MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 6, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0038

Short Communication

PMab-241 Specifically Detects Bear Podoplaninof Lymphatic Endothelial Cells in the Lung of Brown Bear

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Junko Takei,1 Shinji Yamada,1 Satoru Konnai,2,3 Tsuyoshi Ishinazaka,4 Michito Shimozuru,5Mika K. Kaneko,1 and Yukinari Kato1,6

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar
cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for
immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses todetect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish
lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel antibPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in
immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic
endothelial cells specifically in the bear lung tissues.
Keywords: bear podoplanin, PDPN, type I alveolar cells, lymphatic endothelial cells, PMab-241

Introduction

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein,(1) and is also known as T1alpha (2)/gp44(3)/Aggrus,(4) which induces platelet aggregation (PLAG)
through C-type lectin-like receptor-2 (CLEC-2).(5,6) The sialic acid of the PLAG-stimulating domain or the PLAG-likedomain of PDPN is known to be important for PDPN-induced
PLAG.(7,8)
PDPN is expressed in several normal cells, including
pulmonary type I alveolar cells and lymphatic endothelial
cells of every organ.(1) PDPN is used to distinguish lymphatic
endothelial cells from vascular endothelial cells in pathophysiological studies.(9) However, PDPN is expressed in bothtype I alveolar cells and lymphatic endothelial cells in thelung; therefore, it might be difficult to distinguish lymphatic
endothelial cells from type I alveolar cells in the lung tissues.
Materials and Methods
Cell lines

CHO-K1 was obtained from the American Type CultureCollection (ATCC; Manassas, VA). CHO/bear PDPN (bPDPN)

was previously established.(10) CHO-K1 and CHO/bPDPN
were cultured in a Roswell Park Memorial Institute (RPMI)
1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% of heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mLof penicillin, 100 mg/mL of streptomycin, and 25 mg/mL ofamphotericin B (Nacalai Tesque, Inc.). The cells were grownin an incubator at 37C with humidity and 5% CO2 and 95%
air atmosphere.

Flow cytometry

The cells were harvested after a brief exposure to 0.25%
trypsin and 1 mM ethylenediaminetetraacetic acid (NacalaiTesque, Inc.). The cells were washed with 0.1% bovine serum
albumin in phosphate-buffered saline (PBS) and treated withprimary monoclonal antibodies (mAbs) for 30 minutes at4C. Thereafter, the cells were treated with Alexa Fluor 488conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc., Danvers, MA). Then, fluorescence data werecollected using the SA3800 Cell Analyzer (Sony Corp., Tokyo,Japan).

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Sapporo,Hokkaido, Japan.
3
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.
4
Shiretoko Nature Foundation, Shari, Hokkaido, Japan.
5
Laboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Sciences, Faculty of Veterinary Medicine,
Hokkaido University, Sapporo, Hokkaido, Japan.
6
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.
2282

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FIG. 1. Detection of bPDPN by flow cytometry using PMab-241. CHO/bPDPN and CHO-K1 were treated with PMab-241(red line) at a concentration of 1 mg/mL or 0.1% bovine serum albumin in phosphate-buffered saline (gray) for 30 minutes,followed by incubation with secondary antibodies. bPDPN, bear podoplanin.

FIG. 2. Immunohistochemical analyses for the bear lung. The bear lung sections were incubated with 1 mg/mL of PMab241 (A–C), PMab-247 (D–F) or with blocking buffer (G–I), followed by that with the Envision+ Kit. ( J– L) Hematoxylin
and eosin staining. Scale bar = 100 mm. Red arrows indicate lymphatic endothelial cells.
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Immunohistochemical analyses

Four brown bear (Ursus arctos) tissues were collected after
autopsy at Shiretoko Nature Foundation, fixed in 10%
neutral-buffered formalin, and routinely processed to makeparaffin-embedded tissue sections. Histological sections (4-mmthick) were directly autoclaved in citrate buffer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes.
Then, sections were blocked using the SuperBlock T20 (PBS)
Blocking Buffer (Thermo Fisher Scientific, Inc.), incubatedwith PMab-241 or PMab-247 (1 mg/mL) for 1 hour at room
temperature, and treated with the Envision+ Kit for mouse(Agilent Technologies, Inc., Santa Clara, CA) for 30 minutes.
Color was developed using 3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes,
and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

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Results and Discussion

Although we have already established an anti-bPDPN
mAb (PMab-247), which is sensitive and specific for immunohistochemical analyses,(10) it is difficult to distinguish
lymphatic endothelial cells from type I alveolar cells in thebear lung using PMab-247.(11) In this study, we selected anovel anti-bPDPN mAb (PMab-241) of IgG2b subclass frompreviously established anti-bPDPN mAbs.(10) PMab-241 reacted with CHO/bPDPN, not parental CHO-K1 cells in flow
cytometry (Fig. 1).
We performed immunohistochemical analyses against thebear lungs using two anti-bPDPN mAbs, PMab-241 andPMab-247. PMab-241 stained lymphatic endothelial cells of
the bear lung (Fig. 2A, C), but not type I alveolar cells(Fig. 2A, B) although PMab-247 stained both lymphatic endothelial cells (Fig. 2D, F) and type I alveolar cells (Fig. 2D,E). We stained the lungs from the other three bears using
PMab-241 and PMab-247, and obtained the similar staining
results (Supplementary Figs. S1–S3). These results indicatethat PMab-241 could be a specific marker of lymphatic endothelial cells in the bear lung.
Author Disclosure Statement

No competing financial interests exist.
Funding Information

This research was supported in part by AMED under grantnos. JP19am0401013, JP19am0101078, and JP19ae0101028(Y.K.), and by JSPS KAKENHI grant nos. 17K07299 (M.K.K.)
and 19K07705 (Y.K.).
Supplementary Material

Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1 Seiryo-machi, Aoba-ku
Sendai 980-8575
MiyagiJapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: August 21, 2019
Accepted: October 15, 2019