Journal of Histochemistry and Cytochemistry, Vol. 49, 699-710, June 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Immunohistochemical Detection of Interferon-{gamma}: Fake or Fact?

Chris M. van der Loos1,a, Mischa A. Houtkamp1,a, Onno J. de Boera, Peter Teelinga, Allard C. van der Wala, and Anton E. Beckera
a Academic Medical Center, Department of Cardiovascular Pathology, Amsterdam, The Netherlands

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


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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{gamma} immunohistochemistry on tissue sections with a large panel of anti-IFN{gamma} antibodies. Thirteen different commercially available anti-IFN{gamma} 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 IFN{gamma}-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{gamma} antibodies. However, immunohistochemistry of sectioned stimulated T-cells was negative with all. Unstimulated cells were consistently negative. IFN{gamma} 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{gamma}-immunohistochemistry appears unreliable because of lack of specificity to stain T-cells in situ. In fact, depending on the type of anti-IFN{gamma} antibody used, a variety of different cell constituents were nonspecifically stained. Consequently, data based on IFN{gamma}-immunohistochemistry must be interpreted with great caution. (J Histochem Cytochem 49:699–709, 2001)

Key Words: IFN{gamma} protein, immunohistochemistry, immunocytochemistry, T-cells, plasma cells


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

INTERFERON-GAMMA (IFN{gamma}) is a 34–50-kD cytokine exclusively produced by T-cells and natural killer cells (Farrar and Schreiber 1993 ), which plays a key regulatory role in specific immunity. For this reason, IFN{gamma} has been widely investigated in immunological research. In addition to in vitro studies, many investigators also attempted to demonstrate IFN{gamma} protein immunohistochemically in healthy and diseased tissues. For example, studies have been undertaken to localize IFN{gamma} in rheumatoid arthritis (Ulfgren et al. 1995 ; Smeets et al. 1998a , Smeets et al. 1998b ), atherosclerosis (Hansson et al. 1989 ; Frostegard et al. 1999 ), and inflammatory bowel disease (Camoglio et al. 1998 ). Cytokine immunohistochemistry is frequently used in combination with molecular biological techniques such as in situ hybridization (ISH) and polymerase chain reaction (RT-PCR) to gain insight into the biological role of cytokines in diseased tissues. However, our own immunohistochemical experiences with several anti-IFN{gamma} 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{gamma} immunohistochemical staining using a panel of commercially available poly- and monoclonal antibodies.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Human Tissue Samples
Tonsils with hyperplastic reactive changes (n=3, age 3–6 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 62–77 years) were obtained at surgery. These tissue samples were used as positive controls for IFN{gamma} staining, based on the expression of IFN{gamma} mRNA as detected with RT-PCR (Ramshaw et al. 1994 ; Harabuchi et al. 1996 ; Kotake et al. 1997 ). Moreover, IFN{gamma} protein was detected with immunohistochemistry in tonsil and RA synovium (Hoefakker et al. 1993 ; Andersson et al. 1994 ; Dolhain et al. 1996 ; Smeets et al. 1998a , Smeets et al. 1998b ) and ELISA in IAAA (Szekanecz et al. 1994 ). Non-atherosclerotic vessel segments (n=4, age 10–14 years) were used as controls. All tissue samples were snap-frozen in liquid nitrogen and stored at -80C. Six-µm cryostat serial sections were cut, and stored at -80C.

Cell Specimens
Cultured T-cell lines from human aortic atherosclerotic plaques were prepared as previously described (De Boer et al. 1999 ). Cells were cultured (5 x 106 /ml) in Iscoves modification of Dulbecco's medium (IMDM; Gibco BRL Life Technologies, Paisley, Scotland) supplemented with 10% pooled human serum in the presence or absence of 10 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma; St Louis, MO) and 100 ng ionomycin (Calbiochem; La Jolla, CA) in 25-cm2 culture flasks. Monensin (Sigma) was added (2.88 µM/ml in dimethylsulfoxide) to prevent active cytokine excretion, thus resulting in intracellular accumulation of IFN{gamma}. 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{gamma} poly- and monoclonal antibodies, as listed in Table 1. As indicated in Table 1, seven anti-IFN{gamma} antibodies were advertised and/or regularly applied for immunocytochemistry/-histochemistry. Other primary antibodies used in this study are listed in Table 2.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Anti-human IFN{gamma} antibodies used in this study


 
View this table:
[in this window]
[in a new window]
 
Table 2. Other primary antibodies used in this study

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), streptavidin–biotin 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 (Sander et al. 1991 ; Dolhain et al. 1993 ) to all incubation steps. Endogenous peroxidase activity was blocked with 0.1% sodium azide + 0.3% peroxide in 50 mM Tris-HCl-buffered saline, pH 7.8 (TBS) (20 min, RT) (Li et al. 1987 ). Endogenous biotin was blocked with subsequent incubations of 0.1% avidin and 0.01% D-biotin from the DAKO endogenous biotin blocking kit (twice for 15 min at RT). Primary antibodies and antibody–enzyme conjugates were diluted in TBS + 1% bovine serum albumin (BSA). Single immunostaining consisted of overnight incubation at 4C with the selected primary anti-IFN{gamma} 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 (van der Loos 1999 ) was applied for comparison of IFN{gamma} immunostaining with different cellular markers. The staining procedure consisted of the following subsequent incubation steps: NGS (1:10, 15 min), IFN{gamma} 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{gamma} antibody, the first part of the double staining consisted of NSS (1:10, 15 min), IFN{gamma} 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 (van der Loos and Gobel 2000 ), and PE-conjugated CD56 (60 min) (Table 2). For the FITC- and PE-conjugated antibodies, further steps were as follows: rabbit anti-FITC (1:1000, 15 min), or rabbit anti-PE (1:200, 15 min) and PowerVision-GAR/AP (undiluted, 30 min). The biotinylated antibody was followed by streptavidin/AP (1:100, 30 min). The enzymatic activities of AP and HRP were visualized in blue (Fast Blue BB) and red (AEC), respectively (van der Loos 1999 ).

Two triple immunoenzyme stainings using ß-galactosidase (turquoise), AP (red), and HRP (brown) as marker enzymes, and blue nuclear counterstain (van der Loos 1999 ), were performed to monitor the major cell types present in the tissue specimens. The first consisted of anti-{alpha}-actin (mouse IgG2a) and anti-CD68 (mouse IgG1), combined with FITC-conjugated anti-CD3, in a multistep staining procedure. Anti-{alpha}-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-{alpha}-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{gamma} 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 (Sander et al. 1991 ). Final concentrations of the primary antibodies diluted in PBS with 0.1% saponin and 1% BSA (60 min, 4C) were similar as mentioned for immunohistochemistry/cyto-chemistry in Table 1. Depending on the primary antibody, PE-conjugated GAM, GAR, or SAG (all 1:200) was used as second-step reagent (30 min, 4C). Fluorescence of 104 cells was analyzed on a FACS Calibur (Becton Dickinson, Richmond, CA).

Leakage of IFN{gamma} 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{gamma} ELISA. A Pelikine IFN{gamma} kit was used, following the instructions as supplied by the manufacturer (CLB; Amsterdam, The Netherlands).


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

IFN{gamma} 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{gamma} 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).



View larger version (176K):
[in this window]
[in a new window]
 
Figure 1. Acetone-fixed cryostat serial sections from an inflammatory abdominal aorta aneurysm segment, showing a detail of the adventitia. A muscular vessel is surrounded by inflammatory cells and fibrotic tissue. (A) Overview of major cell types. Immunoenzyme triple staining was performed with anti-{alpha}-actin marking smooth muscle cells in turquoise (ß-galactosidase), CD68 marking macrophages in brown (HRP), and anti-CD3 marking T-cells in red (AP). Nuclear counterstain in blue with hematoxylin. Anti-IFN{gamma} clone/source: (B) 45-15, (IQProducts); (C) 25718.11 (RD Systems); (D) MMHG-1 (Accurate; Westbury, NY); (E) MD-2; (F) 25723.11 (Becton Dickinson; San Jose, CA); (G) H21 (Genzyme); (H) B-B1 (Serotec); (I) A section incubated with mouse IgG1 control antibody. (J) Staining pattern of anti-IFN{gamma} receptor antibody, clone MMHGR-1. Media of a non-atherosclerotic normal aorta specimen. (K) Immunostaining with anti-IFN{gamma}, clone MD-2. Note the intense staining of medial smooth muscle cells. (L) Immunostaining with anti-IFN{gamma} receptor is completely negative. Bar = 100 µm.



View larger version (138K):
[in this window]
[in a new window]
 
Figure 2. Acetone-fixed cryostat serial sections from a synovium of a rheumatoid arthritis patient. (A) Section incubated with mouse IgG1 control antibody. Anti-IFN{gamma} clone/source. (B) B-B1, (Serotec). (C) MMHG-1 (Accurate). (D) 35B10G6 (Biosource); nuclear counterstain in blue with hematoxylin. Bar = 100 µm.

Figure 3. PMA/ionomycin-stimulated (D–F) and unstimulated (A–C) cultured T-cells immunostained with anti-IFN{gamma}, clone MMGH-1 (Accurate). (A,D) IFN{gamma} immunocytochemistry on acetone-fixed intact T-cells. (B,E) IFN{gamma} immunocytochemistry on PFA-fixed and saponin-treated intact T-cells. (C,F) IFN{gamma} immunohistochemistry on PFA-fixed and saponin-treated cryostat sections from T-cells. Note the weak IFN{gamma} immuno-staining of stimulated cultured T-cells after acetone-fixation in D compared with PFA/saponin immunocytochemistry in E, and completely negative immunostaining after cryostat sectioning of stimulated cultured T-cells in F. Bar = 20 µm.

Figure 4. Detail of a synovium with rheumatoid arthritis in four adjacent acetone-fixed cryostat sections, showing a focal area with inflammatory cells. (A) IFN{gamma} immuno-single staining using antibody MMHG-1 (Accurate), nuclear counterstaining in blue with hematoxylin. Immunohistochemical double stainings marking IFN{gamma}, clone MMHG-1 in red (HRP) and either CD3 marking T-cells (B), CD68 marking macrophages (C), or anti-syndecan-1, CD138 marking plasma cells (D) in blue (AP). (D) Red and blue reaction products merged to purple indicate co-localization, whereas in B and C no co-localization is observed. (A) Nuclear counterstaining with hematoxylin. (B–D) Weak nuclear counterstaining with methyl green. Bar = 50 µm.

Figure 5. PMA/ionomycin in vitro-stimulated (A) and unstimulated (B) PFA prefixed, frozen, and cryosectioned cultured T-cells, immuno-stained with anti-IFN{gamma}, clone MMGH-1 (Accurate). Cells are counterstained in blue with hematoxylin. Note the characteristic IFN{gamma} staining as a perinuclear Golgi spot in the stimulated T-cells (A), whereas unstimulated T-cells in B are completely negative. Bar = 20 µm.


 
View this table:
[in this window]
[in a new window]
 
Table 3. Anti-human IFN{gamma} antibodies applied in FACS, ICC, and IHCa

An interesting observation was that IFN{gamma} 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{gamma} 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{gamma} protein at 14.4–49.3 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{gamma} values with unfixed sections were too low.

IFN{gamma} Staining Characteristics with Tissue Sections
Comparison of the IFN{gamma} single staining with triple immunostainings marking the major cellular components in tonsil, RA synovium, and IAAA indicated positive IFN{gamma} 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{gamma} 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{gamma} with T-cells was not observed. Remarkably, a strong IFN{gamma} 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{gamma} 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 2B–2D). Negative control experiments did not show any staining (Fig 1I and Fig 2A). Typical results of anti-IFN{gamma} immunohistochemistry of all three tissues are summarized in Table 3 and illustrated in Fig 1A–1H and Fig 2B–2D.

Comparison of acetone and PFA as fixatives before IFN{gamma} 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{gamma} to its concomitant receptor, tissue localization of IFN{gamma} immunostaining was compared with IFN{gamma} receptor staining in serial sections. In RA synovium, IAAA, and tonsil, the anti-IFN{gamma} 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{gamma} staining (Fig 1B–1H). Moreover, medial smooth muscle cells in non-atherosclerotic aortas were IFN{gamma} receptor (Fig 1L) -negative, which contrasted with the strong IFN{gamma} positivity (Fig 1K).

Cellular Specificity of Anti-IFN{gamma} Antibodies
To reveal the characteristic cell type showing positive staining with almost all anti-IFN{gamma} 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{gamma}-positive cell population (Table 3). However, a subpopulation of approximately 5% of all CD138 positive plasma cells co-localized with IFN{gamma}-positive cells, observed as a purple mixed-color in the cytoplasm (Fig 4A–4D).


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

In the present study we have compared the immunohistochemical applicability of 13 different anti-IFN{gamma} antibodies on cryostat tissue sections. IFN{gamma} 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{gamma}-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 1A–1H), despite the fact that the antibodies were capable of detecting IFN{gamma} in cultured and in vitro-stimulated intact T-cells (Table 3). For example, anti-IFN{gamma} clone MD-2 was first described for ELISA application by Van der Meide et al. 1985 and only much later was applied for immunohistochemical purposes (Hoefakker et al. 1993 ). The latter application is subsequently cited by other investigators and has led to the use of this antibody on tissue sections (Dolhain et al. 1996 ; Thepen et al. 1996 ; Van Hoffen et al. 1996 ; Smeets et al. 1998b ).

We also verified the immunoreactivity of the anti-IFN{gamma} antibodies on intact PMA/ionomycin-stimulated T-cells, known to contain substantial amounts of IFN{gamma}. Intact in vitro-stimulated T-cells showed a typical intracellular IFN{gamma} staining pattern (Fig 3E) similar to the findings by others (Sander et al. 1991 ; Dolhain et al. 1993 ; Andersson and Andersson 1994 ; Krouwels et al. 1997 ). In addition, 12 of 13 anti-IFN{gamma} 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{gamma} antibodies have the potential to recognize IFN{gamma} in in vitro-stimulated intact T-cells. In contrast to these intact in vitro-stimulated T-cells, IFN{gamma} 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{gamma} protein than activated T-cells in vivo (Del Prete et al. 1991 ), it may be not surprising that IFN{gamma} 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{gamma} antibodies on cryostat tissue sections, as has been suggested in the literature (Andersson et al. 1994 ; Dolhain et al. 1996 ).

It is remarkable that in the majority of papers the observed IFN{gamma} positivity simply was assumed to represent T-cells. Confirmation by double staining was performed only by Dolhain et al. 1996 , who showed CD3/IFN{gamma} 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{gamma} 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 (Moingeon et al. 1991 ), in contrast to CD3 (Van Lier et al. 1987 ). Furthermore, we have been able to detect IFN{gamma}/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 Dolhain et al. 1996 .

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{gamma} more effectively during the fixation procedure, either with acetone or PFA, than a sectioned cell. Possibly IFN{gamma} protein is lost from the cryosectioned T-cells during the fixation procedure due to extraction of the antigen (Larsson 1993 ) or due to poor fixation. Arguments in favor of this leakage-hypothesis are the observations that (a) IFN{gamma} 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{gamma} is detectable in the overlying buffer, whereas the immunohistochemical visualization of IFN{gamma} 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{gamma} staining result (Fig 3D), whereas the same cells were intensely positive after PFA fixation (Fig 3E).

IFN{gamma} 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{gamma} antibodies revealed staining of a CD138-positive plasma cell subset (Fig 4) but not of T-cells. Non-T-cell positivity with anti-IFN{gamma} antibodies has been reported previously and is considered a result of receptor-bound IFN{gamma} (Dolhain et al. 1996 ). Indeed, some of the IFN{gamma} staining patterns resemble the IFN{gamma} receptor staining pattern (Fig 1J). However, there are indirect arguments that certainly not all non-T-cell IFN{gamma} 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{gamma} positivity, whereas the IFN{gamma} receptor was negative (Fig 1L and Fig 1K). (b) When non-T-cell IFN{gamma} staining in non-atherosclerotic aortas was caused indeed by receptor-bound IFN{gamma}, 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{gamma} receptor staining is perhaps missed because IFN{gamma} blocks the antibody binding site.

Considering the IFN{gamma} positivity of plasma cells, some investigators claimed expression of trace amounts of IFN{gamma} by B-lymphocytes under in vitro conditions (Pang et al. 1992 ; Jelinek and Braaten 1995 ). In our opinion, these findings cannot be matched with the abundant and strong IFN{gamma} 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{gamma}-positivity was considered as a staining artifact. This artifactual staining might be caused by the formation of conditional epitopes (Willingham 1999 ) or redistribution of small molecular weight antigens (Larsson 1993 ), both of which may occur during the fixation procedure.

The nonspecific positive staining of the plasma cell subpopulation was shown not to be unique for anti-IFN{gamma} 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{gamma} antibodies, whereas positive T-cells were not observed. Non-T-cell IFN{gamma} staining may be the basis for conflicting immunohistochemical staining results when different anti-IFN{gamma} antibodies were applied to similar tissue specimens. For example, Andersson et al. 1994 reported in chronic recurrent tonsillitis a few IFN{gamma} positive cells using clone DIK-1, whereas Hoefakker et al. 1993 reported massive IFN{gamma} positivity with clone MD-2 in the same tissue. Moreover, the application of extremely high concentrations of IFN{gamma} antibody (MD-2, 45–100 µg/ml) (Thepen et al. 1996 ; Van Hoffen et al. 1996 ) for immunostaining of cryostat sections may introduce staining artifacts. Not only may the IFN{gamma} antibodies themselves may play a role but also the application of different tissue fixatives before the IFN{gamma} staining procedure may have an effect on the final staining result. For example, acetone fixation was employed by Hoefakker et al. 1993 , Dolhain et al. 1996 , Smeets et al. 1998b , and Thepen et al. 1996 , whereas PFA fixation and saponin treatment were used by Ulfgren et al. 1995 , Smeets et al. 1998a , and Andersson et al. 1994 . In our hands, a comparison of these two popular fixatives showed a dramatic loss of IFN{gamma} staining after acetone fixation of intact T-cells (Fig 3D and Fig 3E), confirming the use of PFA fixation as indicated by Andersson et al. 1994 . However, in considering the non-T-cell IFN{gamma} 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{gamma} immunohistochemical staining shows analogy with the false-positive staining of anti-RAP-5 antibody detecting the ras oncogene product p21 as reported by Samowitz et al. 1988 and Gutheil et al. 1989 . Although these investigators employed immunoprecipitation and Western blotting as contra-evidence, they finally proved that the initial RAP-5 immunohistochemical positivity could be recognized as completely nonspecific. Furthermore, Kaino et al. 2000 described the crossreactivity of anti-rat CD45RA antibody with glucagon-producing islet {alpha}-cells in formalin-fixed and paraffin-embedded rat pancreatic tissue.

We consider further testing of the anti-IFN{gamma} antibody specificity using absorption with IFN{gamma} antigen not to be useful. In our opinion and that of others (Burry 2000 ), an absorption control does not prove the specificity of an antibody for its antigen in a fixed tissue section. For example, anti-IFN{gamma} clone MD-2 was subjected successfully to such an absorption experiment (Hoefakker et al. 1993 ), but our present data clearly show this antibody as not to be specific for tissue IFN{gamma} staining (Table 3; Fig 1L).

Future Perspectives for IFN{gamma} 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 Van Noorden and Polak 1985 , Van Noorden 1986 , and Larsson 1988 , and is especially recommended for detection of small peptides and hormones. However, among the vast majority of reports applying acetone or PFA postfixation of cryosections, we could find only one report using prefixation for the detection of IFN{gamma} (Kakazu et al. 1999 ). Our pilot experiment showed that PFA pre-fixation of in vitro-stimulated T-cells indeed retains and preserves IFN{gamma} 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 (Fox et al. 1985 ) and cannot prevent leaking of IFN{gamma} from cryosections. However, PFA prefixation of a closed cell allows a firm fixation of IFN{gamma}, thus preventing leakage even from tissue sections. Thus far, however, we have not been able to demonstrate IFN{gamma} 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{gamma} and perhaps of other cytokines. Moreover, we suspect that missing the IFN{gamma} 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{gamma} 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{gamma} from the sections. On the basis of the present findings, we conclude that IFN{gamma} immunostaining of cryostat tissue sections may result in significant staining artifacts. As a consequence, IFN{gamma} 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{gamma} positivity and nonspecific staining.


  Footnotes

1 Chris van der Loos and Mischa Houtkamp contributed equally to this work.


  Acknowledgments

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Andersson J, Abrams J, Bjork L, Funa K, Litton M, Agren K, Andersson U (1994) Concomitant in vivo production of 19 different cytokines in human tonsils. Immunology 83:16-24[Medline]

Andersson U, Andersson J (1994) Immunolabeling of cytokine-producing cells in tissues and suspension. In Fradelizi D, ed. Cytokine Producing Cells. Paris, INSERM, 32

Burry RW (2000) Specific controls for immuncytochemical methods. J Histochem Cytochem 48:163-165[Abstract/Free Full Text]

Camoglio L, Te Velde AA, Tigges AJ, Das PK, Van Deventer SJH (1998) Altered expression of interferon-gamma and interleukin-4 in inflammatory bowel disease. Inflam Bowel Dis 4:285-290[Medline]

De Boer OJ, Van der Wal AC, Verhagen CE, Becker AE (1999) Cytokine secretion profiles of cloned T-cells from human aortic atherosclerotic plaques. J Pathol 188:174-179[Medline]

Del Prete GF, De Carli M, Mastromauro C, Biagiotti R, Macchia D, Falagiani P, Ricci M, Romagnani S (1991) Purified protein derivative of Myocbacterium tuberculosis and excretory-secretory antigen(s) of Toxocara canis expand in vitro human T cells with stable and opposite (type 1 T-helper or type 2 T-helper) profile of cytokine production. J Clin Invest 88:346-350[Medline]

Dolhain RJEM, Andersson U, Ter Haar NT, Brinkman BMN, Verweij CL, Daha MR, Breedveld FC, Miltenburg AMM (1993) Detection of intracellular interferon-gamma by light microscopy using an immunoperoxidase technique: correlation with the corresponding mRNA and protein product. J Leukocyte Biol 54:545-551[Abstract]

Dolhain RJEM, Ter Haar NT, Hoefakker S, Tak PP, De Ley M, Claassen E, Breedveld FC, Miltenburg AMM (1996) Increased expression of IFN-gamma together with IFN-gamma receptor in the rheumatoid synovial membrane compared with synovium of patients with osteoarthritis. Br J Rheumatol 35:24-32[Medline]

Farrar MA, Schreiber RD (1993) The molecular cell biology of interferon-gamma and its receptor. Annu Rev Immunol 11:571-611[Medline]

Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845-853[Medline]

Frostegard J, Ulfgren AK, Nyberg P, Hedin U, Swedenborg J, Andersson U, Hansson GK (1999) Cytokine expression in advanced human atherosclerotic plaques: dominance of pro-inflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis 145:33-43[Medline]

Gutheil JC, Mane S, Kapil V, Needleman SW (1989) Immunoprecipitation of cell lysates with RAP-5 does not specifically detect ras oncogene product p21. Hum Pathol 20:1176-1180[Medline]

Hansson GK, Holm J, Jonasson L (1989) Detection of activated T-lymphocytes in the human atherosclerotic plaque. Am J Pathol 135:169-175[Abstract]

Harabuchi Y, Wakashima J, Murakata H, Yoshioka I, Yokoyama Y, Kataura A (1996) Cytokine expression and production by tonsillar lymphocytes. Acta Otolaryngol Suppl 523:75-77[Medline]

Haynes MK, Jackson LG, Tuan RS, Shepley KJ, Smith JB (1993) Cytokine production in first trimester chorionic villi: detection of mRNAs and protein products in situ. Cell Immunol 151:300-308[Medline]

Hoefakker S, Van ‘t Erve EH, Deen C, Van den Eertwegh AJ, Boersma WJ, Notten WR, Claassen E (1993) Immunohistochemical detection of co-localizing cytokine and antibody producing cells in the extrafollicular area of human palatine tonsils. Clin Exp Immunol 93:223-228[Medline]

Jelinek DF, Braaten JK (1995) Role of IL-12 in human B lymphocyte proliferation and differentiation. J Immunol 154:1606-1613[Abstract/Free Full Text]

Kaino Y, Ito T, Hirai H, Kida K (2000) Anti-rat CD45RA monoclonal antibodies cross-react with glucagon. Acta Histochem 102:151-157[Medline]

Kakazu T, Hara J, Matsumoto T, Nakamura S, Oshitani N, Arakawa T, Kitano A, Nakatani K, Kinjo F, Kuroki T (1999) Type 1 T-helper cell predominance in granulomas of Crohn's disease. Am J Gastroenterol 94:2149-2155[Medline]

Kotake S, Schumacher HR, Jr, Yarboro CH, Arayssi TK, Pando JA, Kanik KS, Gourley MF, Klippel JH, Wilder RL (1997) In vivo gene expression of type 1 and type 2 cytokines in synovial tissues from patients in early stages of rheumatoid, reactive, and undifferentiated arthritis. Proc Assoc Am Phys 109:286-301[Medline]

Krouwels FH, Nocker RET, Snoek M, Lutter R, van der Zee JS, Weller FR, Jansen HM, Out TA (1997) Immunocytochemical and flow cytofluorimetric detection of intracellular IL-4, IL-5 and IFNgamma: application in blood- and airway-derived cells. J Immunol Methods 203:89-101[Medline]

Larsson L-I (1988) Immunocytochemistry: Theory and Practice. Boca Raton FL, CRC Press

Larsson L-I (1993) Tissue preparation methods for light microscopic immunohistochemistry. Appl Immunohistochem 1:2-16

Li C-Y, Ziesmer SC, Lazcana–Villareal O (1987) Use of azide and hydrogen peroxide as inhibitor for endogenous peroxidase in the immunoperoxidase method. J Histochem Cytochem 35:1457-1460[Abstract]

Moingeon PE, Lucich JL, Stebbins CC, Recny MA, Wallner BP, Koyaso S, Reinherz EL (1991) Complementary roles for CD2 and LFA-1 adhesion pathway during T cell activation. Eur J Immunol 21:605-610[Medline]

Pang Y, Norihisa Y, Benjamin D, Kantor RRS, Young HA (1992) Interferon-gamma gene expression in human B-cell lines: induction by interleukin-2, protein kinase C activators, and possible effect of hypomethylation on gene regulation. Blood 80:724-732[Abstract]

Ramshaw AL, Roskell DE, Parums DV (1994) Cytokine gene expression in aortic adventitial inflammation associated with advanced atherosclerosis (chronic periaortitis). J Clin Pathol 47:721-727[Abstract]

Samowitz WS, Paull G, Hamilton SR (1988) Reported binding of monoclonal antibody RAP-5 to formalin-fixed tissue sections is not indicative of ras p21 expression. Hum Pathol 19:127-132[Medline]

Sander S, Andersson J, Andersson U (1991) Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure. Immunol Rev 119:65-93[Medline]

Smeets TJ, Dolhain RJEM, Breedveld FC, Tak PP (1998a) Analysis of the cellular infiltrates and expression of cytokines in synovial tissue from patients with rheumatoid arthritis and reactive arthritis. J Pathol 186:75-81[Medline]

Smeets TJ, Dolhain RJEM, Miltenburg AM, De Kuiper R, Breedveld FC, Tak PP (1998b) Poor expression of T-cell-derived cytokines and activation and proliferation markers in early rheumatoid synovial tissue. Clin Immunol Immunopathol 88:84-90[Medline]

Szekanecz Z, Shah MR, Pearce WH, Koch AE (1994) Human atherosclerotic abdominal aortic aneurysms produce interleukin (IL)-6 and interferon-gamma but not IL-2 and IL-4: the possible role for IL-6 and interferon-gamma in vascular inflammation. Agents Actions 42:159-162[Medline]

Thepen T, Langeveld–Wildschut WG, Bihari IC, Wichen DF, Van Reijsen FC, Mudde GC, Bruijnzeel–Koomen CAFM (1996) Biphasic response against aeroallergen in atopic dermatitis showing a switch from an initial Th2 response to a Th1 response in situ: an immunocytochemical study. J Allergy Clin Immunol 97:828-837[Medline]

Ulfgren AK, Lindblad S, Klareskog L, Andersson J, Andersson U (1995) Detection of cytokine producing cells in the synovial membrane from patients with rheumatoid arthritis. Ann Rheum Dis 54:654-661[Abstract]

van der Loos CM (1999) Immunoenzyme Multiple Staining Methods. Oxford, BIOS Scientific Publishers

van der Loos CM, Göbel H (2000) The animal research kit (ARK) can be used in a multistep double staining method for human tissue specimens. J Histochem Cytochem 48:1431-1437[Abstract/Free Full Text]

Van der Meide PH, Dubbeld M, Schellekens H (1985) Monoclonal antibodies to human interferon and their use in a sensitive solid-phase ELISA. J Immunol Methods 79:293-305[Medline]

Van Hoffen E, Van Wichen D, Stuij I, De Jonge N, Klopping C, Lahpor J, Van den Tweel J, Gmelig–Meyling F, De Weger R (1996) In situ expression of cytokines in human heart allografts. Am J Pathol 149:1991-2001[Abstract]

Van Lier RAW, Boot JH, Verhoeven AJ, De Groot ER, Brouwer M, Aarden LA (1987) Functional studies with anti-CD3 heavy chain isotype switch-variant monoclonal antibodies. Accessory cell-independent induction of interleukin 2 responsiveness in T cells by epsilon-anti-CD3. J Immunol 139:2873-2879[Abstract/Free Full Text]

Van Noorden S (1986) Tissue preparation and immunostaining techniques for light microscopy. In Polak JM, Van Noorden S, eds. Immunocytochemistry. Modern Methods and Applications. 2nd ed Bristol, Wright, 26-53

Van Noorden S, Polak JM (1985) Immunocytochemistry of regulatory peptides. In Bullock GR, Petrusz P, eds. Techniques in Immunocytochemistry. Vol 3. London, Academic Press, 115-154

Willingham MC (1999) Conditional epitopes: is your antibody always specific? J Histochem Cytochem 47:1233-1235[Free Full Text]