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Eosinophil Cationic Protein, Expression Levels and Polymorphisms

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Eosinophil Cationic Protein, Expression Levels and Polymorphisms !" #$%& !"'() &'& %*+(*'&'* &,*%* &,& -..- Dissertation for the degree of Doctor of Philosophy (Faculty of Medicine) in Clinical Chemistry presented at Uppsala University in 2002 ABSTRACT Byström, J. 2002. Eosinophil Cationic Protein, Expression levels and Polymorphisms. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1161. 58 pp. Uppsala ISBN 91-554-5336-8. The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted. The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C>T), 434(G>C) and 562(G>C). The two first were located to the mature peptide- coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterised by fibrosis and eosinophils and ECP has been suggested in fibrosis development. Key words: Eosinophils, Neutrophils, Monocytes, Macrophages, mRNA, eosinophil cationic protein, DNA, polymorphism, Hodgkin Lymphoma, asthma, allergy. Jonas Byström, Department of Medical Sciences, Clinical Chemistry, University Hospital, SE- 751 85 Uppsala, Sweden Jonas Byström 2002 ISSN 0282-7476 ISBN 91-554-5336-8 Printed in Sweden by Eklundshofs Grafiska, Uppsala 2002 2 “..to boldly go there no one has gone before” In memorial of my grandfather Pelle Byström, farmers son, agronomist, folk high school teacher and writer 3 This thesis is based on the following papers, which are referred to in the text by their Roman numerals: I. Byström, J., Tenno, T., Håkansson, L., Amin, K., Trulson, A., Högbom, H., Venge, P. Monocytes, but not macrophages, produce the Eosinophil Cationic Protein. APMIS. 2001;109:507–516. II. Byström, J., Garcia, R., Håkansson, L., Karawajczyk, M., Moberg, L., Sukka, J., and Venge, P. Eosinophil Cationic Protein (ECP) is stored in, but not produced by peripheral blood neutrophils. Clin Exp Allergy, 2002(In press). III. Byström, J., Molin, D., Jönsson, U. B., Enblad, G., Sundström, C., Högbom, E., and Venge, P. Identification of polymorphisms in the ECP- gene. Relation to disease activity in Hodgkin lymphoma. Manuscript. IV. Jönsson, U. B., Byström, J., Stålenheim, G., and Venge, P. Polymorphism of the ECP gene is related to the expression of allergic symptoms. Clin Exp Allergy, 2002(In press). Permission for reprinting the articles has been given by the publishers. 4 Table of Contents Abbreviations………………………………………………………………….…6 Introduction………………………………………………………………….……8 Inflammatory cells of the innate immunity system …………………………………8 The eosinophil granulocyte…………………………………………………………8 The neutrophil granulocyte………………….………………………………..……10 The monocyte / macrophage ……………………………………………..….…….11 The eosinophil cationic protein …………………………………………….….…..11 Single nucleotide polymorphisms and the eosinophil……………………...………15 Inflammatory disorders involving the eosinophil…………………………..………16 Hodgkin lymphoma …………………………………………………….………….16 Asthma / Allergy……………………………………………………………………18 Figure 1……………………………………………………………………..………20 The aims of the Present Investigation………………………………………21 Materials and Methods…………………………………………………………22 Subjects…………………………………………………………………………..…22 Cell separation procedures……………….………………………………………..22 Cell line culture ……………………………………………………………………23 Immunohistochemisty………………………………………………………………23 Microscopic techniques…………………………………………………………….24 Flowcytometry for detection of protein inside cells, already present or taken up in vitro……………….……………………………….25 Subcellular fractionations …………………………………………….……………25 Subcellular location of internalised ECP…..………………………………………..26 Protein measurements……………………………………………………………….26 RNA preparation…………………………………………………………….………26 DNA preparation…………………………………………………………………….27 Reverse transcriptase and polymerase chain reaction……………………………….27 Northern Blotting……………………………………………………………………28 DNA sequencing……………………………………………………………………29 Statistical methods……………………………………………………….…………29 Subjects and Clinical Characteristics………………………………….……………30 Results and Discussion………………………………………………………..32 Paper I and II ECP in Neutrophils, monocytes and macrophages………………………………....32 De novo production of protein in circulating cells?…………………………………32 Uptake, storage and release…………………………………………………….……33 Figure 2………………………………………………………………………….…..35 5 Figure 3………………………………………………………………………..35 Paper III and IV ECP gene sequencing…………………………………………………….……36 General population study…………………………………………………..….36 Figure 4……………………………………………………………….………..37 The 434 polymorphism in Hodgkin Lymphoma………………………………38 The 434 polymorphism in allergy and asthma………………………….……..38 Summary and Conclusions………………………………………………40 Aknowledements…………………………………………………………..41 Refereces……………………………………………………………………43 Abbreviations α2M alpha 2 Macroglobulin αxβx integrins βxintegrin B-cell B lyphocyte BPI Bacteria permeability increasing protein C/EBP CAAT box / enhancer binding protein CHL Classical HL CR Complete remission cSNP SNP in coding region EBV Eppstin Barr virus E.coli Escherichia coli ECP Eosinophil cationic protein ECR Erythrocyte sedimentation rate EG-2 ECP specific Mab EPO Eosinophil peroxidase EPX/EDN Eosinophil protein X/Eosinophil derived neurotoxin FcxR Fc receptor GATA Transcriptionfactor G-CSF Granulocyte colony stimulating factor GM-CSF Granulocyte/Macrophage stimulating factor GADPH Glutaraldehyde dehydrogenase GI Gastointestinal H cells Histocytes HL Hodgkin lymphoma H-RS Hodkin Reed Sternberg cells ICAM-1 Intracellular adhesion molecule I Ig Immunoglobulin IGF-1 insulin like growth factor 1 IL Interleukin 6 IPS International prognostic score LDHL Lymphocyte depletion HL LFA-1 Leukocyte function associated antigen 1 L&H cells Lymphocytic and histocytic cells LMP-1 latent membrane protein 1 LRHL Lymphocyte rich HL Mab Monoclonal antibody MAC-1 Monocyte adhesion complex 1 MBP Major basic protein MCHL Mixed cellularity HL MCP-5 Monocyte chemotactic protein 5 M-CSF Macrophage- colony stimulating factor MHC II Major Histocomptability complex class II MIP-1α Macrophage inflammatory protein 1α MPO Myeloperoxidase NFAT Nuclear factor of activated T cells NFkB Nuclear factor kappa B NLPHL Nodular lyphocyte predominance HL NSHL Nodular scleroting HL PAF Platlet activating factor PCR Polymerase chain reaction PU.1 transcription factor RNase Ribonuclease RANTES Regulation upon activation normal T-cell expressed presumed secreted rECP recombinant ECP RSV-B Respiratory syncytial virus B RT Reverse transcriptase S.aureus Staphylococcus aureus SNP single nucleotide polymorphism Th2 cell T Lymphocyte type 2 T cell T lymphocyte TCR T cell recptor TGF-b Transforming growth factor beta TNF-a Tumour Necrosis factor alpha UTR Unstranslated region VCAM-1 Vascular cell adhesion molecule 1 Vla-4 Very late antigen 4 WBC White blood cell count 7 Introduction Inflammatory cells of the innate immunity system The common ancestral tasks of the innate immunity cells are those of phagocytosis of foreign intruders and secretion of destructive peptides and proteins (1), but through evolution different cells have evolved and become specialised in different ways. The leukocytes of the myeloid lineage constitute a separate defence system older than the lymphatic immune system, but a prominent cross talk has evolved between these cells. The cells all have receptors for immunoglobulins (Ig´s), one of the many connections to the lymphatic immune system. In several dysregulatory conditions the cells are recruited and perform actions that damage the tissue. The leukocytes of the innate immunity system are produced in the bone marrow and released into the bloodstream. Cells of the innate immunity system are neutrophils, eosinophils, basophils, mast cells and monocytes/macrophages. In the circulation the neutrophils and monocytes act as guards towards bacteria,
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