ARTICLE |
Correspondence to: Chris M. van der Loos, Academical Medical Center, Dept. of Cardiovascular Pathology (H0-120), Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. E-mail: c.m.vanderloos@amc.uva.nl
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Summary |
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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 IFN immunohistochemistry on tissue sections with a large panel of anti-IFN
antibodies. Thirteen different commercially available anti-IFN
antibodies, including seven advertised and/or regularly applied for immunohistochemistry/-cytochemistry, were tested using a three-step streptavidinbiotinperoxidase technique and a two-step immunofluorescence (FACS) analysis. Immunoenzyme double staining was used to identify the IFN
-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-IFN
antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFN
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. IFN
-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFN
antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFN
-immunohistochemistry must be interpreted with great caution. (J Histochem Cytochem 49:699709, 2001)
Key Words:
IFN protein, immunohistochemistry, immunocytochemistry, T-cells, plasma cells
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Introduction |
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INTERFERON-GAMMA (IFN) is a 3450-kD cytokine exclusively produced by T-cells and natural killer cells (
has been widely investigated in immunological research. In addition to in vitro studies, many investigators also attempted to demonstrate IFN
protein immunohistochemically in healthy and diseased tissues. For example, studies have been undertaken to localize IFN
in rheumatoid arthritis (
antibodies have raised doubts concerning the specificity and the consistency of the staining results obtained. This provides the background for the present study, in which we verified the specificity of anti-IFN
immunohistochemical staining using a panel of commercially available poly- and monoclonal antibodies.
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Materials and Methods |
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Human Tissue Samples
Tonsils with hyperplastic reactive changes (n=3, age 36 years), inflammatory abdominal aortic aneurysms (IAAA) (n=2, age 60 and 75 years), and synovial tissue specimens from knee joint with rheumatoid arthritis (RA synovium) (n=3, age 6277 years) were obtained at surgery. These tissue samples were used as positive controls for IFN staining, based on the expression of IFN
mRNA as detected with RT-PCR (
protein was detected with immunohistochemistry in tonsil and RA synovium (
Cell Specimens
Cultured T-cell lines from human aortic atherosclerotic plaques were prepared as previously described (. After 4 h, the cell suspensions were washed three times with PBS without protein additives and divided into three fractions. The first fraction was adhered overnight at 4C on BioRad adhesion slides (Hercules, CA), 2 x 104 cells per spot. After brief washing with PBS, these cell specimens were stored in PBS at 4C and used for immunocytochemistry within 2 days. The second fraction was concentrated in an Eppendorf tube in 100 µl PBS (2 x 105 cells), mixed gently with 100 µl Tissue-Tek OCT compound (Sakura; Zoeterwoude, The Netherlands), and snap-frozen in liquid nitrogen. Six-µm cryostat sections were cut and handled as described for tissue cryostat sections. The remaining cells were used for FACS analysis.
Immunostaining Reagents
We employed 13 anti-human anti-IFN poly- and monoclonal antibodies, as listed in Table 1. As indicated in Table 1, seven anti-IFN
antibodies were advertised and/or regularly applied for immunocytochemistry/-histochemistry. Other primary antibodies used in this study are listed in Table 2.
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Biotinylated goat anti-mouse Ig (GAM/bio), biotinylated goat anti-rabbit Ig (GAR/bio), alkaline phosphatase-conjugated goat anti-rabbit Ig (GAR/AP), normal mouse serum (NMS), normal goat serum (NGS), normal swine serum (NSS), AP-conjugated streptavidin (streptavidin/AP), streptavidinbiotin complex with HRP (SABC/HRP), rabbit anti-fluorescein (rabbit anti-FITC), and endogenous biotin blocking kit were from DAKO (Glostrup, Denmark). Phycoerythrin (PE)-conjugated GAM, PE-conjugated GAR, biotinylated goat anti-mouse IgG2a (GAM-IgG2a/bio), and HRP-conjugated goat anti-mouse IgG1 (GAM-IgG1/HRP) were from Southern Biotechnology Associates (Birmingham, AL). Biotinylated and PE-conjugated swine anti-goat Ig (SAG) were from BioSource (Nivelles, Belgium). Rabbit anti-phycoerythrin (rabbit anti-PE) was from Biogenesis (Poole, UK). ß-Galactosidase-conjugated streptavidin (streptavidin/GAL) was from Boehringer/Roche (Mannheim, Germany). PowerVision-AP-conjugated goat anti-rabbit Ig (PowerVision-GAR/AP) was from ImmunoVision Technologies (Daly City, CA). 3-Amino-9-ethylcarbazole (AEC), naphthol-AS-MX-phosphate, Fast Blue BB (cat. no. 3378), and saponin (cat. no. 7900) were from Sigma.
Immunocytochemistry and Immunohistochemistry
Cell specimens and cryostat tissue sections were fixed in either acetone (10 min, 4C) or 4% paraformaldehyde (PFA) in PBS (5 min, room temperature). When PFA fixation was used, 0.1% saponin was added for membrane permeabilization ( antibodies (Table 1) and standard SABC/HRP detection. HRP activity was visualized with AEC (0.5 mg/ml) and peroxide (0.01%) in acetate buffer (pH 5.2, 50 mM).
Controls consisted of replacing the primary antibody with a non-immune mouse antibody of identical subclass (DAKO). Specific IgG concentration and Ig isotype/subclass were matched with the specific primary antibody from the original single-staining experiment.
Immunoenzyme Multiple Staining of Tissue Sections
A double-staining procedure based on a multistep technique ( immunostaining with different cellular markers. The staining procedure consisted of the following subsequent incubation steps: NGS (1:10, 15 min), IFN
antibody (clones MMHG-1 1:100, MD-2 1:50 or rabbit antibody Genzyme IP-500 1:5000, all overnight at 4C), GAM/bio (1:200, 30 min) or GAR/bio (1:400, 30 min), SABC/HRP (1:100, 30 min), and NMS (1:10, 15 min). For the goat anti-IFN
antibody, the first part of the double staining consisted of NSS (1:10, 15 min), IFN
goat antibody (1:100, overnight at 4C), SAG/biotin (1:100, 30 min), SABC/HRP (1:100, 30 min), and NGS (1:10, 15 min). Then, a panel of directly conjugated primary antibodies was applied: FITC-conjugated anti-CD3, CD2, CD138, biotinylated anti-CD68 (
Two triple immunoenzyme stainings using ß-galactosidase (turquoise), AP (red), and HRP (brown) as marker enzymes, and blue nuclear counterstain (-actin (mouse IgG2a) and anti-CD68 (mouse IgG1), combined with FITC-conjugated anti-CD3, in a multistep staining procedure. Anti-
-actin and anti-CD68 were incubated in a cocktail, followed by GAM-IgG2a/biotin (1:50), GAM-IgG1/HRP (1:50) in a cocktail (30 min) and streptavidin/GAL (1:40, 30 min). After a blocking step with normal mouse serum (1:10, 15 min), incubation was performed with FITC-conjugated anti-CD3, rabbit anti-FITC (1:1000, 15 min), and GAR/AP (1:20, 30 min). Smooth muscle cells, macrophages, and T-cells were immunostained in turquoise, brown, and red, respectively. The second consisted of anti-
-actin (mouse IgG2a), anti-CD68 (mouse IgG1), and anti-von Willebrand factor (rabbit) in one cocktail, followed by GAM-IgG2a/bio (1:50), GAM-IgG1/HRP (1:50), GAR/AP (1:20) in cocktail (30 min), and streptavidin/GAL (1:40, 30 min). Smooth muscle cells, macrophages, and endothelial cells were immuno-stained in turquoise, brown, and red, respectively.
FACS Analysis
All 13 anti-IFN antibodies were applied for indirect intracellular FACS staining. In vitro PMA/ionomycin-stimulated T-cells were fixed with 1% PFA in PBS (10 min, 4C) and treated with 0.1% saponin for permeabilization (
Leakage of IFN from Tissue Sections
From the frozen stimulated and unstimulated cultured T-cells, 20 6-µm sections were cut and mounted on organosilane-coated slides. Either unfixed, acetone-fixed (10 min, 4C, air-dried) or PFA-fixed (5 min, RT, briefly washed three times with TBS) sections were encircled with a wax pen and covered with 100 µl TBS for 30 min. Fifty-µl samples were taken from the buffer covering the tissue sections and subjected to IFN ELISA. A Pelikine IFN
kit was used, following the instructions as supplied by the manufacturer (CLB; Amsterdam, The Netherlands).
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Results |
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IFN Staining Characteristics with Cultured T-cells
Stimulated intact T-cells on adhesion slides showed a distinct signal in the majority of the cells with 12 of 13 anti-IFN antibodies (Fig 3E). Unstimulated cells were negative or occasionally weakly positive (Fig 3B). Stimulated T-cells showed a much stronger staining intensity after PFA fixation (Fig 3E) than after acetone fixation (Fig 3D). These immunocytochemical staining results were completely confirmed by FACS analysis (Table 3).
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An interesting observation was that IFN immunohistochemistry was consistently negative after cryosectioning of stimulated T-cells, (Table 3; Fig 3F), even after raising the primary antibody concentration to 50 µg/ml. This result was found after both acetone and PFA fixation.
To test the possible leakage of IFN from either unfixed, acetone-, or PFA-fixed cultured T-cell sections, buffer covering the specimens was subjected to ELISA. Buffers covering unfixed sections from stimulated T-cells contained IFN
protein at 14.449.3 pg/ml (n=4) and concentrations below detection limit (± 10 pg/ml) for the unstimulated cells. After acetone or PFA fixation, only values close to or below detection limit were found. Performance of this leakage test with the other tissue specimens failed because IFN
values with unfixed sections were too low.
IFN Staining Characteristics with Tissue Sections
Comparison of the IFN single staining with triple immunostainings marking the major cellular components in tonsil, RA synovium, and IAAA indicated positive IFN
staining of various tissue components with a preference for smooth muscle cells (n=8), endothelial cells (n=4), extracellular matrix (n=4), macrophages (n=1), and nerve bundles (n=2). Considering only those IFN
antibodies that were advertised and/or regularly applied for immunohistochemistry (n=7; Table 1), there was no consistent staining pattern either. The positive cell types included smooth muscle cells (n=4), endothelial cells (n=3), extracellular matrix (n=2), macrophages (n=1), and nerve bundles (n=1). Moreover, distinct expression of IFN
with T-cells was not observed. Remarkably, a strong IFN
smooth muscle cell positivity was also found in the media of non-atherosclerotic and non-inflamed aortic segments (Fig 1K). Apart from these various cellular constituents a distinct, consistently positive cell population was observed with 12 out of 13 antibodies. This characteristic cell population was found exclusively positive without additional staining of other cellular elements with three anti-IFN
antibodies: clones MMHG-1, 35B10G6, and RD Systems goat antibody (Table 3). These positive cells were found in relatively low amounts in tonsil and IAAA but were abundant in the RA synovium (Fig 2B2D). Negative control experiments did not show any staining (Fig 1I and Fig 2A). Typical results of anti-IFN
immunohistochemistry of all three tissues are summarized in Table 3 and illustrated in Fig 1A1H and Fig 2B2D.
Comparison of acetone and PFA as fixatives before IFN immunostaining showed similar results with respect to localization and staining intensity. Raising the antibody concentration (clones MMHG-1, MD-2, Genzyme rabbit antibody, and RD Systems goat antibody) up to 50 µg/ml, revealed over-stained images with intensely positive cells as described above.
To investigate the possible binding of IFN to its concomitant receptor, tissue localization of IFN
immunostaining was compared with IFN
receptor staining in serial sections. In RA synovium, IAAA, and tonsil, the anti-IFN
receptor antibody showed distinct positive staining of endothelial cells, macrophages, and weak staining of smooth muscle cells (Fig 1J), which could only partly be co-localized with the anti-IFN
staining (Fig 1B1H). Moreover, medial smooth muscle cells in non-atherosclerotic aortas were IFN
receptor (Fig 1L) -negative, which contrasted with the strong IFN
positivity (Fig 1K).
Cellular Specificity of Anti-IFN Antibodies
To reveal the characteristic cell type showing positive staining with almost all anti-IFN antibodies, clones MMHG-1, MD-2, Genzyme rabbit antibody, and RD Systems goat antibody were subjected to immunoenzyme double-staining experiments with cellular markers on the RA synovium and tonsil cryostat sections. It appeared that T-cells (CD2, CD3), natural killer cells (CD56), and macrophages (CD68) did not co-localize with the IFN
-positive cell population (Table 3). However, a subpopulation of approximately 5% of all CD138 positive plasma cells co-localized with IFN
-positive cells, observed as a purple mixed-color in the cytoplasm (Fig 4A4D).
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Discussion |
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In the present study we have compared the immunohistochemical applicability of 13 different anti-IFN antibodies on cryostat tissue sections. IFN
immunohistochemical single- and double-staining analysis of the tissue sections showed a large variation in staining patterns, but positive staining of T-lymphocytes was never observed. In this context, it should be emphasized that not all 13 IFN
-antibodies used in this study were originally developed and tested for an immunohistochemical application. This also could explain the highly variable immunohistochemical staining patterns (Fig 1A1H), despite the fact that the antibodies were capable of detecting IFN
in cultured and in vitro-stimulated intact T-cells (Table 3). For example, anti-IFN
clone MD-2 was first described for ELISA application by
We also verified the immunoreactivity of the anti-IFN antibodies on intact PMA/ionomycin-stimulated T-cells, known to contain substantial amounts of IFN
. Intact in vitro-stimulated T-cells showed a typical intracellular IFN
staining pattern (Fig 3E) similar to the findings by others (
antibodies subjected to FACS analysis showed positive staining with in vitro-stimulated T-cells, whereas unstimulated cells were negative. These experiments reveal that 12 of 13 anti-IFN
antibodies have the potential to recognize IFN
in in vitro-stimulated intact T-cells. In contrast to these intact in vitro-stimulated T-cells, IFN
immunohistochemistry performed on cryosections prepared from these cells gave negative results (Fig 3F). Given the fact that these in vitro PMA/ionomycin-stimulated T-cells contain higher levels of IFN
protein than activated T-cells in vivo (
immunostaining of T-cells in tissue sections of IAAA, RA synovium, and tonsil was negative.
The present experiments show that there is a clear discrepancy between the results obtained with cultured intact T-cells and those with cryosectioned T-cells. Apparently, immunocytochemical staining of intact T-cells is not a reliable model system for testing the immunohistochemical applicability of IFN antibodies on cryostat tissue sections, as has been suggested in the literature (
It is remarkable that in the majority of papers the observed IFN positivity simply was assumed to represent T-cells. Confirmation by double staining was performed only by
co-localization in RA synovium samples. In this study we could not reproduce those results, even in the same type of tissue. Attempting to demonstrate the co-localization of IFN
in T-cells, we performed double staining with both CD2 and CD3. Double staining with CD2 was also performed because CD2 is not downregulated after T-cell activation (
/CD3 co-localization in in vitro-stimulated T-cells (not shown). We have no explanation for the different results in our study compared to that of
The obvious difference between in vitro-stimulated intact T-cells and cryostat tissue sections prepared from these cells is the presence of an outer membrane. Therefore, it appears attractive to hypothesize that intact T-cells retain IFN more effectively during the fixation procedure, either with acetone or PFA, than a sectioned cell. Possibly IFN
protein is lost from the cryosectioned T-cells during the fixation procedure due to extraction of the antigen (
is detectable by ELISA in the buffer covering the unfixed in vitro-stimulated T-cell cryostat sections. However, after fixation of the T-cell cryostat sections with either acetone or PFA, almost no IFN
is detectable in the overlying buffer, whereas the immunohistochemical visualization of IFN
in cryosectioned stimulated T-cells was completely negative also (Fig 3F). (b) Acetone fixation of intact T-cells, which completely dissolves the fatty membrane structures, shows a near-negative IFN
staining result (Fig 3D), whereas the same cells were intensely positive after PFA fixation (Fig 3E).
IFN immunohistochemistry on cryostat tissue sections from tonsil, RA synovium, and IAAA revealed highly variable staining patterns with different antibodies. A variety of cellular constituents were positive, with a high preference for smooth muscle cells. Apart from staining of various cellular constituents, 12 of 13 anti-IFN
antibodies revealed staining of a CD138-positive plasma cell subset (Fig 4) but not of T-cells. Non-T-cell positivity with anti-IFN
antibodies has been reported previously and is considered a result of receptor-bound IFN
(
staining patterns resemble the IFN
receptor staining pattern (Fig 1J). However, there are indirect arguments that certainly not all non-T-cell IFN
positivity can be explained as receptor bound. (a) In the non-atherosclerotic aortas, without any morphological signs of inflammation, the medial smooth muscle cells showed massive IFN
positivity, whereas the IFN
receptor was negative (Fig 1L and Fig 1K). (b) When non-T-cell IFN
staining in non-atherosclerotic aortas was caused indeed by receptor-bound IFN
, this would have induced major histocompatibility complex Class II expression (HLA-DR, DP, DQ). However, this was not observed. This absence of HLA-DR, DP, and DQ positivity in non-atherosclerotic aortas also excludes the possibility that IFN
receptor staining is perhaps missed because IFN
blocks the antibody binding site.
Considering the IFN positivity of plasma cells, some investigators claimed expression of trace amounts of IFN
by B-lymphocytes under in vitro conditions (
staining of the plasma cell subpopulation as observed in all three tissues studied. Therefore, apart from the non-T-cell staining of various cellular constituents, also the plasma cell IFN
-positivity was considered as a staining artifact. This artifactual staining might be caused by the formation of conditional epitopes (
The nonspecific positive staining of the plasma cell subpopulation was shown not to be unique for anti-IFN antibodies. Using, for example, anti-IL-2 or anti-IL-4 antibodies, known to be secreted by T-cells upon activation, we also observed a strong positivity in RA synovium of the same plasma cell population that was found positive with anti-IFN
antibodies, whereas positive T-cells were not observed. Non-T-cell IFN
staining may be the basis for conflicting immunohistochemical staining results when different anti-IFN
antibodies were applied to similar tissue specimens. For example,
positive cells using clone DIK-1, whereas
positivity with clone MD-2 in the same tissue. Moreover, the application of extremely high concentrations of IFN
antibody (MD-2, 45100 µg/ml) (
antibodies themselves may play a role but also the application of different tissue fixatives before the IFN
staining procedure may have an effect on the final staining result. For example, acetone fixation was employed by
staining after acetone fixation of intact T-cells (Fig 3D and Fig 3E), confirming the use of PFA fixation as indicated by
staining of tissue sections after either acetone or PFA fixation, there was no difference regarding localization and intensity. This observation is another indication that the non-T-cell staining in tissue sections after acetone fixation has a nonspecific basis. The present description of nonspecific IFN
immunohistochemical staining shows analogy with the false-positive staining of anti-RAP-5 antibody detecting the ras oncogene product p21 as reported by
-cells in formalin-fixed and paraffin-embedded rat pancreatic tissue.
We consider further testing of the anti-IFN antibody specificity using absorption with IFN
antigen not to be useful. In our opinion and that of others (
clone MD-2 was subjected successfully to such an absorption experiment (
staining (Table 3; Fig 1L).
Future Perspectives for IFN Immunostaining?
At the request of one of the reviewers and after discussions at the latest Congress of Histochemistry and Cytochemistry, we tested the option of prefixation by perfusion or immersion in PFA before freezing. This method has been applied, for example, by (
after freezing and sectioning (Fig 5A and Fig 5B), in contrast to PFA postfixation (Fig 3F). Apparently, PFA postfixation of a cryosection is too slow (
from cryosections. However, PFA prefixation of a closed cell allows a firm fixation of IFN
, thus preventing leakage even from tissue sections. Thus far, however, we have not been able to demonstrate IFN
immunohistochemically in a few PFA-prefixed and 20% sucrose freeze-protected tissue blocks tested. Nevertheless, it could be that PFA prefixation is the key to successful immunohistochemical visualization of IFN
and perhaps of other cytokines. Moreover, we suspect that missing the IFN
staining of activated T-cells in cryosections from PFA prefixed tissue blocks, in contrast to prefixed in vitro-stimulated T-cells, may be a sensitivity problem of current detection systems even when tyramide amplification is used.
Conclusion
Using the present panel of anti-IFN antibodies, specific staining of activated T-cells (Th-1 type) in tissue sections is not observed with generally applied staining procedures, a phenomenon most likely due to either the lack of sensitivity and/or to leakage of IFN
from the sections. On the basis of the present findings, we conclude that IFN
immunostaining of cryostat tissue sections may result in significant staining artifacts. As a consequence, IFN
immunostaining results on cryostat tissue sections should be interpreted with great caution. The performance of double staining is highly recommended, at least for the distinction between "true" IFN
positivity and nonspecific staining.
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Footnotes |
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1 Chris van der Loos and Mischa Houtkamp contributed equally to this work.
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Acknowledgments |
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We wish to express our gratitude to Prof Dr P.P. Tak and Mr T. Smeets (Academic Medical Center, Dept. of Rheumatology) for providing us with synovial tissue from patients with rheumatoid arthritis.
Received for publication July 28, 2000; accepted January 8, 2001.
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