Immunohistochemical detection of interferon-gamma: fake or fact?

Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNgamma immunohistochemistry on tissue sections with a large panel of anti-IFNgamma antibodie...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry Vol. 49; no. 6; p. 699
Main Authors: van der Loos, C M, Houtkamp, M A, de Boer, O J, Teeling, P, van der Wal, A C, Becker, A E
Format: Journal Article
Language:English
Published: United States 01.06.2001
Subjects:
Aged
Antibody Specificity
Aortic Aneurysm - immunology
Aortic Aneurysm - pathology
Arthritis, Rheumatoid - immunology
Arthritis, Rheumatoid - pathology
Child
Child, Preschool
Flow Cytometry
Humans
Immunoenzyme Techniques - methods
Immunohistochemistry - methods
Indicators and Reagents - standards
Interferon-gamma - immunology
Interferon-gamma - isolation & purification
Middle Aged
Palatine Tonsil - immunology
Palatine Tonsil - pathology
Reproducibility of Results
Th1 Cells
ISSN:0022-1554
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Summary:Immunohistochemistry is a widely accepted tool to investigate the presence and immunolocalization of cytokines in tissue sections at the protein level. We have tested the specificity and reproducibility of IFNgamma immunohistochemistry on tissue sections with a large panel of anti-IFNgamma antibodies. Thirteen different commercially available anti-IFNgamma antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidin-biotin-peroxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFNgamma-positive cells. Serial cryostat sections were used of human reactive hyperplastic tonsils, rheumatoid synovium, and inflammatory abdominal aortic aneurysms, known to possess a prominent Th1-type immune response. In vitro phorbol myristate acetate/ionomycin-stimulated T-cells served as positive control; unstimulated cells served as negative control. Cultured T-cells were used adhered to glass slides (immunocytochemistry), in suspension (FACS), or snap-frozen and sectioned (immunohistochemistry). Immunocytochemistry and FACS analysis on stimulated cultured T-cells showed positive staining results with 12 of 13 anti-IFNgamma antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFNgamma immunohistochemical single- and double staining analysis of the tissue sections showed huge variations in staining patterns, including positivity for smooth muscle cells (n = 8), endothelial cells (n = 4), extracellular matrix (n = 4), and CD138+ plasma cells (n = 12). Specific staining of T-cells, as the sole positive staining, was not achieved with any of the 13 antibodies. IFNgamma-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFNgamma antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFNgamma-immunohistochemistry must be interpreted with great caution.
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ISSN:0022-1554
DOI:10.1177/002215540104900604