Development of Sensitive Monoclonal Antibody PMab-2 Against Rat Podoplanin

Abstract

Podoplanin (PDPN) is a platelet aggregation-inducing factor, which is known as an endogenous ligand of C-type lectin-like receptor-2 (CLEC-2). PDPN is also expressed in several normal tissues, such as lung type I alveolar cells and kidney podocytes. Although many monoclonal antibodies (MAbs) against human PDPN (hPDPN) or mouse PDPN (mPDPN) have been established, anti-rat PDPN (rPDPN) MAbs, especially against platelet aggregation-stimulating (PLAG) domain (29-54 amino acids) of rPDPN, have not been developed. Therefore, functional analysis of rPDPN in normal tissues has been limited. Here, we immunized mice with rPDPN peptides (38-51 amino acids) and developed a novel mouse anti-rPDPN MAb, PMab-2 (IgG₁, kappa), which possesses high affinity compared with anti-hPDPN or mPDPN MAbs. The K_D of PMab-2 was determined to be 5.9 × 10⁻¹⁰ M. PMab-2 is useful, not only in flow cytometry and Western blot analysis against endogenous rPDPN, which is expressed in rat dermal fibroblast, but also in immunohistochemistry against normal tissues. PMab-2 showed extraordinarily high sensitivity in immunohistochemistry, indicating that PMab-2 is very advantageous for functional analysis of rPDPN.

Introduction

Podoplanin (PDPN/PA2.26/T1α/Aggrus) is a type I transmembrane O-glycoprotein that possesses platelet aggregating activity.^(1–4) We recently performed crystallographic studies of human PDPN (hPDPN) in complex with C-type lectin-like receptor-2 (CLEC-2).⁽⁵⁾ The interaction with CLEC-2 was mainly observed at Glu47 and Asp48 in the platelet aggregation-stimulating (PLAG) domain of hPDPN (29-54 amino acids) and the α2-6 linked sialic acid at Thr52 of hPDPN. The platelet aggregation via PDPN-CLEC-2 interaction is critical for embryonic blood-lymphatic vascular separation.⁽⁶⁾ In adult tissues, PDPN is very useful for detecting lymphatic endothelial cells.⁽⁷⁾ PDPN also possesses many important functions. Interestingly, both PDPN-CLEC-2 axis and CLEC-2-PDPN axis have been reported. PDPN binds to CLEC-2 and rearranges the actin cytoskeleton in dendritic cells to promote efficient motility along stromal surfaces.⁽⁸⁾ The development of ectopic lymphoid follicles is dependent on Th17-expressing PDPN.⁽⁹⁾ The signal from CLEC-2 to PDPN controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.⁽¹⁰⁾ The physical elasticity of lymph nodes is maintained by PDPN of stromal fibroblastic reticular cells.⁽¹¹⁾ Expression of PDPN has also been reported in many cancers, including oral cancers, malignant brain tumors, esophageal cancers, lung cancers, malignant mesotheliomas, testicular tumors, and osteosarcomas.^(3,12–23) Moreover, PDPN expression in cancer-associated fibroblasts (CAFs) is involved in poor prognosis of cancers.^(24–28) PDPN is also reported in advanced atherosclerotic lesions of human aortas.⁽²⁹⁾ Although anti-PDPN MAbs with high sensitivity and specificity are necessary to investigate the physiological function of PDPN in normal tissues, almost all anti-PDPN MAbs have been developed against hPDPN^(17,30–39) and mouse PDPN (mPDPN),^(40–42) not against rat PDPN (rPDPN).⁽¹⁾ Therefore, we immunized mice with PLAG domain of rPDPN and developed high-sensitive MAbs against rPDPN.

Materials and Methods

Cell lines

Chinese hamster ovary (CHO)-K1 and P3U1 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Rat dermal fibroblast was obtained from Cell Applications (San Diego, CA). CHO-K1 was transfected with rPDPN, hPDPN, or mPDPN plasmids (CHO/rPDPN, CHO/hPDPN, CHO/mPDPN) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA).⁽³⁾ CHO-K1, CHO/rPDPN, CHO/hPDPN, CHO/mPDPN, and P3U1 were cultured in RPMI 1640 medium including RPMI 2 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B (Nacalai Tesque), and rat dermal fibroblast was cultured in Fibroblast Growth Medium 2, including FBS, basic fibroblast growth factor (1 ng/mL), and insulin (5 μg/mL; PromoCell, Heidelberg, Germany) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Animals

Female BALB/c mice (four-weeks-old) were purchased from CLEA Japan (Tokyo, Japan). Animals were housed under pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein.

Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.) injection of 100 μg of synthetic peptides (rpp3851: GDDMV NPGLEDRIEC), conjugated with KLH together with Imject Alum (Thermo Fisher Scientific). After several additional immunizations, a booster injection was given i.p. 2 days before spleen cells were harvested. The spleen cells were fused with P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN). The hybridomas were grown in RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific). The culture supernatants were screened using enzyme-linked immunosorbent assay (ELISA) for binding to rpp3851 peptides.

ELISA

Synthetic peptides were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientific) at 1 μg/mL for 30 min. After blocking with SuperBlock T20 (PBS) Blocking Buffer, the plates were incubated with culture supernatant followed by 1:1000 diluted peroxidase-conjugated anti-mouse IgG (Dako, Glostrup, Denmark). The enzymatic reaction was conducted with a 1-Step Ultra TMB-ELISA (Thermo Fisher Scientific). The optical density was measured at 655 nm using an iMark microplate reader (Bio-Rad Laboratories, Philadelphia, PA). These reactions were performed with a volume of 50 μL at 37°C.

Western blot analyses

Cell lysates (10 μg) were boiled in SDS sample buffer (Nacalai Tesque). The proteins were electrophoresed on 5–20% polyacrylamide gels (Wako Pure Chemical Industries, Osaka, Japan) and were transferred onto a PVDF membrane (EMD Millipore, Billerica, MA). After blocking with 4% skim milk (Nacalai Tesque), the membrane was incubated with primary antibodies,⁴³ and then with peroxidase-conjugated secondary antibodies (Dako; 1:1000 diluted), and developed with Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific) or ImmunoStar LD Chemiluminescence Reagent (Wako Pure Chemical Industries) using a Sayaca-Imager (DRC Co., Tokyo, Japan).

Flow cytometry

Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA (Nacalai Tesque). After washing with PBS, the cells were treated with primary MAbs (1 μg/mL) for 30 min at 4°C, followed by treatment with Oregon green-conjugated anti-mouse IgG or anti-rat IgG (Thermo Fisher Scientific). Fluorescence data were collected using a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

Determination of binding affinity by flow cytometry

Binding affinity was determined as described previously.⁽⁴⁴⁾ Briefly, cells (2 × 10⁵ cells) were resuspended at 100 μL of serially diluted antibodies (0.02–100 μg/mL), followed by secondary antibodies (Thermo Fisher Scientific). Fluorescence data were collected using a Cell Analyzer EC800. The dissociation constants (K_D) were obtained by fitting the binding isotherms using the built-in one-site binding models in GraphPad PRISM 6 (GraphPad Software, La Jolla, CA).

Immunohistochemical analyses

Four-μm-thick histologic sections were deparaffinized in xylene and rehydrated. Sections were incubated with PMab-2 for 1 h at room temperature followed by treatment with Envision+ kit (Dako). Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB, Dako) for 5 min, and then the sections were counterstained with hematoxylin (Wako Pure Chemical Industries).

Results

Production of monoclonal antibody against rPDPN

We first immunized mice with a synthetic peptide of rat PDPN; then ELISA screening was performed. Among 23 ELISA-positive wells, 11 wells reacted with CHO/rPDPN in flow cytometry. Furthermore, three wells recognized a specific band in Western blot analysis. After limiting dilution, clone PMab-2 (mouse IgG₁, kappa) was established. PMab-2 detected about 40 kDa band of rPDPN-transfected CHO cells (CHO/rPDPN), not with hPDPN-transfected CHO cells (CHO/hPDPN), mPDPN-transfected CHO cells (CHO/mPDPN), and CHO cells in Western blot analysis (Fig. 1A). In contrast, NZ-1 and PMab-1 detected about 40 kDa bands of CHO/hPDPN and CHO/mPDPN, respectively, indicating that three MAbs are species-specific. PMab-2 reacted with CHO/rPDPN, not with CHO/hPDPN, CHO/mPDPN, and CHO cells in flow cytometry (Fig. 1B). NZ-1 and PMab-1 specifically reacted with CHO/hPDPN and CHO/mPDPN, respectively. Taken together, PMab-2 is a specific MAb against rPDPN, and is very useful in Western blot and flow cytometric analyses. We next investigated whether PMab-2 can detect endogenous rPDPN. Because PDPN has been reported to be expressed in mouse or human fibroblast cells, we checked the PMab-2 reactivity against rat dermal fibroblast. As shown in Figure 2A, PMab-2 detected not only exogenous rPDPN of CHO/rPDPN but also an endogenous one, which is expressed in rat dermal fibroblast. PMab-2 also reacted with rat fibroblast in flow cytometry (Fig. 2B). These results indicate that PMab-2 is useful for detecting endogenous rPDPN. We further performed a kinetic analysis of the interaction of PMab-2 with CHO/rPDPN using flow cytometry. As shown in Figure 3, K_D of PMab-2 was determined to be 5.9 × 10⁻¹⁰ M using CHO/rPDPN. In contrast, K_D of NZ-1 was determined to be 2.9 × 10⁻⁹ M using CHO/hPDPN and K_D of PMab-1 was determined to be 3.2 × 10⁻⁹ M using CHO/mPDPN, indicating that the binding affinity of PMab-2 is much higher than that of NZ-1 and PMab-1.

FIG. 1.

Specific binding of anti-PDPN MAbs. (A) Western blot analysis by PMab-2, NZ-1, and PMab-1. AC-15 (anti-β-actin) and RcMAb-1 (anti-IDH1) were used as internal control. Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 0.1 μg/mL of MAbs (PMab-2, NZ-1, PMab-1) or 1 μg/mL of MAbs (AC-15 and RcMAb-1), and then with peroxidase-conjugated secondary antibodies; the membrane was detected using a Sayaca-Imager. (B) Flow cytometry. Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, cells were treated with PMab-2, NZ-1, and PMab-1, followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800.

FIG. 2.

Endogenous rPDPN is detected by PMab-2. (A) Western blot analysis. AC-15 (anti-β-actin) and RcMAb-1 (anti-IDH1) were used as internal control. Total cell lysate were electrophoresed on 5–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, the membrane was incubated with 1 μg/mL of MAbs, and then with peroxidase-conjugated secondary antibodies; the membrane was detected using a Sayaca-Imager. (B) Flow cytometry. Cells were harvested by brief exposure to 0.25% Trypsin/1 mM EDTA. After washing with PBS, the cells were treated with PMab-2, followed by treatment with secondary antibodies. Fluorescence data were collected using a Cell Analyzer EC800.

FIG. 3.

Determination of binding affinity using flow cytometry. CHO = rPDPN, CHO = hPDPN, and CHO = mPDPN (2 × 10s cells) were re-suspended at 100 μL of serially diluted PMab-2, NZ-1, and PMab-1 (0.02–100 μg/mL, respectively. Fluorescence data were collected using a Cell Analyzer EC800. K_D was obtained by fitting binding isotherms using built-in one-site binding models in Prism software.

Immunohistochemical analysis using PMab-2

We next investigated whether PMab-2 can detect rPDPN of normal tissues in immunohistochemistry. PMab-2 reacted with type I alveolar cells of lung, lymphatic endothelial cells of colon, podocytes, and Bowman's capsule epithelial cells of kidney (Fig. 4). Because PMab-2 showed high affinity against rPDPN, we next checked the dose dependency of PMab-2 in immunohistochemistry using three different tissues. As shown in Figure 5, PMab-2 detected rPDPN of lung type I alveolar cells at 0.001 μg/mL, rPDPN of colon lymphatic endothelial cells at 0.005 μg/mL, and rPDPN of kidney podocytes at 0.01 μg/mL, indicating that PMab-2 is very sensitive against rPDPN.

FIG. 4.

Immunohistochemical analysis by PMab-2. Sections of rat lung, colon, and kidney were incubated with 0.5 μg/mL of PMab-2 or control PBS, followed by Envision+ kit, and color was developed using DAB and counterstained with hematoxylin. Arrows, lymphatic vessels; arrowheads, glomerulus. Scale bar, 100 μm.

FIG. 5.

Immunohistochemical analysis by PMab-2 in a dose-dependent manner. Sections of rat lung, colon, and kidney were incubated with 0.001-0.1 μg/mL of PMab-2, followed by Envision+ kit, and color was developed using DAB and counterstained with hematoxylin. Arrows, lymphatic vessels; arrowheads, glomerulus. Scale bar, 100 μm.

Discussion

In this study, we immunized mice with a synthetic peptide (rpp3851), which is included in platelet aggregation-stimulating (PLAG) domain of rPDPN. We previously produced an anti-hPDPN MAb (NZ-1)⁽¹⁷⁾ and an anti-mPDPN MAb (PMab-1)⁽³⁹⁾ by immunizing rats with PLAG domain of hPDPN and mPDPN, respectively. Because these MAbs are not only very sensitive and specific but also very useful for almost all experiments including flow cytometry, Western blot, immunoprecipitation, immunocytochemistry, and immunohistochemistry, MAbs against PLAG domain of rPDPN was thought to be advantageous. As expected, established PMab-2 is a specific MAb against rPDPN, and is very useful in Western blot and flow cytometric analyses. Furthermore, PMab-2 detected not only exogenous rPDPN but also an endogenous one of dermal fibroblast in both Western blot and flow cytometry, which leads to the advantage in immunohistochemistry. By kinetic analysis of the interaction of PMab-2 using flow cytometry, K_D of PMab-2 was determined to be 5.9 × 10⁻¹⁰ M, indicating that the binding affinity of PMab-2 is extraordinarily high. In rat normal tissues, rPDPN was reported to be highly expressed in lymphatic endothelial cells, lung type I alveolar cells, or kidney podocytes.⁽¹⁾ Expectedly, PMab-2 reacted with type I alveolar cells of lung, lymphatic endothelial cells of colon, and podocytes of kidney in immunohistochemistry. A previous study showed that PMab-1 detects mPDPN in immunohistochemistry; however, antigen retrieval procedure was necessary.⁽³⁹⁾ Furthermore, NZ-1 detects hPDPN in immunohistochemistry without antigen retrieval; however, it does not react with type I alveolar cells of lung and podocytes of kidney.⁽²⁰⁾ In contrast, PMab-2 does not need antigen retrieval and detects normal rPDPN, which is expressed in various tissues. Surprisingly, PMab-2 detected rPDPN of lung type I alveolar cells at a low concentration of 1 ng/mL, indicating that PMab-2 is extraordinarily sensitive against rPDPN.

Taken together, PMab-2 is very useful for all experiments such as flow cytometry, Western blot, and immunohistochemistry. Furthermore, the epitope of PMab-2 is PLAG domain of rPDPN, indicating that PMab-2 is advantageous for investigating the platelet aggregation activity of rPDPN or rPDPN-CLEC-2 interaction.

Footnotes

Acknowledgments

We thank Takuro Nakamura, Noriko Saidoh, Hazuki Kanno, and Kanae Yoshida for their excellent technical assistance. This work was supported in part by the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from Japan Agency for Medical Research and Development, AMED (Y.K.); by the Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED (Y.K.); by the Regional Innovation Strategy Support Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Y.K.); and by JSPS KAKENHI Grant Number 26440019 (M.K.K.) and Grant Number 25462242 (Y.K.).

Author Disclosure Statement

The authors have no financial interests to disclose.

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