Detection and localization of non-HLA-ABC antigenic sites relevant to kidney rejection on endothelial cells

• Published in: Journal of immunological methods Vol. 251; no. 1; pp. 73 - 80
• Main Authors: Pontes, L.F.S., Carvalho, L., Stumbo, A.C., Porto, L.C.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2001; Elsevier
• Subjects: Adult; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Cells, Cultured; Diverse techniques; Endothelial cell; Endothelium, Vascular - immunology; Female; Fluorescent Antibody Technique, Indirect; Fundamental and applied biological sciences. Psychology; Graft Rejection - etiology; Graft Rejection - immunology; histocompatibility antigen HLA; HLA; HLA Antigens; Humans; Immunohistochemistry - methods; Isoantibodies - metabolism; Kidney transplant; Kidney Transplantation - adverse effects; Kidney Transplantation - immunology; Male; Microbiology; Microscopy, Immunoelectron; Middle Aged; Molecular and cellular biology; Rejection; ADCC, antibody-dependent cell cytotoxicity; HLA, human leukocyte antigen; PR, patient with previous renal graft rejection; Endothelial cell; BSA, bovine serum albumin; HUVEC, human umbilical vein endothelial cells; Ig, immunoglobulin; FITC, fluorescein isothiocyanate; PBS, phosphate buffered saline; ELISA, enzyme-linked immunosorbent assay; TRITC, tetramethylrhodamine isothiocyanate; CDC, complement dependent cytotoxicity; Rejection; DMEM, Dulbecco’s modified Eagle’s medium; Kidney transplant; PHS, pooled normal human serum; HLA; FCS, fetal calf serum; Human; HLA-System; Homograft; Kidney; Endothelium; Antigen; Urinary system; Antigenicity; Target cell; Detection; Localization; Method study
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/S0022-1759(01)00309-X

Immunological rejection of kidney allografts is usually attributed to presensitization to HLA antigens. However, data on HLA identical transplant rejections indicate that non-HLA antigens may also be involved, and it has been suggested that vascular endothelium represents the main target cell. The purpose of the present study is to describe a method of detecting non-HLA antibodies immunocytochemically. We showed the molecular independence between HLA-ABC molecules identified by the monoclonal antibody w6/32, and antigenic sites identified by a kidney rejection patient serum, previously characterized, on cultured endothelial cells isolated from human umbilical cords by collagenase digestion. Single immunofluorescence staining indicated the molecular independence between these antigenic sites, as the first serum showed a granular pattern, diffused throughout the cytoplasm and the other a reticular pattern restricted to the same cytoplasmic region. This result was confirmed by double labeling. Immunoelectronmicroscopy study also confirmed site independence, showing labeling patterns with different intensities and distinct localizations, using 10- and 20-nm colloidal gold particles to reveal HLA-ABC and non-HLA-ABC determinants, respectively. In conclusion, cultured endothelial cells may be used immunocytochemically to detect non-HLA-ABC determinants of antibody reactivity in renal graft recipients, and the indirect immunofluorescence may be the methodology of choice, since it is easy, reliable and low cost.