JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Imunologia e Infecção

使用流细胞测定测量红细胞补充受体1

Published: May 19, 2020
doi:
10.3791/60810
, , , , ,
1Oncogeriatric Coordination Unit,Reims University Hospitals, Maison Blanche Hospital, 2Faculty of Medicine,University of Reims Champagne-Ardenne, 3URCACyt, Flow cytometry technical platform,University of Reims Champagne-Ardenne, 4Department of Immunology,Reims University Hospitals, Robert Debre Hospital, 5Department of Internal Medicine and Geriatrics,Reims University Hospitals, Maison Blanche Hospital, 6Faculty of Medicine,University of Reims Champagne-Ardenne

Summary

该方法的目的是通过与已知红细胞CR1密度的三个受试者进行比较，确定任何受试者红细胞CR1密度的CR1密度。该方法使用抗CR1单克隆抗体在受试者红细胞免疫染色后使用流细胞学，并结合使用植物素（PE）的放大系统。

Abstract

CR1（CD35，C3b/C4b的补色受体1型）是一种高分子量膜糖蛋白，约200 kDa，控制补充活化，运输免疫复合物，并参与体液和细胞免疫反应。CR1 存在于许多细胞类型的表面上，包括红细胞，在长度、结构（Knops 或 KN、血群）和密度方面表现出多态性。每红细胞（CR1/E）CR1的平均密度为每红细胞500个分子。此密度因人而异（100~1，200 CR1/E），同一个体从一个红细胞到另一个红细胞。我们在这里提出了一种可靠的流细胞测量方法，用于测量CR1/E的密度，包括在表达低密度的受试者中，借助放大免疫染色系统。这种方法使我们能够显示CR1红细胞表达在疾病，如阿尔茨海默氏病（AD），全身红斑狼疮（SLE），艾滋病，或疟疾的降低。

Introduction

CR1（补充受体类型1，CD35）是一种200 kDa跨膜糖蛋白存在于许多细胞类型的表面，如红细胞1、B淋巴细胞2、单细胞细胞、一些T细胞、卵泡变性细胞3、胎儿星斑细胞4和球状球状细胞5。CR1干扰其配体C3b、C4b、C3bi6,6、7、8、9，第一补分组C1q7,8,910和MBL（曼南结合语）11的子单元抑制补品的激活，并参与体液和细胞免疫反应。

在灵长类动物，包括人类，红细胞CR1参与将免疫复合物输送到肝脏和脾脏，以净化血液，防止它们在脆弱的组织中积累，如皮肤或肾脏12、13、14。12,13,14免疫复合物和红细胞之间的免疫粘附现象取决于CR1分子的数量15。在人类中，CR1/E的平均密度只有500（即，每红细胞500个CR1分子）。此密度因人而异（100~1，200 CR1/E），同一个体从一个红细胞到另一个红细胞。一些”空”表型的个体表达少于20 CR1/E16。

CR1/E的密度由两个共同主导体体等位基因调节，这些等位基因编码为CR1_117，18,18的intron 27中。这种突变为HindIII酶产生额外的限制位点。在这种情况下，使用 HindIII 消化后获得的限制片段为 7.4 kb，用于与 CR1（H： 高等位基因）的强表达相连的等位基因，与低 CR1 表达式（L：低等位基因）相连的等位机为 6.9 kb。这种联系在白种人和亚洲人中被发现，但在19岁的非洲人后裔中却找不到。

红细胞CR1的表达水平也与exon 13编码SCR 10（I643T）和exon 19编码SCR16（Q981H）中存在点核苷酸突变有关。其高在同型643I/981Q和低同源643T/981H个体20。因此，”低”个人表达约150 CR1/E，”中等”个人表达约500CR1/E，”高”个人表达约1，000CR1/E。

除了这种红细胞密度多态性外，CR1 还具有与四种不同尺寸的异位对应的长度多态性：CR1+1 （190 kDa）、CR1+2 （220 kDa）、CR1+3 （160 kDa） 和 CR1+4 （250 kDa）21和对应于血型 KN22的抗原多态性。

我们提出基于流细胞测定的方法，以确定CR1/E的密度。 使用已知CR1/E密度的三个受试者，表达低密度水平（180 CR1/E）、中等密度水平（646 CR1/E）和高密度水平（966 CR1/E），使用流式细胞仪进行抗CR1免疫后，很容易测量其红细胞或红血球（RBC）的平均荧光强度（MFI）。然后，可以将表示 MFI 的标准线绘制为 CR1/E 密度的函数。测量其CR1/E密度不为人知的受试者的MFI，并将其与该标准线进行比较，可以确定个体的CR1/E密度。这项技术已在实验室使用多年，并使我们能够发现红细胞CR1在许多疾病的表达减少，如系统性红斑狼疮（SLE）23，后天免疫机能丧失综合症（艾滋病）24，疟疾25，最近阿尔茨海默病（AD）26，27。26,27开发针对CR1的药物与红细胞耦合，如抗血栓药物28需要评估CR1/E密度，以及提供量化CR1的可靠技术。

呈现的协议以唱歌的方式运行。它适用于确定CR1/E的密度，许多个人使用特定的商业上可用的96孔板（见材料表）。为此，我们很容易将方法适应任何96井板。对于每个样本，红细胞细胞的细胞悬浮液（0.5 x 106×106红细胞）分布在每口井。对于每一口井，首先添加主要的抗CR1抗体，然后是链球菌PE，二级抗链球菌类抗体，再次使用与方法相同的稀释剂，但通过调整体积和尊重相称性。

应同时抽取来自受试者的血液样本和用于CR1的受试者的血液样本，在4°C下储存在冰箱中，并在4°C（冰上和/或冰箱）处理。

Protocol

《人类血液收集和处理议定书》经区域道德委员会（CPP Est II）审查和批准，协议编号为2011-A00594-37。由于以下议定书描述了人类血液的处理，因此应遵循生物有害物质处理的制度准则。实验室安全设备，如实验室外套和手套，应穿。 1. 红细胞洗涤 注：处理前一天，准备一个含有0.15%牛血清白蛋白（BSA）的磷酸盐缓冲盐水（PBS）的PBS-BSA缓冲液，并将其放入?…

Representative Results

已知CR1密度的三个受试者的红细胞（”低”受试者[180 CR1/E]，”中等”受试者[646 CR1/E]和”高”受试者[966 CR1/E]）和需要确定CR1密度的两个受试者的红细胞被抗CR1抗体与使用植物氟色素的扩增系统耦染免疫。一开始，低高范围受试者的CR1密度由Scatchard方法29使用放射性标记抗体确定。确定的标准（低、中、高）用于校准曲线，并使得通过我们的细胞测定方法30来量化新标准…

Discussion

有多种技术可用于确定红细胞CR1（CR1/E）的密度。使用的第一种技术是抗CR1抗体31凝固红血球，以及红细胞在红细胞中涂有C3b32时形成红血球。这些基本技术很快被使用放射性标记的抗CR1抗体11，3333的免疫染色方法所取代。也可以通过酶相关免疫吸附剂测定（ELISA）34来测量膜提取物中CR1的浓度。虽然?…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢URCACyt、流细胞学技术平台的所有成员、免疫学系的工作人员以及内科和老年病学部的工作人员，他们为优化和验证协议做出了贡献。这项工作由兰斯大学医院资助（赠款号AOL11UF9156）。

Materials

1000E Barrier Tip Thermo Fischer Scientific , F-67403 Illkirch, France 2079E sample pipetting
1-100 µL Bevelled, filter tip Starlab GmbH, D-22926 Ahrenburg, Germany S1120-1840 sample pipetting
Biotinylated anti-CR1 monoclonal antibody (J3D3) Home production of non-commercial monoclonal antibody, courtesy of Dr J. Cook immunostaining
Blouse protection
Bovin serum albumin (7,5%) Thermo Fischer Scientific , F-67403 Illkirch, France 15260037 cytometry
Centrifuge Thermo Fischer Scientific , F-67403 Illkirch, France 11176917 centrifugation
Clean Solution BD, F-38801 Le Pont de Claix, France 340345 cytometry
Comorack-96 Dominique DUTSCHER SAS, F-67172 Brumath 944060P rack
Cytometer Setup & Tracking Beads Kit BD, F-38801 Le Pont de Claix, France 655051 cytometry
Formaldehyde solution 36.5 % Sigma Aldrich, F-38070 Saint Quentin Fallavier, France F8775-25ML Fixation
10 µL Graduated, filter tip Starlab GmbH, D-22926 Ahrenburg, Germany S1121-3810 sample pipetting
LSRFORTESSA Flow Cytometer BD, F-38801 Le Pont de Claix, France 647788 cytometry
Microman Capillary Pistons Dominique DUTSCHER SAS, F-67172 Brumath 067494 sample pipetting
Micronic 1.40 mL round bottom tubes Dominique DUTSCHER SAS, F-67172 Brumath MP32051 mix
Micropipette Microman – type M25 – Dominique DUTSCHER SAS, F-67172 Brumath 066379 sample pipetting
Phosphate buffered Saline (PBS) Thermo Fischer Scientific , F-67403 Illkirch, France 10010031 cytometry
Pipette PS 325 mm, 10 mL Dominique DUTSCHER SAS, F-67172 Brumath 391952 sample pipetting
powder-free Nitrile Exam gloves Medline Industries, Inc, Mundelein, IL 60060, USA 486802 sample protection
Reference 2 pipette, 0,5-10 µL Eppendorf France SAS, F-78360 Montesson, France 4920000024 sample pipetting
Reference 2 pipette, 20-100 µL Eppendorf France SAS, F-78360 Montesson, France 4920000059 sample pipetting
Reference 2 pipette, 100-1000 µL Eppendorf France SAS, F-78360 Montesson, France 4920000083 sample pipetting
Rinse Solution BD, F-38801 Le Pont de Claix, France 340346 cytometry
Round bottom tube Sarstedt, F-70150 Marnay, France 55.1579 cytometry
Safe-Lock Tubes, 1.5 mL Eppendorf France SAS, F-78360 Montesson, France 0030120086 mix
streptavidin R-PE Tebu Bio, F-78612 Le Perray-en-Yvelines, France AS-60669 immunostaining
Tapered Centrifuge Tubes 50 mL Thermo Fischer Scientific , F-67403 Illkirch, France 10203001 mix
Vector anti streptavidin biotin Eurobio Ingen, F-91953 Les Ulis, France BA-0500 immunostaining
Vortex-Genie 2 Scientific Industries, Inc, Bohemia, NY 111716, USA SI-0236 mix
DOWNLOAD MATERIALS LIST

Referências

  1. Fearon, D. T. Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte. Journal of Experimental Medicine. 152 (1), 20-30 (1980).
  2. Ross, G. D., Winchester, R. J., Rabellino, E. M., Hoffman, T. Surface markers of complement receptor lymphocytes. Journal of Clinical Investigation. 62 (5), 1086-1092 (1978).
  3. Reynes, M., et al. Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens. The Journal of Immunology. 135 (4), 2687-2694 (1985).
  4. Gasque, P., et al. Identification and characterization of complement C3 receptors on human astrocytes. The Journal of Immunology. 156 (6), 2247-2255 (1996).
  5. Pascual, M., et al. Identification of membrane-bound CR1 (CD35) in human urine: evidence for its release by glomerular podocytes. Journal of Experimental Medicine. 179 (3), 889-899 (1994).
  6. Fearon, D. T. Regulation of the amplification C3 convertase of human complement by an inhibitory protein isolated from human erythrocyte membrane. Proceedings of the National Academy of Sciences of the United States of America. 76 (11), 5867-5871 (1979).
  7. Dobson, N. J., Lambris, J. D., Ross, G. D. Characteristics of isolated erythrocyte complement receptor type one (CR1, C4b-C3b receptor) and CR1-specific antibodies. The Journal of Immunology. 126 (2), 693-698 (1981).
  8. Schreiber, R. D., Pangburn, M. K., Muller-Eberhard, H. J. C3 modified at the thiolester site: acquisition of reactivity with cellular C3b receptors. Bioscience Reports. 1 (11), 873-880 (1981).
  9. Ross, G. D., et al. Generation of three different fragments of bound C3 with purified factor I or serum. II. Location of binding sites in the C3 fragments for factors B and H, complement receptors, and bovine conglutinin. Journal of Experimental Medicine. 158 (2), 334-352 (1983).
  10. Klickstein, L. B., Barbashov, S. F., Liu, T., Jack, R. M., Nicholson-Weller, A. Complement receptor type 1 (CR1, CD35) is a receptor for C1q. Immunity. 7 (3), 345-355 (1997).
  11. Ghiran, I., et al. Complement receptor 1/CD35 is a receptor for mannan-binding lectin. Journal of Experimental Medicine. 192 (12), 1797-1808 (2000).
  12. Cornacoff, J. B., et al. Primate erythrocyte-immune complex-clearing mechanism. Journal of Clinical Investigation. 71 (2), 236-247 (1983).
  13. Waxman, F. J., et al. Complement depletion accelerates the clearance of immune complexes from the circulation of primates. Journal of Clinical Investigation. 74 (4), 1329-1340 (1984).
  14. Waxman, F. J., et al. Differential binding of immunoglobulin A and immunoglobulin G1 immune complexes to primate erythrocytes in vivo. Immunoglobulin A immune complexes bind less well to erythrocytes and are preferentially deposited in glomeruli. Journal of Clinical Investigation. 77 (1), 82-89 (1986).
  15. Horgan, C., Taylor, R. P. Studies on the kinetics of binding of complement-fixing dsDNA/anti-dsDNA immune complexes to the red blood cells of normal individuals and patients with systemic lupus erythematosus. Arthritis & Rheumatology. 27 (3), 320-329 (1984).
  16. Pham, B. N., et al. Analysis of complement receptor type 1 expression on red blood cells in negative phenotypes of the Knops blood group system, according to CR1 gene allotype polymorphisms. Transfusion. 50 (7), 1435-1443 (2010).
  17. Wilson, J. G., et al. Identification of a restriction fragment length polymorphism by a CR1 cDNA that correlates with the number of CR1 on erythrocytes. Journal of Experimental Medicine. 164 (1), 50-59 (1986).
  18. Rodriguez de Cordoba, S., Rubinstein, P. Quantitative variations of the C3b/C4b receptor (CR1) in human erythrocytes are controlled by genes within the regulator of complement activation (RCA) gene cluster. Journal of Experimental Medicine. 164 (4), 1274-1283 (1986).
  19. Herrera, A. H., Xiang, L., Martin, S. G., Lewis, J., Wilson, J. G. Analysis of complement receptor type 1 (CR1) expression on erythrocytes and of CR1 allelic markers in caucasian and african american populations. Clinical Immunology and Immunopathology. 87 (2), 176-183 (1998).
  20. Birmingham, D. J., et al. A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b. Immunology. 108 (4), 531-538 (2003).
  21. Dykman, T. R., Hatch, J. A., Aqua, M. S., Atkinson, J. P. Polymorphism of the C3b/C4b receptor (CR1): characterization of a fourth allele. The Journal of Immunology. 134 (3), 1787-1789 (1985).
  22. Moulds, J. M., Moulds, J. J., Brown, M., Atkinson, J. P. Antiglobulin testing for CR1-related (Knops/McCoy/Swain-Langley/York) blood group antigens: negative and weak reactions are caused by variable expression of CR1. Vox Sanguinis. 62 (4), 230-235 (1992).
  23. Cohen, J. H., Lutz, H. U., Pennaforte, J. L., Bouchard, A., Kazatchkine, M. D. Peripheral catabolism of CR1 (the C3b receptor, CD35) on erythrocytes from healthy individuals and patients with systemic lupus erythematosus (SLE). Clinical & Experimental Immunology. 87 (3), 422-428 (1992).
  24. Jouvin, M. H., Rozenbaum, W., Russo, R., Kazatchkine, M. D. Decreased expression of the C3b/C4b complement receptor (CR1) in AIDS and AIDS-related syndromes correlates with clinical subpopulations of patients with HIV infection. AIDS. 1 (2), 89-94 (1987).
  25. Waitumbi, J. N., Donvito, B., Kisserli, A., Cohen, J. H., Stoute, J. A. Age-related changes in red blood cell complement regulatory proteins and susceptibility to severe malaria. The Journal of Infectious Diseases. 190 (6), 1183-1191 (2004).
  26. Mahmoudi, R., et al. Alzheimer’s disease is associated with low density of the long CR1 isoform. Neurobiology of Aging. 36 (4), 5-12 (2015).
  27. Mahmoudi, R., et al. Inherited and Acquired Decrease in Complement Receptor 1 (CR1) Density on Red Blood Cells Associated with High Levels of Soluble CR1 in Alzheimer’s Disease. International Journal of Molecular Sciences. 19 (8), 2175 (2018).
  28. Zaitsev, S., et al. Human complement receptor type 1-directed loading of tissue plasminogen activator on circulating erythrocytes for prophylactic fibrinolysis. Blood. 108 (6), 1895-1902 (2006).
  29. Scatchard, G. The attractions of proteins for small molecules and ions. Annals of the New York Academy of Sciences. 51 (4), 660-672 (1949).
  30. Cohen, J. H., et al. Enumeration of CR1 complement receptors on erythrocytes using a new method for detecting low density cell surface antigens by flow cytometry. Journal of Immunological Methods. 99 (1), 53-58 (1987).
  31. Minota, S., et al. Low C3b receptor reactivity on erythrocytes from patients with systemic lupus erythematosus detected by immune adherence hemagglutination and radioimmunoassays with monoclonal antibody. Arthritis & Rheumatology. 27 (12), 1329-1335 (1984).
  32. Miyakawa, Y., et al. Defective immune-adherence (C3b) receptor on erythrocytes from patients with systemic lupus erythematosus. The Lancet. 2 (8245), 493-497 (1981).
  33. Lida, K., Mornaghi, R., Nussenzweig, V. Complement receptor (CR1) deficiency in erythrocytes from patients with systemic lupus erythematosus. Journal of Experimental Medicine. 155 (5), 1427-1438 (1982).
  34. Tao, K., Nicholls, K., Rockman, S., Kincaid-Smith, P. Expression of complement 3 receptors (CR1 and CR3) on neutrophils and erythrocytes in patients with IgA nephropathy. Clinical Nephrology. 32 (5), 203-208 (1989).
  35. Nickells, M., et al. Mapping epitopes for 20 monoclonal antibodies to CR1. Clinical and Experimental Immunology. 112 (1), 27-33 (1998).
  36. Oi, V. T., Glazer, A. N., Stryer, L. Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. The Journal of Cell Biology. 93 (3), 981-986 (1982).
  37. Chaiet, L., Wolf, F. J. The properties of streptavidin, a biotin-binding protein produced by streptomyces. Archives of Biochemistry and Biophysics. 20 (106), 1-5 (1964).
  38. Cockburn, I. A., Donvito, B., Cohen, J. H., Rowe, J. A. A simple method for accurate quantification of complement receptor 1 on erythrocytes preserved by fixing or freezing. Journal of Immunological Methods. 20 (271), 59-64 (2002).
  39. Chen, C. H., et al. Antibody CR1-2B11 recognizes a non-polymorphic epitope of human CR1 (CD35). Clinical & Experimental Immunology. 148 (3), 546-554 (2007).
  40. Ripoche, J., Sim, R. B. Loss of complement receptor type 1 (CR1) on ageing of erythrocytes. Studies of proteolytic release of the receptor. Biochemical Journal. 235 (3), 815-821 (1986).
  41. Moldenhauer, F., Botto, M., Walport, M. J. The rate of loss of CR1 from ageing erythrocytes in vivo in normal subjects and SLE patients: no correlation with structural or numerical polymorphisms. Clinical & Experimental Immunology. 72 (1), 74-78 (1988).
  42. Cohen, J. H., Lutz, H. U., Pennaforte, J. L., Bouchard, A., Kazatchkine, M. D. Peripheral catabolism of CR1 (the C3b receptor, CD35) on erythrocytes from healthy individuals and patients with systemic lupus erythematosus (SLE). Clinical & Experimental Immunology. 87 (3), 422-428 (1992).
  43. Nickells, M. W., Subramanian, V. B., Clemenza, L., Atkinson, J. P. Identification of complement receptor type 1-related proteins on primate erythrocytes. The Journal of Immunology. 154 (6), 2829-2837 (1995).
  44. Hebert, L. A., Birmingham, D. J., Shen, X. P., Cosio, F. G. Stimulating erythropoiesis increases complement receptor expression on primate erythrocytes. Clinical Immunology and Immunopathology. 62 (3), 301-306 (1992).
  45. Davis, K. A., Abrams, B., Iyer, S. B., Hoffman, R. A., Bishop, J. E. Determination of CD4 antigen density on cells: Role of antibody valency, avidity, clones, and conjugation. Cytometry. 33 (2), 197-205 (1998).
  46. Pannu, K. K., Joe, E. T., Iyer, S. B. Performance evaluation of QuantiBRITE phycoerythrin beads. Cytometry. 45 (4), 250-258 (2001).
  47. Barnett, D., Storie, I., Wilson, G. A., Granger, V., Reilly, J. T. Determination of leucocyte antibody binding capacity (ABC): the need for standardization. Clinical Laboratory Haematology. 20 (3), 155-164 (1998).
  48. Bikoue, A., et al. Quantitative analysis of leukocyte membrane antigen expression: normal adult values. Cytometry. 26 (2), 137-147 (1996).
  49. Serke, S., van Lessen, A., Huhn, D. Quantitative fluorescence flow cytometry: a comparison of the three techniques for direct and indirect immunofluorescence. Cytometry. 33 (2), 179-187 (1998).

Tags

ErythrocyteComplement Receptor 1Flow CytometryCell Receptor DensityImmunostainingBiotinylated AntibodyStreptavidin Phycoerythrin
check_url/pt/60810?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Kisserli, A., Audonnet, S., Duret, V., Tabary, T., Cohen, J. H. M., Mahmoudi, R. Measuring Erythrocyte Complement Receptor 1 Using Flow Cytometry. J. Vis. Exp. (159), e60810, doi:10.3791/60810 (2020).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image