Phycoerythrin (PE) is a major phycobiliprotein of light-harvesting complex found in cyanobacteria and red algae, which functions as a light energy transfer molecule (1). PE shows the distinct property of excitation and emission spectra via covalent binding with phycoerythrobilins (2). Because PE has a high absorbance coefficient and fluorescence yield, it has been considered suitable for biotechnical uses and therefore, the antibody-conjugated form has been developed for biologically specific analysis (3). Three classes of PE, namely R-PE, B-PE, and C-PE, have been reported and each of them exhibits slightly different absorption spectra (4). Until now, R-PE has been frequently used to label antibodies for several immunoassays, including cytofluorometric analysis and immunofluorescence microscopy.
During experimental analysis using target-specific antibodies, “nonspecific binding,” a term used for antibody binding excluding antibody-epitope binding, decreases analytic accuracy, and causes serious misunderstandings. Because basic experimental methods, such as western blotting, flow cytometry, enzyme-linked immunosorbent assay, and immunohistochemistry, have been performed with antibodies, numerous studies have been undertaken to understand and solve the “nonspecific binding” problems. For example, IgM antibody has been demonstrated to bind nonspecifically to galactocerebroside (5). There is also a kinetic study about how nonspecific binding affects specific binding for biosensors (6). However, few cases of nonspecific binding in specific cell types by a fluorescent dye-conjugated form of antibody have been reported.
In the present study, we found that PE-conjugated antibodies, but not allophycocyanin (APC)-conjugated antibodies, selectively and strongly stained murine plasma cells from small intestinal lamina propria cells (LPCs). This nonspecific intracellular staining of PE-antibodies to plasma cells was not due to interactions of antibody and epitope, or antibody and Fc receptor. This unexpected staining was also detected in plasma cells isolated from other immune organs, including bone marrow, spleen, and mesenteric lymph nodes. Furthermore, we demonstrated that this phenomenon occurred in in vitro activated B cells and newly generated plasma cells in vivo using germ-free mice.
Materials and Methods
Mice and Media
A 7- to 9-week old female C57/BL6, C3H/HeN, and BALB/c mice were from OrientBio (Seongnam, Korea), and 6-week old female germ-free Swiss Webster mice were from Taconic (Hudson, NY). The animals were housed in a specific pathogen-free facility. The experiments were performed according to the guidelines of Korea University Institutional Animal Care and Use Committee. All murine cells were cultured in RPMI medium (Thermo Scientific, Rockford, IL) with 10% FBS (WelGENE, Daegu, Korea), 50 μM of 2-ME (Sigma–Aldrich, St Louis, MO), 10 mM of HEPES (WelGENE), 1 mM of sodium pyruvate, 100 U ml−1 of penicillin, and 0.1 mg ml−1 of streptomycin (all from Invitrogen, Carlsbad, CA).
PE-conjugated anti-B220 (RA3-6B2, Cat. No. 553090), FITC-conjugated anti-CD4 (RM4-5, Cat. No. 553046), FITC-conjugated anti-CD8 (53-6.7, Cat. No. 553030), PE-conjugated anti-CD8 (53-6.7, Cat. No. 553032), APC-conjugated anti-CD25 (PC-61, Cat. No.557192), and APC-conjugated anti-CD138 (281-2, Cat. No. 561705) were from BD Biosciences (San Diego, CA). APC-conjugated anti-CD19 (MB19-1, Cat. No. 17-0191), PE-conjugated anti-Foxp3 (NRRF-30, Cat. No. 12-4771), FITC-conjugated anti-IgA (mA-6E1, Cat. No. 11-4204), PE-conjugated anti-IL-17 (eBio17B7, Cat. No. 12-7177), APC-conjugated anti-IL-17 (eBio17B7, Cat. No. 17-7177), FITC-conjugated anti-human CD123 (6H6, Cat. No. 11-1239), PE-conjugated anti-human CD123 (6H6, Cat. No. 12-1239), PE-conjugated control antibody (eBR2a, Cat. No. 12-4321), and anti-CD40 (1C10, Cat. No. 16-0401) were obtained from eBioscience (San Diego, CA). F(ab')2 fragment anti-mouse IgM was from Jackson ImmunoResearch (Cat. No. 115-006-020, West Grove, PA).
Preparation of Single Cell Suspensions
Spleen cells, lymph node cells, mesenteric lymph node cells, and Peyer's patch cells were obtained by homogenization of the tissues, followed by passage through a 40-μm cell strainer. Bone marrow cells and bone marrow-derived dendritic cells (BMDC) were prepared as previously described (7). Lamina propria cells (LPCs) were recovered from the small intestine using previously described protocols (8). In brief, small intestines were removed from mice, followed by feces clearance in cold PBS. Fat tissues and Peyer's patches were resected and the intestines were cut longitudinally. After washing with cold PBS, the intestines were cut into 2–3 cm pieces and transferred into RPMI medium containing 1 mM EDTA. The intestines were incubated for 20 min in the medium with gentle stirring at 37°C in medium containing EDTA, followed by washing with warm PBS. Incubation was done twice. Afterward, the tissues were cut into fine pieces and incubated for 40 min in RPMI medium containing 0.1 mg ml−1 of collagenase D (Roche, Indianapolis, IN) and DNase I (Sigma-Aldrich) at 37°C with gentle stirring. After incubation, the cells were collected and passed through a 40 μm cell strainer, and the unfractionated cells were centrifuged as 400g. Lamina propria lymphocytes were isolated using 40 and 85% Percoll gradient media (Amersham Biosciences, Uppsala, Sweden).
In Vitro B Cell Activation
CD19+ B cells were isolated from spleen, peripheral lymph nodes, or mesenteric lymph nodes using FACSAriaII (BD Biosciences). Briefly, total cells from each organ were stained with APC-conjugated anti CD19 antibody. Positively stained cells were sorted at 45 psi with 85 μm nozzle and 1.2–1.6 flow rate with maximum 1,000 events s−1. The sorting efficacy was determined by FACSCalibur. Isolated B cells were seeded in 96-well plate (1 × 105 cells well−1) and cultured for 3 days in the presence of LPS (20 μg ml−1, Sigma-Aldrich), anti-IgM antibody (40 μg ml−1), or anti-CD40 antibody (20 μg ml−1). IgA class switching was induced by coculture with lamina propria stromal cells (LP-SC) as previously described (9). CD19+ B cells from mesenteric lymph nodes were cocultivated with LP-SCs or BMDCs in the presence of IL-5 (PeproTech, Rocky Hill, NJ, 10 ng ml−1), TGF-β (PeproTech, 1 ng ml−1), and anti-CD40 antibody (eBioscience, 10 μg ml−1).
Small intestines were removed and flushed with cold PBS, followed by flushing with 5 mM DTT. The intestine was then opened longitudinally and washed with PBS. The sample was fixed overnight in 4% paraformaldehyde solution and then incubated for 18 h in 30% sucrose solution. The tissue was embedded in OCT compound, sectioned with a cryotome (Leica CM3050S, Leica, Berlin, Germany) at 6 μm, and attached on slide glasses. For staining, slide glasses were washed two times with PBS, followed by blocking with staining buffer (0.5% Triton X-100 (Sigma–Aldrich) and 5% FBS in PBS) for 1 h at room temperature. Tissue samples were then washed two times with PBS and fluorescence-conjugated monoclonal antibodies (0.5 μg ml−1) were applied overnight at 4°C. After PBS washing, specimens were stained with DAPI (300 nM in PBS) for 3 min. Slides were mounted with Permount mounting medium (Fisher Scientific, Pittsburgh, PA). Immunohistochemical analysis was performed in a fluorescence microscope (Olympus IX71, Olympus, Tokyo, Japan) equipped with fluorescence attachment (IX-FLA, Olympus) and CoolSNAP-Pro (Media Cybernetics, Silver Spring, MD). U-MWU (330–385 nm), U-MWIBA (460–490 nm), and U-MWG (510–550 nm) filter cubes were used for fluorescence detection. Pearson's Correlation Coefficient was obtained by ImageJ software.
Colony Transfer from the Feces of SPF Mice into Germ-Free Mice
Bacteria from feces of SPF C57BL/6 mice were transferred into germ-free Swiss Webster mice using protocols from the Taconic website (www.taconic.com). In brief, germ-free Swiss Webster mice were cohoused with C57BL/6 donors and water bottles of co-housed cages were withdrawn overnight. In the following morning, 100–150 mg of feces was collected from donors and mixed in 50 ml of autoclaved water by shaking vigorously in a water bottle. Germ-free recipients in every cage were allowed to drink the mixed water for 5 min, and the remaining was aliquoted into new bottles. After the addition of 150 ml of water, each cage was supplied with one of the aliquots. This process was performed three times each week at 2–3 day intervals.
Enumeration of Fecal Bacteria from Colony Transferred Mice After Anaerobic Culture
The transferred bacteria after colony transfer were monitored every week by colony counting from the feces of the recipients. Briefly, feces of the donors or the recipients were collected every 7 days and mixed in autoclaved water. Nearly 5 ng of feces diluted in water was spread on Schaedler agar (Difco) plate and cultured at 37°C under anaerobic condition using anaerobe container system (BD, Spark, MD). After overnight culture, the number of colony was determined.
For cell surface staining, single-cell suspensions were washed and stained in 100 μl of 1× FACS buffer for 20 min at room temperature. Fixation and intracellular staining were performed in Cytofix/Cytoperm and Perm/Wash solutions (BD Biosciences), according to the manufacturer's instructions. Antibodies were used at 1:100 dilution for surface and intracellular staining. For R-PE staining, 1 μg of PE (Sigma–Aldrich) was added at the beginning of extracellular or intracellular staining. For machine set-up and compensation, unstained controls, single-stained cells, fluorescence minus one controls, and isotype antibody stained cells were used as described previously (10). Compensation beads (BD Biosciences, Cat. No. 552845) were used for quality control. All of flow cytometric analyses were performed using FACSCalibur (BD Biosciences) with 488 nm argon ion for FITC (520 V) and PE (510 V) and 635 nm Red diode lasers for APC (710 V). The compensation matrix was FITC-PE 2.0%; PE-FITC 16.0%. Data were analyzed using CellQuest Pro software (BD Biosciences). Compensation for FITC-APC and PE-APC was not applicable.
A Student's t test was used to compare experimental groups and control groups. P values < 0.05 were considered to be statistically significant.
PE-Conjugated Antibodies Stain Lamina Propria Plasma Cells from the Murine Small Intestines via Nonspecific Binding
During flow cytometric analysis, we found that a large population of non-CD4 T cells was stained with PE-conjugated anti-mouse Foxp3 antibody in LPCs isolated from the small intestine of C57BL/6 mice (Fig. 1A). The unknown population was also detected by intracellular staining with PE-conjugated isotype control antibody (Fig. 1A), which indicated that the staining was not done via antibody–epitope interaction. As shown (Supporting Information Fig. 1), the nonspecific binding of Foxp3-PE antibody to non-T cells was not due to cell death during enzymatic cell isolation. Surprisingly, PE-stained non-CD4 T cells showed phenotypes that were typical of intestinal plasma cells. For example, they expressed surface IgA and high levels of intracellular IgA (Fig. 1B). In addition, >95% of LPCs stained with PE-conjugated isotype control antibody were IgA+ lamina propria plasma cells (LP-PCs) (Supporting Information Fig. 2). Because PE-conjugated antibodies did not stain plasma cells via surface staining (Supporting Information Fig. 3), we investigated whether this phenomenon was caused by intracellular staining with another fluorescence-conjugated antibody, including APC-conjugated antibody. For this, we used antibodies that are not related to plasma cells such as anti-CD8 and anti-IL-17 antibodies for intracellular staining. Unexpectedly, FITC- or APC-conjugated antibodies failed to stain LP-PCs, while each of the same clonal antibodies conjugated with PE significantly stained LP-PCs (Fig. 1C), indicating that this phenomenon was not due to nonspecifically trapped antibody inside LP-PCs. These results demonstrated that plasma cells were stained selectively among LPCs by PE-conjugated antibodies via nonspecific binding. Therefore, we investigated whether the unconjugated form of R-PE could label LP-PCs selectively. As shown (Supporting Information Fig. 4), R-PE selectively stained LP-PCs via nonspecific binding by intracellular challenge, indicating that the non-specific binding of PE-antibodies to LP-PCs was caused by R-PE. In the extracellular staining, R-PE didn't bind LPCs. Next, we confirmed the plasma cell-selective staining by PE-conjugated isotype control antibody using antibodies as various lineage markers. The results again showed that PE-stained LPCs were surface CD138- and Ly-6c-expressing plasma cells, which were negative for B220 expression (Fig. 1D). We also confirmed again that the nonspecific staining was not due to technical mistakes. The nonspecific binding was not reduced by excessive blocking with FBS, washing with Triton X-100 containing buffer (Supporting Information Fig. 5), or increased washing steps (data not shown). We next examined whether the nonspecific staining was due to high concentration of staining antibody via titration. We induced IFNγ expression in LP T cells by TCR stimulation, and compared nonspecific staining of LP-PCs with specific staining of CD4 T cells using PE-conjugated anti-IFNγ antibody. We observed reduced nonspecific LP-PC staining only at extremely low concentration, where specific staining of CD4 T cells were also dramatically reduced (Supporting Information Fig. 6). Therefore, we concluded that LP-PCs were stained selectively and intracellularly among LPCs by PE-conjugated antibodies, which was not due to antibody–epitope interaction.
LP-PC-Selective Staining by PE-Conjugated Antibodies is a “Pan-Specific” Phenomenon
We next determined whether LP-PC-selective staining by PE-antibodies occurred only in the LP-PCs of C57BL/6 mice. Although there were differences in the relative percentages, almost all IgAhigh LP-PCs from C3H/HeN and Balb/c mice were also selectively stained with PE-conjugated antibody via nonspecific binding (Fig. 2A), suggesting that this phenomenon occurs in the intestinal LP-PCs of various mouse strains. To investigate whether the unexpected staining occurred only in the flow cytometric analysis, we next performed immunohistochemistry using IgA-specific antibody and PE-conjugated antibody. IgA-expressing LP-PCs were visible in the small intestinal villi after fluorescent immunohistochemical staining of the tissue sections, and LP-PCs were also positive for PE staining (Fig. 2B). Furthermore, mouse LP-PCs were positively stained with PE-conjugated mouse anti-human CD123 antibody, but not with the same clonal antibody conjugated with FITC (Fig. 2C), strongly suggesting that staining of LP-PC by PE-conjugated antibody was not restricted by origin or target species of the antibodies. Our results also showed that the LP-PC-selective staining by PE-antibodies was not due to Fc receptor–antibody interaction because we used the same clonal mouse antibodies conjugated with different fluorescence. Taken together, these results strongly indicate that LP-PC-selective intracellular staining by PE-conjugated antibodies was not limited by the mouse strain of LP-PCs, certain technical procedures, or origin and target molecule species of antibodies used.
Plasma Cells in Immune Organs and Activated B Cells are Nonspecifically and Selectively Stained by PE-Conjugated Antibodies
Intestinal LP is one of the richest sites of plasma cells and LP-PCs are polarized to secret IgA. As LP-PCs are stained selectively by PE-conjugated antibodies, we next investigated whether plasma cells from other immune organs were also nonspecifically stained by PE-conjugated antibodies. As a general site for germinal center reaction, spleen cells were isolated and analyzed. Although only few CD138+ plasma cells existed in splenocytes, most CD138high cells were positively stained with PE-conjugated control antibody (Fig. 3A). In addition, bone marrow plasma cells, which migrated from secondary immune organs via chemokine responses (11), were also detected positively by PE-conjugated antibody via nonspecific binding (Fig. 3B). Interestingly, less number of CD138-expressing plasma cells from mesenteric lymph nodes was stained by PE-antibody compared to other organs (Fig. 3C). Because activated B cells are known to migrate from mesenteric lymph nodes to LP and fully differentiate into plasma cells in LP (12), we hypothesized that B cell activation induced the unexpected staining with PE-antibodies. After 3 days of in vitro activation of splenic and lymph node B cells with LPS, CD19+ B cells were stained by PE-conjugated antibody (Fig. 4A). Activated mesenteric lymph node B cells by LPS, anti-IgM, or anti-CD40 antibody also showed the same phenomenon (Fig. 4B). Furthermore, most IgA+ B cells induced by coculture with LP-SC were positively detected by PE-conjugated antibody (Fig. 4C). These results demonstrated that plasma cell-selective intracellular staining by PE-antibody via nonspecific binding was strongly related to B cell activation and stages of plasma cell differentiation.
Newly Generated LP-PCs In Vivo are Strongly Stained by PE-Conjugated Antibodies
To further confirm the plasma cell-selective staining by PE-antibodies, we tested the phenomenon in newly generated plasma cells in vivo. Relatively few IgA+ plasma cells are present in the intestinal LP of germ-free mice, and bacterial exposure induces the generation of LP-PCs (13). Therefore, we performed intestinal colony transfer from SPF mice to germ-free mice and determined whether newly generated LP-PCs were stained by PE-conjugated antibody. After 1 week from colony transfer, efficient transfer was confirmed by colony counting from the anaerobic culture of feces component (Fig. 5A). As expected, the number of IgA+ LP-PCs was increased in the LP of the recipients compared with that of the control mice (Fig. 5B). Weekly analysis of LPCs from colony transferred mice revealed a time-dependent increase of LP-PC generation and significant staining of most newly generated IgA+ LP-PCs were detected by PE-conjugated antibody (Fig. 5C). In addition, PE-antibody staining was also detected in IgA+ or CD138+ cells of Peyer's patches, mesenteric lymph nodes, spleen, and bone marrow from colony transferred mice (Supporting Information Fig. 7A and B). These data suggest that the unexpected staining by PE-conjugated antibodies, which is not due to antibody–epitope interaction, occurs in activated B cells and newly generated LP-PCs by bacterial exposure. Therefore, we concluded that plasma cell-selective staining by PE-antibody via nonspecific binding occurred in most plasma cells of the body.
In this study, we identified the non-specifically stained LPCs with PE-conjugated antibodies in the murine intestinal LP as IgA+CD138+ plasma cells. More than 95% of LPCs stained by PE-conjugated rat isotype control antibody was LP-PCs, indicating that staining with PE-conjugated antibodies was selective to LP-PCs. We also demonstrated that LP-PC-selective staining with PE-antibody was not caused by interactions of antibody and epitope or Fc receptor. The phenomenon was not dependent on the mouse strain of LP-PCs, experimental methods, or origin and target molecule species of antibody, but dependent on PE. In addition, this unexpected phenomenon was also observed in plasma cells from bone marrow, spleen, and mesenteric lymph nodes. The in vitro activated B cells and in vivo generated LP-PCs were also stained selectively by PE-conjugated antibody.
Possible mechanisms of the unexpected staining by PE-conjugated antibody could be varied since non-specific binding of antibody has not been fully understood. We confirmed the most possible mechanisms, such as Fc receptors or simply trapping of antibody inside the cell membrane. However, our results revealed that the phenomenon was not due to Fc receptors or antibody trapping. In addition, the plasma cell-selective staining by PE-antibody was not dependent on the origins of antibody or company that provides antibodies. Furthermore, distinct structural features of plasma cells, such as the large size of endoplasmic reticulum, could not explain nonspecific staining by PE-antibody, not by FITC- or APC-antibody. Unexpectedly, the unconjugated form of PE also exhibited similar results, plasma cell-selective intracellular staining. Therefore, molecular interactions between plasma cell-specific molecules and PE are suspected.
Further studies will be focused on mechanisms of plasma cell-selective intracellular staining by PE-conjugated antibody, including search for molecules interacting with PE in plasma cells. Because we observed the selective staining by PE-antibody only in activated B cells or plasma cells, the phenomenon seems to be related with processes of plasma cell differentiation. LPS induces class switch recombination in B cells and switched type is detectable after 60 h from LPS treatment (14). Because the staining of activated B cells by PE-antibody was detected 3 days after LPS treatment, we can suspect that class switching may be involved in the staining process. However, there are difficulties in molecular studies using activated B cells or plasma cells. It has been found that genome-wide mutations are caused by activation-induced deaminase, a key molecule for class switching, in germinal center B cells during somatic hypermutation, indicating that genome-wide mutations and repairs occur frequently in activated B cells (15). Activated B cells can produce not only antibodies, but also chemokines and cytokines (16). Therefore, the intracellular phase of activated B cells is thought to be more complicated than any other cells. Although CD138+ plasma cells were also stained by PE-conjugated antibody, it is difficult to isolate enough numbers of plasma cells for molecular works or differentiate B cells into abundant plasma cells.
In the present study, we found that PE-conjugated antibodies, including isotype control, selectively stained activated B cells and plasma cells. This phenomenon may cause serious missinterpretation of experimental data in various fields of life sciences because activated B cells and plasma cells exist in various tissues, such as bone marrow, secondary immune organs, gastrointestinal tracts, respiratory tracts, and blood. In addition, in the experiments using fluorescence-conjugated antibodies, as isotype control antibodies have been considered as one of solutions to evaluate nonspecific binding or background intensity (17), researchers are needed to confirm whether this kind of unexpected staining by fluorescence-conjugated antibodies occurs in their target cells.
The authors thank JH Song, MY Jung, and HJ Hong for helpful discussion and technical assistance, and also EP Cohen (University of Illinois) for editing the manuscript.