprotech) for 48 hours, lysed, and concentration of total protein was measured using a Bradford assay (Bio-Rad Laboratories, Hercules, CA). Fifty μg of each protein sample was electrophoresed on sodium dodecyl sulfate gels containing 15% polyacrylamide and transferred to nitrocellulose filters. Equal loading and transfer of proteins was ensured by staining with Ponceau S. Bcl-2 protein was detected using a mouse anti-human bcl-2 mAb (clone 100; dilution 1:200; Santa Cruz Biotechnology) and bcl-xL was detected using a mouse anti-human bcl-xL mAb (clone H-5; dilution 1:200; Santa Cruz Biotechnology), followed by a horseradish peroxidase-conjugated rabbit anti-mouse antibody (DakoCytomation). Immunoblots were visualized with an ECL chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). Optical densities of immunoreactive bands were monitored using Quantity One software (Bio-Rad Laboratories). Statistical differences were evaluated using Student's t-test for unpaired values. P values were determined by a two-sided calculation, and a P value of less than 0.05 was considered significant. To study the expression of apoptosis-preventing molecules in cutaneous biopsies of mastocytosis patients, paraffin-embedded sections of 32 patients with different forms of mastocytosis and 7 healthy controls were analyzed by immunohistochemistry using antibodies against bcl-2 and bcl-xL (Table 1; Figure 1, A and B). In patients with mastocytosis, the expression of bcl-2 protein in mast cells was significantly enhanced compared to controls (Table 1; Figure 1A), suggesting that prolonged survival of mast cells may, at least in some patients, be associated with increased mast cell numbers. In contrast, mast cell immunoreactivity for bcl-xL was comparable between mastocytosis and control skin (Table 1; Figure 1B), although a high variation of bcl-xL expression was observed with up-regulation in about half of all patients. Evaluating different subgroups of patients (Table 1), all groups showed an increased re
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