Immunohistochemistry in Human Placental TissuePitfalls of Antigen Detection
Department of Obstetrics and Gynecology, University of Wuerzburg, Germany
Correspondence to: Ulrike Kämmerer, PhD, Department of Obstetrics and Gynecology, Josef-Schneider-Strasse 4, D-97080 Würzburg, Germany. E-mail: frak057{at}mail.uni-wuerzburg.de
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Summary |
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Key Words: human placenta IgG2b isotype nonspecific binding HLA-G human IgG Fc receptor endothelial cells immunohistochemistry
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Introduction |
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We investigated frozen samples of human first trimester placenta with a mouse monoclonal antibody against CD83 (clone HB15a), which is specific for a subset of the dendritic cell (DC) lineage. As anticipated, decidual DC showed a clear and reproducible staining pattern. However, to our surprise, vessels in the placental villi were also stained. Because CD83 is a highly specific surface marker for DCs at a certain stage of their maturation, its expression on endothelial cells (EC) did not seem plausible (Zhou and Tedder 1995; Banchereau and Steinman 1998
).
We obtained similar unexpected staining results with the antihuman leukocyte antigen (HLA)-G antibody clone BFL-1 (Bensussan et al. 1995). HLA-G is an atypical major histocompatibility complex class 1 antigen with limited polymorphism. It has been described to be specifically expressed by extravillous cytotrophoblast (Kovats et al. 1990
; McMaster et al. 1995
; Hiby et al. 1999
), although staining of EC of villous vessels for this antigen has also been described (Blaschitz et al. 1997
). Because of the similarity of the two unexpected results, we hypothesized that the staining pattern observed with the two antibody clones on frozen sections of placental tissue was unrelated to CD83 and HLA-G antigen expression. Both the antibody clones were of the IgG2b isotype. Because staining of placental EC was not observed with numerous antibodies of the IgG1 and IgG2a isotypes, the aberrant staining pattern observed seemed to be restricted to IgG2b antibodies. To confirm this hypothesis, we investigated the "specificity" of this binding of antibodies to placental villous EC with several different antibodies. In addition, various established blocking procedures known to prevent nonspecific binding of antibodies in immunohistochemistry and the application of purified human immunoglobulin were tested in an attempt to overcome the problem of nonspecific binding in placental sections.
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Materials and Methods |
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Immunohistochemistry
The antibodies applied in the study are listed in Table 1. The specificity of the primary antibodies was confirmed by staining of frozen sections of human lymph nodes and skin. Serial frozen sections were cut at 4 µm and placed onto APES (3-amino-propyltriethoxy-silane; Roth, Karlsruhe, Germany)-coated slides. They were air-dried overnight to destroy endogenous peroxidases, then fixed in acetone and rehydrated in TBS (25 mM TRIS/HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl) for 10 min each.
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Additional Blocking Procedures and Solutions
Several different blocking procedures were applied to prevent the postulated nonspecific binding: before the application of the primary antibodies, the sections were incubated for 30 min at room temperature in one of the following blocking solutions: (a) 5% bovine serum albumin (Sigma), (b) 1% heat-inactivated (30 min 56C) fetal calf serum (PAA Laboratories; Cölbe, Germany), (c) 2.5% human AB serum (PAA Laboratories), and (d) 20% normal goat serum (Dako). All four blocking reagents were diluted in Dulbecco's PBS (without Ca2+ and Mg2+; PAA Laboratories).
Preincubation with purified IgG was performed with a mixture of human IgG immunoglobulins (stock solution: 30 mg/ml; Beriglobin; Behringwerke, Marburg, Germany) diluted in PBS, with which the sections were incubated for 30 min at room temperature. A series of dilutions (1:10, 1:50, 1:100, 1:500) was tested. After the blocking procedure, excess liquid was removed and the standard immunohistochemical procedure was performed immediately as described previously.
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Results |
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We hypothesized that one or more Fc receptors could be responsible for the nonspecific binding and applied purified and concentrated human IgG immunoglobins diluted in PBS (at 1:10, 1:50, 1:100, 1:500) before the sections were incubated with the primary antibody. After this preincubation step, the nonspecific binding of all antibodies (both isotype control antibodies, anti-CD83 clone HB15a, and anti-HLA-G clone BFL-1) to villous vessels at dilutions of 1:10 and 1:50 totally disappeared (Figures 1B, 1D, 1F, and 1H). There was still nonspecific background staining at a dilution of 1:50, and no blocking effect was seen at a dilution of 1:100 (not shown). To optimize the cost:effect ratio, a dilution of 1:50 (corresponding to 0.6 mg/ml human immunoglobulin) was selected as standard for our blocking protocol. This blocking step did not interfere with the specific detection of CD83-positive dendritic cells with the HB15a clone (Figures 3E and 3F) or HLA-Gpositive extravillous trophoblast cells (Figures 3G and 3H) in the decidua basalis.
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No staining for FcRIII was observed in fetal endothelial cells or placental Hofbauer cells (Figures 2A and 2B). Figures 2C and 2D demonstrate that placental vessel EC are clearly positive for Fc
RII (CDw32), even if incubation with the primary antibody is preceded by incubation with human IgG immunoglobulin (Figure 2D). Decidual macrophages showed moderate staining for Fc
RII. The antibody against CD64, stained only Hofbauer cells in the placental villous stroma (Figures 2E and 2F).
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The antibodies directed against FcRI (CD64) (Figures 2E and 2F), Fc
RIII (CD16) (data not shown), and CD9 are known to detect antigens that are expressed on monocytes. Macrophage staining with these antibodies in decidual areas attached to the placenta was not altered by preincubation with purified IgG (data not shown). Likewise, specific staining of endothelial cells for CD9 was not affected and for CD34 staining intensity decreased only slightly by the IgG-blocking procedure (Figures 3B and 3D). In summary, the specific staining of endothelial cells, macrophages, mature dendritic cells (Figure 3F), and extravillous cytotrophoblast (Figure 3H) in the decidua was not affected by preincubation with IgG, but nonspecific binding of IgG2b isotype antibodies disappeared.
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Discussion |
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To test this hypothesis in experiments, we applied mouse monoclonal IgG2b isotype control antibodies and found that this indeed led to endothelial cell staining in chorionic villi. That all these staining reactions vanished after purified human IgG was applied as a preincubation step suggested that they were nonspecific.
We used antibodies against CD34 (IgG1) and CD9 (IgG2b), which belong to different IgG subtypes, to investigate whether this preincubation would interfere with specific endothelial staining. In these experiments, specific detection of EC was found to be largely unaffected by preincubation. Further experiments revealed that preincubation with IgG does not interfere with other specific IHC reactions.
We can only speculate as to which structure in human placenta is responsible for the nonspecific binding of mouse IgG2b isotype antibodies observed. The obvious candidates are Fc receptors, which are known to mediate maternofetal IgG transport. Because of the known homology of mouse and human placental transport mechanisms, it is possible that human Fc receptors might have an affinity for mouse antibody (Lyden et al. 2001; Simister 2003
). Both species have a hemochorial placenta and exhibit parallels in terms of IgG transport from mother to fetus. It is therefore conceivable that mouse IgG2b could bind to human placental Fc receptors that normally bind human IgG in the course of maternofetal immunoglobulin transport. Such crossreactivity could result in stable Fc binding of these antibodies, which would explain the immunohistochemical results we obtained.
In keeping with the results of Kameda et al. (1991) and Lyden et al. (2001)
, we found the human placenta to be negative for Fc
RIII (CD16). Our findings were also consistent with those of Wainwright and Holmes (1993)
and Bright et al. (1994)
in that Fc
RII (CDw32) was clearly detectable on vascular endothelium in the villi. After the preincubation step, the intensity of staining with anti-CDw32 (IgG2b) decreased. The more intense staining of this antibody without preincubation is most probably the result of additional binding of antibodies via this Fc receptor in the human placenta. Our results with Fc
RI (CD64) confirmed the findings of Lyden et al. (2001)
with regard to vascular endothelium, which was negative for this receptor.
Conflicting research findings concerning the presence of Fc receptors in the placenta have been published (Simister and Story 1997; Saji et al. 1999
; Gafencu et al. 2003
). Fc
RII detection nicely exemplifies how challenging the immunohistochemical investigation of the placenta can be. Five different investigators used clone IV3, a mouse monoclonal antibody, of the IgG2b subclass to detect Fc
RII expression by fetal EC in term placenta. Three studies found expression of Fc
RII (Kristoffersen et al. 1990
; Micklem et al. 1990
; Sedmak et al. 1991
), whereas two did not (Stuart et al. 1989
; Kameda et al. 1991
), which underlies the necessity for improvements in immunohistochemistry techniques in human placenta.
Hofbauer cells are fetal macrophages that are known to express multiple Fc receptors (Saji et al. 1999; Lyden et al. 2001
). Although one of the IgG2b isoform control antibodies stained these cells, nonspecific IgG preincubation prevented this staining, suggesting that receptors responsible for nonspecific binding could be expressed on these macrophages.
Whether one of the fetal Fc receptors known to be expressed on placental vessels or other, still to be identified receptors mediate crossreactivity with mouse IgG2b has not yet been fully elucidated. One potential candidate is FcRIIb2, which was found by Lyden et al. (2001)
to be expressed on fetal EC.
In conclusion, our study has shown that the interpretation of immunohistochemical studies performed on frozen sections of human placental tissue is a demanding task. The difficulties arise from one of the organ's functions, maternofetal IgG transport. There is an obvious tendency of this tissue to absorb antibodies in an antigen-independent manner. This nonspecific binding of antibodies is not seen in formalin-fixed placental tissue. However, numerous primary antibodies fail to stain fixed (and paraffin-embedded) tissue, so the problem of nonspecific binding associated with the use of frozen tissue needs to be solved. As demonstrated in the present study, this nonspecific staining can be eradicated by the application of purified human IgG before the primary antibody. The blocking procedure with immunoglobulin could also be appropriate for frozen tissues other than placenta, such as the tissue of the immune system and certain epithelia, in which Fc receptors are expressed (Stuart et al. 1989). However, even when this reliable tool is employed to improve the specificity of immunohistochemical staining, suitable controls on serial sections should still always be included. These controls should involve staining with antibodies of the same IgG isotype but irrelevant specificity, which are applied at the same concentrations as the original primary antibodies.
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Footnotes |
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Literature Cited |
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