MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 1, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0047

Immunohistochemical Analysis of Inflammatory
Rheumatoid Synovial Tissues Using Anti-Human
Podoplanin Monoclonal Antibody Panel

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Tomoto Suzuki,1 Yuya Takakubo,1 Hiroharu Oki,1 Xing Liu,1 Ryusuke Honma,1 Yasushi Naganuma,1Stuart B. Goodman,2 Mika K. Kaneko,3 Yukinari Kato,3,4 and Michiaki Takagi1

Podoplanin (PDPN) is a transmembrane sialoglycoprotein, which is expressed in several normal tissues andmalignant tumors. Although PDPN expression in rheumatoid arthritis (RA) has been reported, the role of PDPN
in RA and other arthritic conditions has not been fully elucidated. In this study, we examined PDPN expression
in inflammatory synovial tissues using an anti-human PDPN (hPDPN) monoclonal antibody (mAb) panel toselect the most useful one for evaluation of synovitis. Synovial tissue samples were obtained from 11 RA
patients and 9 osteoarthritis (OA) patients undergoing joint surgery. PDPN-positive cells were immunostainedby a panel of PDPN mAbs (NZ-1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17),
followed by cell grading of inflammation and cell counting of PDPN-positivity by a quantitative analyzer.
Immunohistochemistry showed that PDPN was markedly expressed in both macrophage-like type A andfibroblast-like type B lining cells of the hyperplastic synovial lining cell layer, and macrophages and fibroblasts
in the stroma of RA. Among anti-PDPN mAbs, LpMab-12 showed the highest score. In inflammatory OA
synovium, PDPN expression was also detectable. Although LpMab-12 also showed the highest score in OA, thedifference was not statistically significant. The inflammatory synovitis score of RA was significantly higher
than that of OA. PDPN was expressed in inflammatory lining cells and sublining stroma of RA and OA
synovium. In the seven anti-hPDPN antibodies examined, LpMab-12 was the most stainable antibody for PDPN
in RA synovitis. Thus, LpMab-12 for PDPN has a possible and promising specific biomarker for evaluating
synovitis in RA and inflammatory OA.
Keywords: podoplanin, rheumatoid arthritis, synovitis, inflammation

Recently, marked expression of PDPN in fibroblast-likesynoviocytes of the active inflammatory synovium of rheumatoid arthritis (RA) was reported. It has been suggested that
PDPN can increase the migratory potential of activatedfibroblast-like synoviocytes.(10) RA is a chronic inflammatory joint disease, often leading to cartilage and bone damage,resulting in musculoskeletal disability.(11) Many anti-RA
drugs, such as conventional disease-modifying antirheumatic
drugs (cDMARDs), corticosteroids, and nonsteroidal antiinflammatory drugs, have been used for their treatment. In
contrast, clinical applications of antibody drugs have been
spread globally and have contributed to the long-term remission of RA.(12–17) However, resistance to present antibody
drugs has been found in 20%–40% of patients with RA.(18,19)
Accordingly, the development of novel antibody drugs with

IntroductionP

odoplanin (PDPN) is a small mucin-type transmembrane sialoglycoprotein, which is expressed in several
normal tissues, including the endothelium of lymphatic
vessels, type-I alveolar cells, and glomerular podocytes.(1–4)
PDPN is also expressed by malignant tumors such as
squamous cell carcinomas, glioblastomas, and osteosarcomas.(5–7) In malignant tumors, PDPN is associated withtumor cell migration and cancer metastasis.(8) Anti-human
PDPN (hPDPN) monoclonal antibody (mAb), NZ-1, also
inhibited pulmonary metastasis in an experimental metastasis model.(9) Thus, it has been anticipated that anti-hPDPN
antibody has the potential application for the treatment ofvarious cancers.
1

Department of Orthopaedic Surgery, Faculty of Medicine, Yamagata University, Yamagata, Japan.
Department of Orthopaedic Surgery, Stanford University, Stanford, California.
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
4
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2
3

12

PODOPLANIN IN RHEUMATOID ARTHRITIS

13

newly mechanistic properties for the treatment of RA patients, who were resistant to preexisting antibodies, has beenin strong demand.
As PDPN is markedly expressed in inflammatory synovium, it has been anticipated as a therapeutic target to control
synovitis.(10,18) However, little is known about the potential
role of PDPN in inflammation and synovitis in RA patients.
Recently, six new anti-hPDPN mAbs were established.(20–25)
Comparative studies for the evaluation of inflammatory synovitis using those newly developed anti-hPDPN mAbs havenot been performed. Therefore, the aim of this study is toelucidate PDPN expression in inflammatory synovial tissues
from surgically treated joint diseases, and to select the mostuseful PDPN mAb for evaluating synovitis or the degree andproperty of synovial inflammation.

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Materials and Methods
Patients and specimens

Eleven RA patients who underwent surgery (total knee arthroplasty: 4, total hip arthroplasty: 3, wrist synovectomy: 2,
total shoulder arthroplasty: 1, and metacarpophalangeal joint
arthroplasty: 1) for upper and lower limb joint disease wereenrolled in this study. Mean age at surgery was 56.7 years(range: 32–79 years). There were four males and seven females. Mean history of RA at surgery was 10.3 years (range: 5–
19 years). Eight patients used cDMARDs. Five patients usedmethotrexate (mean: 9.6 mg/week, range: 6–16 mg/week).
Eight patients used steroids (mean: 5.4 mg/day, range: 4–9 mg/
day). Eight patients used biologics (Etanercept: 6, Abatacept: 1,
Tocilizumab: 1). Accordingly, most of the RA patients were
drug-resistant. Mean C-reactive protein at surgery was 1.3 mg/
dL (range: 0.1–4.3 mg/dL). All the RA patients fulfilled the1987 American College of Rheumatology(26) or the 2010 ACR/
European League Against Rheumatism (EULAR) Criteria for
RA.(27) Synovial tissue samples were obtained at joint surgeries. Nine osteoarthritis (OA) synovial samples were collected
for comparison. Mean age of the patients at surgery (total knee
arthroplasty: 3, total hip arthroplasty: 6) was 70.1 years (57–82). There were three males and six females (Table 1). This
study was approved by the Yamagata University Faculty of
Medicine Ethics Committee (approval number: 369, December
13, 2016) and written informed consent was obtained from all
the patients.

Table 1. Characteristics of Rheumatoid
Arthritis Patients and Osteoarthritis Patients

Age, mean year (range)
Gender M/F
Disease duration, mean
years (range)
cDMARDs, %Steroids, %Biologics, %CRP at surgery (mg/dL),mean (range)

RA (n = 11)

OA (n = 9)56.7 (32–79)
4/7
10.3 (5–19)

70.1 (57–82)
3/6
—

72.70
72.70
72.70
1.3 (0.1–4.3)———

cDMARDs, conventional disease-modifying antirheumatic drugs;
CRP, C-reactive protein; OA, osteoarthritis; RA, rheumatoid
arthritis.

Assessment of synovitis

The degree of synovitis was evaluated under a light microscope (DMD108; Leica Microsystems, Wetzlar, Germany)by hematoxylin and eosin staining and by Krenn histopathological grading system (0–9 points).(28)
Immunohistochemistry

Synovial samples were fixed in 4% paraformaldehyde.
Four micrometers-thick serial sections were deparaffinized inxylene and rehydrated. After blocking of endogenous peroxidase activity with 0.3% H2O2 in phosphate-buffered saline for 10 minutes, they were autoclaved at +121C in citratebuffer (pH 6.0) for 20 minutes. The sections were incubated
in 5% bovine serum albumin for 20 minutes to block nonspecific binding and then incubated overnight at +4C with
anti-human CD68 antibody, which can recognize heavily
glycosylated 110-kDa membrane protein, and was used as a
macrophage marker (5 mg/mL, Agilent Technologies Inc.,
Santa Clara, CA), and an anti-human CD248 antibody, which
can recognize highly sialylated cell surface type I transmembrane glycoprotein, was used as a stromal fibroblast marker(29)(0.5 mg/mL; abcam, Cambridge, MA), anti-hPDPN mAbs (NZ1: 5 mg/mL, LpMab-3: 5 mg/mL, LpMab-7: 5 mg/mL, LpMab10: 5 mg/mL, LpMab-12: 1 mg/mL, LpMab-13: 5 mg/mL,
LpMab-17: 1 mg/mL). After treatment with biotinylated antirat IgG antibody (Vector Laboratories, CA) or anti-mouse
IgG antibody (Nichirei Biosciences, Tokyo, Japan) for
1 hour, the immunolabeling was visualized by the biotin–
streptavidin–horseradish peroxidase complex (Nichirei Biosciences) and 3,3¢-diaminobenzidine tetrahydrochloride(DOJINDO, Kumamoto, Japan). The sections were counterstained with hematoxylin and analyzed under a light microscope (DMD108).(20)
Immunofluorescence colocalization studies

The sections were incubated with the primary antibodies,monoclonal mouse anti-human CD68 antibody (5 mg/mL;
Agilent Technologies, Inc.), monoclonal rabbit anti-humanCD248 antibody (0.5 mg/mL; abcam), and with a rat antihPDPN mAb (NZ-1), followed by incubation with secondary
antibodies, Alexa Fluor 488-conjugated goat anti-mouse
IgG antibodies (Thermo Fisher Scientific, Inc., Waltham,MA), Alexa Fluor 568-conjugated goat anti-rat IgG antibodies (Thermo Fisher Scientific, Inc.), Alexa Fluor 488conjugated donkey anti-rabbit IgG antibodies (Thermo
Fisher Scientific, Inc.), or Alexa Fluor 546-conjugateddonkey anti-rabbit IgG antibodies (Thermo Fisher Scientific,Inc.) according to the manufacturer’s instructions. Nuclei
were counterstained with DAPI (Thermo Fisher Scientific,Inc.). The images were observed under a fluorescence digital
microscope (BZ-X 710 digital microscope; Keyence, Osaka,
Japan).
Quantitative histomorphometric analyses

PDPN immunopositive cells that typically inflamed
synovial lining layers in both RA and OA were scanned
using a BZ-X 710 digital microscope. The area of immunopositive cells per reference area was calculated digitally using BZ H3C Hybrid Cell Count Software (Keyence,
Osaka, Japan).(30)

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Statistical analysis

Mann–Whitney U test and Kruskal–Wallis test were performed using EZR (Saitama Medical Center, Jichi Medical
University, Saitama, Japan), which was a graphical user interface for R (The R Foundation for Statistical Computing,
Vienna, Austria). More precisely, it was a modified version of
R commander designed to add statistical functions frequently
used in biostatistics. Values of p < 0.05 were considered toindicate statistical significance.
Results

CD248-positive (CD248+) fibroblast-like type B lining cells
and stromal fibroblasts were observed (Fig. 1B). In synovium
of OA with inflammatory change, CD68+ macrophage-liketype A lining cells and CD248+ fibroblast-like type B lining
cells were also observed, but immunoreactivity of macrophage and fibroblast in stroma were modest (data not shown).
Inflammatory synovitis scores of RA were significantly
higher than for OA (Fig. 1C, RA: 7.4 – 1.0, OA: 4.3 – 1.7,
p < 0.01).
Immunohistochemistry showed that PDPN was markedly
expressed in the hyperplastic synovial lining layer and stromal macrophages and fibroblasts of RA (Fig. 1D–J). In OA
samples with inflammatory reaction, immunoreactivity of
PDPN was modestly observed in the superficial lining layerand slightly in the stromal macrophages and fibroblasts

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In actively inflamed synovium of RA, CD68-positive(CD68+) macrophage-like type A lining cells and macrophage infiltrates of the sublining stroma (Fig. 1A) and

SUZUKI ET AL.

FIG. 1. Immunohistochemistry of RA synovium. (A) CD68+ macrophage-like type A lining cells and macrophage
infiltrates of the sublining stroma were observed. (B) CD248+ fibroblast-like type B lining cells and fibroblasts of the stroma
were also observed. (C) Inflammatory synovitis score of RA using Krenn histopathological grading system was significantly
higher than that of OA (RA: 7.4 – 1.0, OA: 4.3 – 1.7, p < 0.01). Immunohistochemistry using anti-human PDPN mAbs of RA
and OA synovium (RA: D–J, OA: K–Q). Marked expression of PDPN was detected in the hyperplastic synovial lining layerand stromal fibroblasts and macrophages of RA (D: NZ-1, E: LpMab-3, F: LpMab-7, G: LpMab-10, H: LpMab-12, I:
LpMab-13, J: LpMab-17). PDPN was modestly observed in the superficial lining layer and slightly in the macrophages andfibroblasts of the sublining stroma of OA (K: NZ-1, L: LpMab-3, M: LpMab-7, N: LpMab-10, O: LpMab-12, P: LpMab13, Q: LpMab-17). mAb, monoclonal antibody; OA, osteoarthritis; PDPN, podoplanin; RA, rheumatoid arthritis.

PODOPLANIN IN RHEUMATOID ARTHRITIS

and those in CD248+ fibroblast-like type B lining cells wereobserved (Fig. 2S–X).
Evaluation of immunoreactivity using BZ H3C showedthat LpMab-12 showed the highest score and was significantly higher than that of NZ-1, LpMab-3, and LpMab-17 in
RA (NZ-1: 28.8% – 5.6%, LpMab-3: 29.5% – 7.6%, LpMab7: 31.5% – 6.7%, LpMab-10: 30.1% – 9.9%, LpMab-12:
41.3% – 6.6%, LpMab-13: 37.5% – 7.9%, LpMab-17: 30.4% –7.4%) (Fig. 3A). Similarly, LpMab-12 had the highest score in
OA. In contrast to RA, the difference was not statisticallyDownloaded by Yukinari Kato from online.liebertpub.com at 02/24/18. For personal use only.

(Fig. 1K–Q). Immunofluorescence showed cellular colocalization of NZ-1 in CD68+ macrophage-like type A lining
cells and infiltrating macrophages of the stroma (Fig. 2A–D),
and in CD248+ fibroblast-like type B lining cells, stromal
fibroblasts were observed (Fig. 2E–H). Moreover, CD68+ andCD248+ cells were observed in the inflamed synovial lining
layer of RA (Fig. 2I–L), but only in the superficial lining layerof OA (data not shown). Similarly, colocalization of LpMab-3,
LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17
in CD68+ macrophage-like type A lining cells (Fig. 2M–R)

15

FIG. 2. Immunohistochemical analysis. Double-immunofluorescence staining of CD68 for macrophage (A, I), CD248 forfibroblast (E, J), NZ-1 for PDPN (B, F), and DAPI for nuclei (C, G, K) was performed to reveal colocalizations of CD68/
NZ-1 (D), CD248/NZ-1 (H), or CD68/CD248 (L) with yellow color in overlays. Double-immunofluorescence staining of
CD68 and LpMab-3 (M), LpMab-7 (N), LpMab-10 (O), LpMab-12 (P), LpMab-13 (Q), and LpMab-17 (R). Green for
PDPN, red for CD68, and blue for nuclei. Double-immunofluorescence staining of CD248 and LpMab-3 (S), LpMab-7 (T),
LpMab-10 (U), LpMab-12 (V), LpMab-13 (W), and LpMab-17 (X). Green for PDPN, red for CD248, and blue for nuclei.

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16

FIG. 3. The result of evaluation of immunoreactivity using BZ H3C Hybrid Cell Count Software. (A) LpMab-12
has the highest score, which was significantly higher thanthat of NZ-1, LpMab-3, and LpMab-17 in RA ( p < 0.05).
(B) LpMab-12 has the highest score, but the difference was
not statistically significant in OA.
significant in OA (NZ-1: 29.3% – 5.6%, LpMab-3: 25.4% –
7.5%, LpMab-7: 28.6% – 7.4%, LpMab-10: 24.9% – 9.5%,
LpMab-12: 36.3% – 8.5%, LpMab-13: 28.9% – 10.5%, and
LpMab-17: 33.8% – 7.1%) (Fig. 3B).
Discussion

To the best of our knowledge, this was the first comparative study of PDPN stainability in RA and OA synovium
using an anti-hPDPN mAb panel. We demonstrated that
LpMab-12 was the most stainable anti-hPDPN mAb for inflamed synovial lining layer of RA patients in immunohistochemistry. Boilard et al. demonstrated that platelets play animportant role in the pathophysiology of inflammatory arthritis.(31) Semple et al. proposed that platelets may contribute to the acceleration of the joint destruction in patients with
inflammatory arthritis, such as RA.(32) Platelet aggregation
stimulating (PLAG) domain is an important site for hPDPN on
its function of platelet aggregation.(33) PLAG domain locates on
29–54 amino acid of hPDPN.(34) The minimum epitope of NZ1, LpMab-3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and
LpMab-17 was Met43-Asp49, Thr76-Glu81, Alg79-Leu83,
Glu33-Gly45, Asp49-Pro53, Ala42-Asp49, and Gly77-Asp82
of hPDPN, respectively.(20–25) In particular, LpMab-12 detects
the glycosylation on Thr52 of hPDPN.(23) It was reported thatSUZUKI ET AL.

sialylated O-glycan on Tnr52 was required for the platelet aggregating activity of hPDPN.(34,35) The reason of superiority of
LpMab-12 in immunohistochemistry for synovial tissues may
be derived from its interaction with platelets which plays animportant role in inflammatory synovitis.
In this study, anti-hPDPN mAbs, including NZ-1, LpMab3, LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab17, showed immunoreactivity in inflamed the hyperplastic
synovial lining layer of RA tissues. In contrast to previous
reports, our results showed that PDPN was also observed in
OA synovial tissues. Ekwall et al. reported that synovium in
OA patients were predominantly PDPN-negative, and immunohistochemistry staining scores of PDPN in RA wassignificantly higher than OA.(10) In their report, D2-40(mouse IgG1) was used as anti-PDPN antibody.(10) The difference for the binding affinity of D2-40, NZ-1, LpMab-3,
LpMab-7, LpMab-10, LpMab-12, LpMab-13, and LpMab-17
may lead to the discrepancy of immunohistochemistry.
However, in primary cultured fibroblast-like synoviocytes,
average PDPN expression was similar in RA and OA(52% – 24% and 64% – 6%, respectively).(10) In addition, thereason for the difference of positivity of PDPN between RA
and OA among surgically resected fibroblast-like synoviocytes and cultured fibroblast-like synoviocytes was not still
elucidated. In contrast to in-vivo, cellular activity of fibroblasts may be altered, leading to the increased expression of
PDPN in culture conditions. Further investigation is necessary to clarify the discrepancy between in -vivo and in -vitro.
The synovial lining layer is mainly constituted bymacrophage-like type A lining cells and fibroblast-like type
B lining cells.(36) Kerrigan et al. reported that PDPN was
expressed on thioglycolate-elicited inflammatory peritoneal
macrophages and lipopolysaccharide-treated RAW264.7
macrophages, but not bone marrow-derived macrophages,
alveolar macrophages, and peritoneal macrophages.(37) Croftet al. reported that PDPN was expressed predominately on thelining layer of the synovium, but was seen only on a small
number of cells within the sublining layer, whereas CD248
was superiorly expressed in the sublining fibroblasts.(38)Conversely, our results showed that CD248 was clearly
positive in synovial lining cells (Fig. 2B). It is possible thatthe difference between Croft’s study and ours with respect tograde of inflammation of synovitis or drugs used at the timeof tissue sampling affected the location of CD248-positive
cells and their immunoreactivity.
Clinically, 20%–40% of RA patients using biologics arestill resistant to the treatment.(18,19) Del Rey et al. reported
that there was a strong association between PDPN expression and synovial lymphoid neogenesis in RA synovium,
which was reduced after anti-TNF-alpha therapy.(39) Thus,
PDPN has been suggested as one of the biomarkers fortreatment-resistant inflammatory synovitis, because its
expression correlated with local inflammatory grading of
the synovitis in groups with biologics or cDMARDs byimmunohistochemical analyses using NZ-1.(18) Dakin et al.
reported PDPN was increased in diseased compared tohealthy tendon tissues and cells, thus concluding thatstromal fibroblast activation is a potential therapeutic target
in the chronic inflammation.(40) Mechanisms of antibody
drugs are as follows; activation of antibody-dependent
cellular cytotoxicity (ADCC) or complement-dependent
cytotoxicity (CDC), blocking of signal transduction byDownloaded by Yukinari Kato from online.liebertpub.com at 02/24/18. For personal use only.

PODOPLANIN IN RHEUMATOID ARTHRITIS

means of binding to the receptor, and antibody drug conjugates (ADCs).(41–44) Because RA is an inflammatory
disease, not a malignant neoplastic disease, blocking signaltransduction or ADCs for abrogation of the activation of
inflammatory cascade of synovium may be suitable rather
than ADCC or CDC. In our study, LpMab-12 was the moststainable anti-hPDPN antibody in RA synovitis. PDPN,
especially LpMab-12, seems not only to be a promisingbiomarker for inflammatory arthritis both in RA and OA but
also may have the potential of being a therapeutic target in
RA synovitis by blocking signal transduction or ADCs forsuppressing inflammation. Further investigation on PDPN
as a therapeutic target molecule for antirheumatic drug is
mandatory.
Inflammatory synovium generates proinflammatory mediators, which act as cartilage-degrading factors such as
matrix metalloproteinases, tumor necrosis factor, interleukin1 (IL-1), IL-6, prostaglandin E2, and nitric oxide.(45–48) Inhibition of the cell activation in inflammatory synovial
tissues by anti-hPDPN mAbs may have the potential to protect joint destruction leading to the loss of daily function inarthritic patients.
Recently, OA has been reported to be an inflammatory
joint disease.(49) In early OA, synovial tissue demonstrated
the features of inflammation such as lining layer hyperplasia,
CD68+ macrophage infiltration, and vascular proliferation.(50) Synovitis had relationships with not only pain but
also physical function in knee OA patients.(51) A recent studyof 514 patients without radiographic knee OA revealed thatjoint effusion and synovitis predicted cartilage loss in the
future.(52) In our study, immunoreactivity of PDPN was observed in the lining layer of OA synovium, which was a small
number of RA. Thus, PDPN may be also one of therapeutictargets in inflammatory OA synovial tissue as well as in active and/or treatment-resistant RA synovitis.
There are three limitations in this study. First, the study
populations were relatively small. LpMab-12 may have astatistically higher score of PDPN positivity in OA synovium
from larger samples. Second, synovial samples in this study
were collected from joint surgery, which was done for terminally destructive joints. Therefore, it was not possible tocompare PDPN expression in early-stage inflamed joint andsevere inflamed joint of RA patients. Third, it is likely thatsteroids may influence PDPN positivity and function in the
inflamed synovial lining layer. In fact, 72.7% of patients inthis study used oral steroids at the time of tissue sampling.
Comparative analyses between steroids naive and steroids
user are anticipated.
In conclusion, PDPN was expressed in inflammatory lining
cells and sublining stroma of RA and OA synovium. In theseven anti-hPDPN antibodies examined, LpMab-12 was themost stainable antibody for PDPN in RA synovitis. Thus,
LpMab-12 has a possible and promising specific biomarker
for evaluating synovitis in RA and inflammatory OA. Furthermore, additional analyses of LpMab-12 for the therapeutic target of treatment resistance inflammatory arthritis
are required.
Acknowledgments

We sincerely thank Ms. Eiko Saito for her skillful technical support for immunohistochemistry. This work was

17

supported, in part, by the Basic Science and Platform Technology Program for Innovative Biological Medicine fromJapan Agency for Medical Research and Development,
AMED (Y.K.) and by Platform Project for Supporting Drug
Discovery and Life Science Research (Basis for Supporting
Innovative Drug Discovery and Life Science Research,
BINDS) from AMED (Y.K.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. The other authors have no conflicts of interest.
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Address correspondence to:
Tomoto Suzuki
Department of Orthopaedic Surgery
Faculty of Medicine
Yamagata University
2-2-2 Iida Nishi
Yamagata 990-9585
JapanE-mail: tomoto@med.id.yamagata-u.ac.jp
Received: September 25, 2017
Accepted: November 16, 2017