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Immunohistochemical detection of interferon-gamma: fake or fact?
C M van der Loos1, M A Houtkamp, O J de Boer
1Academic Medical Center, Department of Cardiovascular Pathology, Amsterdam, The Netherlands. c.m.vanderloos@amc.uva.nl
Insights
Immunohistochemistry for interferon-gamma (IFNγ) is unreliable for detecting T-cells in tissues. Most tested antibodies showed poor specificity, leading to non-specific staining in various cell types, not T-cells.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Immunohistochemistry (IHC) is a common method for detecting proteins like cytokines in tissues.
- Interferon-gamma (IFNγ) is a key cytokine in immune responses, particularly Th1-mediated immunity.
- Accurate detection of IFNγ in situ is crucial for understanding inflammatory and autoimmune diseases.
Purpose of the Study:
- To evaluate the specificity and reproducibility of commercially available anti-IFNγ antibodies for immunohistochemistry.
- To determine if IFNγ-IHC can reliably identify IFNγ-producing T-cells in human tissue samples.
- To assess the reliability of IFNγ-IHC data in disease contexts.
Main Methods:
- Thirteen anti-IFNγ antibodies were tested using immunohistochemistry, immunocytochemistry, and flow cytometry (FACS).
- Tests were performed on stimulated and unstimulated T-cells in vitro and on tissue sections from human reactive tonsils, rheumatoid synovium, and aortic aneurysms.
- Immunoenzyme double staining was employed to identify IFNγ-positive cells.
Main Results:
- While 12 out of 13 antibodies showed positive results in immunocytochemistry and FACS on cultured T-cells, none were successful in immunohistochemistry on sectioned T-cells.
- IFNγ-IHC on tissue sections revealed significant non-specific staining in smooth muscle cells, endothelial cells, extracellular matrix, and plasma cells.
- No antibody achieved specific staining of T-cells as the sole positive cell type in tissue sections.
Conclusions:
- Current anti-IFNγ antibodies lack the specificity required for reliable detection of IFNγ-producing T-cells in situ using immunohistochemistry.
- The widespread use of IFNγ-IHC may lead to misinterpretation of results due to non-specific staining.
- Data derived from IFNγ-immunohistochemistry should be interpreted with extreme caution, and alternative methods may be necessary.
Abstract:
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.
