Honda et al. Arthritis Research & Therapy(2021) 23:214
https://doi.org/10.1186/s13075-021-02597-6

RESEARCH ARTICLE

Open Access

Pathogenic roles and therapeutic potentialof the CCL8–CCR8 axis in a murine modelof IgG4-related sialadenitis
Fumika Honda1, Hiroto Tsuboi1* , Yuko Ono1, Saori Abe1, Hiroyuki Takahashi1, Kiyoaki Ito2, Kazunori Yamada2,
Mitsuhiro Kawano2, Yuya Kondo1, Kenichi Asano3, Masato Tanaka3, Marie Malissen4, Bernard Malissen4,
Isao Matsumoto1 and Takayuki Sumida1

Abstract
Background: Our previous studies reveal that CCL18-CCR8 chemokine axis is upregulated in patients ofimmunoglobulin G4-related disease (IgG4-RD), suggesting that the CCL18–CCR8 axis is implicated in the etiology of
IgG4-RD, although whether this axis has a potential as a therapeutic target remains unclear. Our purpose was toclarify the pathogenic roles and therapeutic potential of the murine CCL8 (analog of human CCL18)–CCR8 axis byusing an animal model of IgG4-RD (LAT Y136F knockin mice; LAT mice).
Methods: We compared the infiltration of inflammatory cells and the fibrosis of the salivary glands of 6-week-old
LAT mice and littermate mice. The expressions of Ccl8 and Ccr8 were also compared. Next, we investigated thetherapeutic effects of intravenous administration of anti-CCL8 neutralizing antibody in LAT mice against
inflammation and fibrosis of the salivary glands. We also investigated the effects of stimulation with recombinantmouse CCL8 on the collagen production in a mouse fibroblast cell line (NIH/3 T3) in vitro.
Results: When compared with the littermates, the LAT mice showed apparent infiltration of inflammatory cells andfibrosis in the salivary glands. The focus and fibrosis score in the salivary glands were significantly higher in the LAT
mice than in the littermates. The expression levels of Ccl8 in the spleen and of Ccr8 in the salivary glands weresignificantly higher in the LAT mice than in the littermates. Anti-CCL8 antibody significantly improved the focus andfibrosis score in the salivary glands of the LAT mice. In vitro, stimulation with recombinant mouse CCL8 significantly
increased the expression of collagen and ERK1/2 phosphorylation in NIH/3 T3.
Conclusion: We clarified the overexpression and therapeutic potential of the mouse CCL8–CCR8 axis in LAT mice,
which could play a crucial role in fibrosis via ERK1/2 phosphorylation, as well as the chemotaxis of inflammatory
cells. The human CCL18–CCR8 axis might be a novel therapeutic target for IgG4-RD.
Keywords: IgG4-related disease, Animal model, Chemokine, CCL18, CCL8, Fibrosis* Correspondence: Hiroto-Tsuboi@md.tsukuba.ac.jp
1
Departments of Internal Medicine, Faculty of Medicine, University ofTsukuba, 1-1-1 Tennodai, Tsukuba-city, Ibaraki 305-8575, Japan
Full list of author information is available at the end of the article© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Honda et al. Arthritis Research & Therapy(2021) 23:214

Background
Immunoglobulin G4-related disease (IgG4-RD) is characterized by elevation of serum IgG4 levels and infiltration of IgG4-positive plasmacytes and fibrosis ofmultiple organs [1, 2]. Especially in IgG4-RD sialadenitis,the cytokine environment of the salivary glands (SGs) is
predominantly composed of type-2 T helper (Th2) cytokines and IL-10 expression is elevated when comparedwith the cytokine environment of Sjögren’s syndrome,
which also presents with sialadenitis [3]. In clinical practice, glucocorticoid treatment is effective for inducing
clinical remission, but relapse occurs frequently with tapering of the steroid dose [4]. Therefore, a diseasespecific treatment strategy based on the etiology isrequired.
In our previous studies, we revealed that the chemokine (C-C motif) ligand 18 (CCL18) and its receptor
chemokine (C-C motif) receptor 8 (CCR8) in labial salivary glands (LSGs) of IgG4-RD patients are upregulated
when compared with those of healthy controls and of
Sjögren’s syndrome patients [5]. Both IgG4-RD and
Sjögren’s syndrome can cause sialadenitis and are important for differential diagnosis [6], while the upregulation of CCL18-CCR8 axis is considered to be specific to
IgG4-RD. CCL18 (hCCL18) is produced mainly by macrophages and dendritic cells. It has a chemotactic function for T cells and B cells and induces the fibrosis [7,8]. We also reported that in the LSGs of IgG4-RD patients, some macrophages and dendritic cells expressed
hCCL18 [9]. In addition, the number of hCCL18positive macrophages and dendritic cells in the LSGs of
IgG4-RD patients was higher than in those of healthy
controls and Sjögren’s syndrome patients [9]. Furthermore, hCCL18 specifically enhanced IgG4 production bystimulating peripheral blood mononuclear cells in vitro[9]. It was reported that serum hCCL18 concentration
was correlated with disease activity and the number of
involved organs [10]. These findings suggest that thehCCL18–CCR8 axis is implicated in the etiology of
IgG4-RD, although whether this axis has a potential as atherapeutic target remains unclear.
Animal models of disease are essential for the development of therapeutic agents, especially in rare diseases of
unknown etiology. However, rodents do not possess the
IgG4 subtype, making it difficult to replicate IgG4-RD ina mouse model. The linker for activation of T cells(LAT) Y136F knockin mouse (LAT mouse) has been reported as a mouse with elevated type-2 T helper (Th2)cytokine and serum IgG1 (human IgG4-equivalent)
levels [11]. In recent years, some reports showed that
the LAT mouse could be an animal model of IgG4-RD[12, 13]. LAT mice showed IgG1-positive plasmacyte infiltration and fibrosis in some organs, including the SGs,
as well as elevated Th2 cytokine and serum IgG1 [12].

Page 2 of 12

These characteristics are held in common by human
IgG4-RD patients. Furthermore, the tissue lesions of
LAT mice were reduced by therapeutic administrationof corticosteroid, also in common with human IgG4-RD[12]. Interestingly, it has been reported that CCR8,
which is a receptor for hCCL18 and murine CCL8(mCCL8; analog of hCCL18), could be upregulated in T
cells in a Th2-polarized environment [14] and that IL-4
and IL-10 could induce the production of hCCL18 andmCCL8 from macrophages [14].
In this study, we analyzed the expression levels of themCCL8 (analog of hCCL18)–CCR8 axis in LAT mice asa murine model of IgG4-RD and injected the mice with
anti-mCCL8 antibody to explore whether mCCL8blocking therapy would reduce the development of tissue lesions, especially in sialadenitis, in which the elevated expression of hCCL18 has already been
demonstrated in human IgG4-RD.

Materials and methods
Animals

LAT Y136F knockin mice with a C57BL/6j backgroundwere kindly provided by Dr. Malissen (Marseille Université, Marseille, France) and maintained under specificpathogen-free conditions. All the animal protocols wereapproved by the Institutional Animal Experiment Committee of the University of Tsukuba (Permit No.20-189),
and all the experiments were performed in accordancewith relevant guidelines and regulations.
Cell culture and treatment

The NIH/3 T3 mouse fibroblast cell line was purchasedfrom the Cell Engineering Division of the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). The cells were
cultured in Dulbecco’s modified Eagle’s medium(DMEM) containing antibiotics and 10% fetal bovine
serum (FBS) at 37 °C in a humidified incubator containing 5% CO2. For all the experiments, the cells were usedwithin 3 to 7 passages after the reception. For in vitro
functional analysis of CCL8, NIH/3 T3 cells were seeded
in a 96-well plate at a density of 1 × 105 cells/mL. Somegroups of cells were cultured with varying concentrations of recombinant mouse CCL8 (BioLegend, San
Diego, CA) for 24 h. The concentration of FBS was reduced to 1% at 24 h before testing. Total collagen was
detected by the Sirius Red Collagen Detection Kit(Chondrex, Woodinville, WA) according to the manufacturer’s instructions.
Histopathologic analysis

The SGs were surgically excised, fixed in neutralized
10% formalin, and embedded in paraffin, and 3-μm-thickslices were prepared. The slices were stained withhematoxylin and eosin (H&E) and with Masson’sHonda et al. Arthritis Research & Therapy(2021) 23:214

trichrome (M&T) according to the standard techniques.
The inflammatory lesions were graded histologicallyusing the focus score method proposed by Greenspan
et al. [15] as follows: the focus score was described as
the number of a focus per 4 mm2 of each section. One
focus was defined as > 50 mononuclear cells foundaround the SG ducts. The fibrotic lesions were graded as
occupancy of fibrosis per 4 mm2 of each section: 0, without fibrosis; 1, 1 to 24%; 2, 25 to 49%; and 3, higher than
50%. The histopathologic evaluation was performed bymeans of light microscopy in a blinded manner.
Immunofluorescence and immunohistochemical staining

For confocal microscopy, the organs were embedded in
optimum cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan) and frozen at −80 °C. A cryostat was used to generate 5-μm slices,which were kept at − 20 °C. On the day of staining, theslices were thawed and dried and then fixed with coldacetone for 10 min. The slices were rehydrated inphosphate-buffered saline (PBS), blocked in PBS containing 1% bovine serum albumin (BSA; FujiFilm Wako
Pure Chemical, Tokyo, Japan) for 30 min at 20 °C–22 °C,
and stained with Alexa Fluor 488-conjugated rat antimouse CD4 antibody (Thermo Fisher Scientific, Waltham, MA) or with Alexa Fluor 647-conjugated rat antimouse B220, Alexa Fluor 488-conjugated rat anti-mouse
F4/80 antibody, and Alexa Fluor 488-conjugated rat
anti-mouse CD11c antibody (all from BioLegend). For
double staining of CCL8 and cell surface molecules, theslices were blocked in 3% H2O2 in methanol to block endogenous biotin and stained with biotinylated antimouse MCP-2/CCL8 antibody (R&D Systems, Minneapolis, NE). The primary antibody was left to incubate
overnight at 4 °C. Streptavidin-Alexa Fluor 594 (BioLegend) was used as a secondary antibody and was left toincubate for 30 min at 20 °C–22 °C. For staining of
CCR8, the cells were applied to glass slides by use of
Smear Gel (GenoStaff, Tokyo, Japan) according to the
manufacturer’s instructions. After being blocked in PBS
containing 1% BSA, the slices were stained with antimouse CCR8 antibody (Abcam, Cambridge, UK) and incubated overnight at 4 °C. Alexa Fluor 546 goat antirabbit IgG (H + L) (Thermo Fisher Scientific) was used
as a secondary antibody. The control slides were incubated with a dilution buffer containing isotype-matchedantibodies instead of primary antibodies. All the dilutions were made in Solution B (Toyobo, Osaka, Japan).
The nuclei were counterstained with 4′,6-diamidino-2phenylindole (DAPI). All the slides were mounted in
Fluorescence Mounting Medium (Dako Carpinteria,
Santa Barbara, CA). For immunohistochemical staining,
5-μm slices were cut from formalin-fixed and paraffinembedded tissues, deparaffinized, rehydrated, blocked inPage 3 of 12

PBS containing 1% BSA for 30 min at 20 °C–22 °C, andthen stained with anti-mouse CD138 antibody (BioLegend). After incubation overnight at 4 °C, the slices were
washed in PBS 5 times and incubated with Histofine
Simple Stain MAX-PO (Nichirei Bio, Tokyo, Japan) for
30 min at 20 °C–22 °C. Peroxidase activity was detectedwith diaminobenzidine (Nichirei Bio). The slices werecounterstained with hematoxylin and dehydrated. A
Fluoview FV10i confocal laser scanning microscope(Olympus, Tokyo, Japan) or Biozero BZ-8100 (Keyence,
Osaka, Japan) was used to acquire images.
RNA extraction and quantitative real-time reversetranscription polymerase chain reaction (qRT-PCR)

Cervical lymph nodes (cLNs) and SGs were submerged
in approximately 5 volumes of RNAlater solution(Thermo Fisher Scientific) immediately after sampling.
Spleens were strained through a 40-μm filter and subjected to red blood cell (RBC) lysis with 0.16 M of
NH4Cl solution for 5 min. These samples or cultured
NIH/3 T3 mouse fibroblast cells were suspended in Isogen (Nippon Gene, Tokyo, Japan). Total RNA was extracted using the Isogen method. Complementary DNA(cDNA) was synthesized at 37 °C for 15 min using a
Prime Script reverse transcriptase master mix (Takara
Bio, Shiga, Japan), and the reaction mixture was used for
the PCR. qRT-PCR reactions were performed using a
Taqman gene expression assay and normalized toglyceraldehyde-3-phosphate dehydrogenase (Gapdh)
abundance. The primers and probes, all from Thermo
Fisher Scientific, were as follows: Gapdh, Mm99999915_
g1; Ccr8, Mm99999115-s1; Ccl8, Mm01297183-m1; IL4,
Mm00445259_m1; IL10, Mm00439614_m1; and Col1a2,
Mm00483888_m1.
Enzyme-linked immunosorbent assay (ELISA)

Serum levels of mouse CCL8 were measured using acommercially available ELISA (R&D systems) according
to the manufacturer’s instructions.
Cell preparation for FACS staining

To obtain tissue-infiltrating mononuclear cells in theSGs, the SGs from LAT mice were harvested, cut into
small pieces with scissors, and incubated at 37 °C for 30
min with 2 mg/mL collagenase D (Roche Diagnostics
GmbH, Mannheim, Germany) and 5 mM CaCl2. Afterincubation, they were disrupted by grinding between
frosted glass coverslips. After straining through a 40-μm
filter and washing with the PBS, infiltrating mononuclear
cells were isolated from parenchyma cells by OptiPrep(Cosmo Bio, Tokyo, Japan) with gradient centrifugationaccording to the manufacturer’s instructions.

Honda et al. Arthritis Research & Therapy(2021) 23:214

Page 4 of 12

Fluorescence-activated cell sorting (FACS) staining

mice were significantly higher than those of the littermates(Fig. 1B, C). Immunostaining revealed infiltration of CD4+
T cells, B220+ B cells, and CD138+ plasma cells into theSGs of the LAT mice (Fig. 1D, E). Angiogenesis of the SGs
was less noticeable in LAT mice. This is consistent with description in the 2019 American College of Rheumatology/
European League Against Rheumatism classification criteria
for IgG4-related disease in which angiogenesis is not
adopted in the histopathology section [17].

For FACS analyses, the cells were washed in FACS buffer (2% FBS in PBS) and then incubated with FACS antibodies. Staining for mouse CD4 and CD25 (all from
BioLegend) was performed for 20 min using a mixture ofantibodies. Dead cells were excluded by Fixable Viability
Dye eFluor 780 (eBioscience, San Diego, CA). Intracellular staining for Foxp3, IFN-γ, IL-17, and IL-4 (all from
BioLegend) was performed after fixation andpermeabilization using a Foxp3/Transcription Factor
Staining Buffer Set (eBioscience) according to the manufacturer’s instructions. Intracellular staining of IFN-γ,
IL-17, and IL-4 (BioLegend) was performed after 4 h ofstimulation by phorbol myristate acetate (PMA) (50 ng/mL), ionomycin (0.5 μg/ml), and GolgiStop (eBioscience)
were added during the stimulation.
For phosphoextracellular signal-regulated kinase(ERK)1/2 (BioLegend) staining, Phosflow Lyse/Perm buffer and Perm Buffer III (Becton, Dickinson and Company [BD], Franklin Lakes, NJ) were used according to
the manufacturer’s instructions. The samples were analyzed with a FACSVerse flow cytometer (BD), and thedata were analyzed using FlowJo software (Tree Star,
Ashland, OR). The relative mean fluorescence intensity(MFI) ratio was calculated on the basis of the ratio of
the MFI for a marker to the MFI of its isotype control.
In vivo inhibition of CCL8

One hundred micrograms of anti-mouse CCL8 neutralizing antibody (clone 17D6, generated by the Tokyo University of Pharmacy and Life Sciences, Tokyo) or isotype
control (BioLegend) was intravenously injected on days
1 and 2 in 5-week-old LAT mice on the basis of a previous study [16]. On day 8 (6 weeks of age), the effects oftreatment on the development of tissue lesions (spleen,cLNs, and SGs) were assessed.
Statistical analysis

All the data were expressed as means ± SEMs. Differences between groups were examined for significanceusing a 2-tailed unpaired t test or Kruskal-Wallis test.
Probability values lower than 0.05 were considered to indicate significant differences. Statistical analysis was performed using GraphPad Prism version 9 (GraphPad
software, San Diego, CA).

Results
Histopathologic analysis of the salivary glands of 6-weekold LAT mice and littermates

We histopathologically analyzed the SGs of 6-week -old
LAT mice and the control littermates. All the analyzed
LAT mice displayed inflammatory lesions showing infiltration of mononuclear cells with fibrosis (Fig. 1A). The
histologic focus scores and fibrosis scores of the LAT

Upregulation of the CCL8–CCR8 axis in the salivary glandsof LAT mice

To clarify whether Th2 cytokines and the CCL8–CCR8
axis are upregulated in LAT mice as they are in IgG4-RD
patients, we compared Il4, Il10, Ccl8, and Ccr8 mRNA expressions in the spleen, cLNs, and SGs of the LAT miceand the littermates. Our results showed that Il4 expression
in the spleen, cLNs, and SGs of the LAT mice was significantly higher than that in the littermates (Fig. 2A). Il10
and Ccl8 expressions were significantly higher in the
spleen (Fig. 2B, C), and Il10 and Ccr8 expressions weresignificantly higher in the SGs of the LAT mice (Fig. 2D).
Besides, serum CCL8 levels were significantly higher inthe LAT mice than in the littermates (Fig. 2E). The proportion of IL-4+ cells in splenic CD4+ cells was significantly higher in LAT mice compared to littermates,
whereas comparable in IFN-γ+ and IL-17+ cells (Fig. 2F
and Figure S1[A][B]). There was no significant differencein the production of these cytokines between the spleen
and SGs of LAT mice (Fig. 2G), while, in LAT mice,
CD4+CD25+Foxp3+ cells significantly decreased comparedwith littermates (Figure S2[A][B]), which was consistent
with the previous report [18].
CCL8-expressing cells in the salivary glands of LAT mice

To confirm that mCCL8-expressing cells were exactly
present in the SGs, we immunofluorescently stained theinfiltrating cells. The results revealed that CCL8expressing F4/80-positive macrophages (Fig. 3A) andCD11c-positive dendritic cells (Fig. 3B) were predominantly present in the SGs of the LAT mice. In contrast,
these cells were not detected in the littermates.
Suppression of salivary gland inflammation and fibrosis
in LAT mice by administration of anti-mCCL8 neutralizing
antibody

To determine whether the mCCL8–CCR8 pathway
could be a therapeutic target for IgG4-RD, we evaluatedthe SG pathology of anti-mCCL8 neutralizing antibody(anti-CCL8 Ab) and control immunoglobulin- (controlIg) treated LAT mice. The development of sialadenitis
was found to be inhibited in the anti-CCL8 Ab-treated
LAT mice when compared with the control Ig-treatedmice (Fig. 4A). The focus scores of the anti-CCL8 Ab-

Honda et al. Arthritis Research & Therapy(2021) 23:214

Page 5 of 12

Fig. 1 Histologic analysis of the salivary glands of 6-week-old LAT mice and of the littermates. A H&E staining and M&T staining of salivary glands.
The boxed areas in the upper panels are shown in detail in the lower panels. Scale bars, 200 μm. Representative images of 3 mice per group.
H&E, hematoxylin and eosin stain; M&T, Masson’s trichrome stain. B Focus score of salivary glands. Histologic evaluation was performed in ablinded manner (littermate, n = 3; LAT mice, n = 3). C Fibrosis score of salivary glands. Histologic evaluation was performed in a blinded manner(littermate, n = 3; LAT mice, n = 3). D Images are representative of salivary gland slices of LAT mice stained for CD4 (green), B220 (red), and 4′6diamino-2-phenylindole (DAPI) (blue). Top: low magnification images; second row from the top: the part focused on CD4+ cells; third row fromthe top: the part focused on B220+ cells; bottom row: images stained for isotype control. Scale bars (top, 50 μm; second and third rows
from the top, 10 μm; bottom row, 50 μm). E Salivary gland slices of LAT mice stained for CD138 or isotype control. Scale bar,100 μm. *p< 0.05, unpaired t test

treated mice were significantly lower than those of thecontrol Ig-treated mice (Fig. 4B). Interestingly, the development of SG fibrosis was also found to be inhibited in
the anti-CCL8 Ab-treated LAT mice (Fig. 4C). The fibrosis scores of the anti-CCL8 Ab-treated mice were significantly lower than those of the control Ig-treated mice
(Fig. 4D). Immunostaining revealed that the number of
CD4+ cells that had infiltrated the region around the SG
ducts of the anti-CCL8 Ab-treated mice was significantly
reduced when compared with that of the control Ig-

treated mice (Fig. 4E, F). The number of B220+ cells andCD138+ cells also tended to be reduced, although thedifference between the treatment groups did not reachstatistical significance (Fig. 4E, G, H, I).
Effects of CCL8-blocking therapy on mRNA expression of
Il4 and Il10 in LAT mice

Next, we investigated whether anti-mCCL8 neutralizing
therapy affected the cytokine environment in LAT mice.
The mRNA expression of Il4 and Il10 tended to beHonda et al. Arthritis Research & Therapy(2021) 23:214

Page 6 of 12

Fig. 2 Upregulation of Th2 cytokines and the CCL8–CCR8 axis in 6-week-old LAT mice. A Il4 mRNA levels in spleens, cervical lymph nodes (cLNs),
and salivary glands (SGs) were determined by qRT-PCR. The expression levels were calculated as relative amounts normalized to Gapdh(littermate, n = 3; LAT, n = 3). B Il10 mRNA levels in spleens, cLNs, and SGs were determined by qRT-PCR. The expression levels were calculated as
relative amounts normalized to Gapdh (littermate, n = 3; LAT, n = 3). C Ccl8 mRNA levels in each tissue were determined by means of qRT-PCR.
The expression levels were calculated as relative amounts normalized to Gapdh (littermate, n = 3; LAT, n = 3). D Ccr8 mRNA levels in each tissue
was determined by means of qRT-PCR. The expression levels were calculated as relative amounts normalized to Gapdh (littermate, n = 3; LAT, n =3). E Serum CCL8 levels were determined by ELISA (littermate, n = 4; LAT, n = 5). F Splenocytes from littermates and LAT mice were assessed byflow cytometry for cytokine production after stimulation with phorbol myristate acetate and ionomycin for 4 h. The proportion of CD4+ cells that
producing each cytokine was quantified (littermate, n = 3; LAT, n = 3). G Splenocytes and mononuclear cells infiltrated in SGs from LAT mice
were assessed by flow cytometry for cytokine production after stimulation with phorbol myristate acetate and ionomycin for 4 h. The proportionof CD4+ cells that producing each cytokine was quantified. *p < 0.05, Unpaired t test. The data are presented as means ± SEMs

reduced in the anti-CCL8 Ab-treated LAT mice whencompared with the control Ig-treated group, especiallyin the spleen, although the difference between thetreatment groups did not reach statistical significance(Fig. 5A, B). These results could suggest that theattenuated Th2 phenotypes were not the main reason
for the improved fibrosis. The following in vitro experiment was conducted to investigate the possibility thatmCCL8 acts directly on fibroblasts and has a mechanism
involved in fibrosis.

Honda et al. Arthritis Research & Therapy(2021) 23:214

Page 7 of 12

Fig. 3 CCL8-expressing cells in salivary glands of LAT mice. A Salivary gland slices from 6-week-old control littermates or LAT mice were stainedfor F4/80 (green) and CCL8 (red). B Salivary gland slices from 6-week-old control littermates or LAT mice were stained for CD11c (green) and
CCL8 (red). DAPI (blue), 4′6-diamino-2-phenylindole. Scale bars, 50 μm

Effects of mCCL8 on collagen production of fibroblasts
in vitro

To further analyze the association between mCCL8 andfibrosis, we cultured a mouse fibroblast cell line (NIH/3
T3) with or without recombinant mouse CCL8
(rmCCL8). First, we confirmed the protein expression of
CCR8 in NIH/3 T3 cells by means of immunofluorescence staining (Fig. 6A). Then, the NIH/3 T3 cells were
stimulated with various concentrations of rmCCL8.
Twenty-four hours after the stimulation with rmCCL8,
we investigated the mRNA expression level of Col1a2
and the total collagen protein production in the NIH/3
T3 cells. The mRNA expression levels of Col1a2 and thetotal collagen protein production were increased in
rmCCL8 dose-dependent manner (Fig. 6B, C). To investigate whether ERK1/2 phosphorylation is involved in

upregulation of collagen production by rmCCL8, theNIH/3 T3 cells were treated with 300 ng/mL of rmCCL8.
After 12 min of stimulation with rmCCL8, phosphorylation of ERK1/2 in the NIH/3 T3 cells was significantly
increased in both the MFI and the positive cell ratio
when compared with no stimulation (Fig. 6D, E, F).
Thus, our data suggested that the mCCL8–CCR8 pathway is involved in the induction of collagen production
by fibroblasts via phosphorylation of ERK1/2.

Discussion
In this study, we showed that LAT mice had plasmacyte
infiltration and fibrosis of the SGs similar to human
IgG4-RD as well as an enhanced CCL8–CCR8 axis.
Neutralization of mCCL8 showed improvement of sialadenitis including fibrosis, and it was shown that mCCL8

Honda et al. Arthritis Research & Therapy(2021) 23:214

Page 8 of 12

Fig. 4 Suppression of salivary gland inflammation and fibrosis in LAT mice by CCL8 neutralizing therapy. A H&E staining of salivary glands in LAT
mice treated with anti-CCL8 neutralizing antibody (anti-CCL8 Ab) or control immunoglobulin (control Ig). The boxed areas in the upper panels
are shown magnified in the lower panels (scale bars: upper; 200 μm; lower, 100 μm). Representative images of 3 mice. B Comparison of focus
score in salivary glands between anti-CCL8 Ab-treated LAT mice and control Ig-treated LAT mice. C M&T staining of salivary glands in LAT micetreated with anti-CCL8 Ab or control Ig. The boxed areas in the upper panels are magnified in the lower panels (scale bars: upper, 200 μm; lower,
100 μm). Representative images of 3 mice. D Comparison of fibrosis score in salivary glands of anti-CCL8 Ab-treated LAT mice and control Igtreated LAT mice. E The salivary glands slices of LAT mice treated with anti-CCL8 Ab or control Ig were stained for CD4 (green) and B220 (red).
DAPI (blue), 4′6-diamino-2-phenylindole (scale bars, 50 μm). F The average of CD4-positive cells per 60× magnification area. Three areas per
mouse were counted. G The average of B220-positive cells per × 60 magnification area. Three areas per mouse were counted. H Salivary glands
slices of LAT mice treated with anti-CCL8 Ab or control Ig were stained for CD138. Counterstaining was done with hematoxylin (scale bars: upper,
200 μm; lower, 100 μm). I The average of CD138-positive cells per high power field. Three areas per mouse were counted. The data are presented
as the means ± SEMs of 3 mice. *p < 0.05, unpaired t test

Honda et al. Arthritis Research & Therapy(2021) 23:214

Page 9 of 12

Fig. 5 Effects of CCL8-blocking therapy on mRNA expression of Il4 and Il10 in LAT mice. A Comparison of mRNA expression levels of Il4 in antiCCL8 Ab-treated LAT mice and control Ig-treated LAT mice. B Comparison of mRNA expression levels of Il10 in anti-CCL8 Ab-treated LAT miceand control Ig-treated LAT mice. cLN, cervical lymph nodes; SG, salivary glands. The data are presented as means ± SEMs of 3 mice. *p < 0.05,
unpaired t test

may enhance collagen production in fibroblasts via
phosphorylation of ERK1/2. Through our investigation,
the following 3 clinicopathologic findings emerged.
First, commonalities between LAT mice and IgG4-RD
have been further demonstrated. We confirmed that
LAT mice had a type 2 cytokine-dominant immune environment that reported in IgG4-RD including IgG4ralated sialadenitis [3], while Treg cells were decreasedin LAT mice. Thus, we could interpret that LAT mice
did not reflect enhanced Treg response, but Th2 response of IgG4-RD as one of the mimicking micemodels for IgG4-RD. The expression levels of themCCL8–CCR8 axis were upregulated in LAT mice, in
common with those of hCCL18–CCR8 in IgG4-RD.
Therefore, we thought that the potential of the mCCL8(hCCL18)–CCR8 pathway as a therapeutic target could
be clarified by using LAT mice as a model of IgG4-RD.
The chemokine–chemokine receptor system in mice differs from that in humans [7]. In mice, CCL1 and CCL8
have been reported as ligands for CCR8 [14]. The relationship between hCCL18 and mCCL8, not mCCL1, hasbeen proven to be a functional analog [19]. Focusing on
the induction mechanism, IL-4 and IL-10 induce
mCCL8 production from macrophages, and the samecan be said for hCCL18 [14]. In LAT mice, infiltration of
mCCL8-producing macrophages was observed in theSGs. Furthermore, both Il4 and Il10 were highly
expressed in the SGs of LAT mice, and this environment
may induce mCCL8 production.
Second, our results suggest that the CCL8–CCR8 axis
may have a potential as a therapeutic target for IgG4-RD.
The improvement of inflammatory cell infiltration was
thought to be due to the suppression of chemotaxis of T
cells and B cells to the SGs in which mCCL8 was produced by macrophages and dendritic cells. Expression of
CCR8 in T cells was reportedly upregulated in the Th2
cytokine environment [14]. Since LAT mice showed a
Th2 cytokine-dominant phenotype, like that of IgG4-RD[11], it is an environment in which CCR8 expression in T
cells is likely to be enhanced. Even more interesting, antiCCL8 Ab improved not only the infiltration of inflammatory cells but also the fibrosis of the SGs. Thus, theHonda et al. Arthritis Research & Therapy(2021) 23:214

Page 10 of 12

Fig. 6 Effects of CCL8 on collagen production of fibroblasts in vitro. A Immunofluorescence staining of CCR8 in a mouse fibroblast cell line (NIH/3 T3). Upper panels: Alexa Fluor 546-conjugated anti-mouse CCR8 antibody staining. Lower panels: isotype control antibody staining. Original
magnification × 40 (scale bar, 50 μm). DAPI, 4′6-diamino-2-phenylindole. B mRNA expression levels of Col1a2 in fibroblasts treated withrecombinant mouse CCL8 for 24 h. C Total collagen protein production from fibroblasts treated with recombinant mouse CCL8 for 24 h. D Flowcytometric analysis of phosphorylated ERK1/2 (pERK) in fibroblasts treated with recombinant mouse CCL8 at 300 ng/mL of concentration for theindicated times (min). The panels show a representative histogram of each of the 3 wells for the indicated times. E The bar graph shows the rateof pERK1/2-positive cells. F The bar graph shows the mean fluorescence intensity (MFI) of pERK1/2 in fibroblasts treated with recombinant mouse
CCL8. The data are presented as means ± SEMs of 5 (B and C) and 3 (E and F) wells in each condition. *p < 0.05, Kruskal-Wallis test

CCL8–CCR8 axis may have pathogenic roles beyond thechemotaxis of lymphocytes, such as induction of fibrosis.
Third, we showed for the first time that mCCL8 could
induce collagen production in fibroblasts via ERK1/2

phosphorylation. Although many unclear points about
the downstream pathway of CCR8 remained, it was reported that ERK phosphorylation occurred when a signal
from the ligand was input in this pathway [20, 21]. ERKHonda et al. Arthritis Research & Therapy(2021) 23:214

was contained in mitogen-activated protein kinase(MAPK) [22], which transmitted extracellular stimuli
into cells, and ERK1/2 has been reported to induce
fibroblast proliferation [23]. Still, only a limited amountof evidence is available related to the function of
hCCL18 in inducing collagen production in fibroblasts[8], and it was reported that the Th2 cytokine environment itself was involved in the induction of fibrosis [24].
Importantly, our data suggest that mCCL8 could induce
collagen production in fibroblasts via an ERK-dependent
pathway, independently of Th2 cytokines.
The following has been reported regarding the fibrotic
mechanism in human IgG4-RD. CD4+ T cells with cytotoxic activity (CD4+CTL), which were clonally proliferated in the affected organs, expressed cytotoxic proteinssuch as granzyme B and perforin [25]. These cells produce IL-1β, IFNγ, and TGFβ, which activates fibroblasts
and alternatively activated (M2) macrophages, thereby
inducing fibrosis in IgG4-related dacryoadenitis and sialadenitis [26]. Moreover, the number of infiltrating M2
macrophages has been reported to correlate with the fibrosis score in IgG4-related sialadenitis and that thesemacrophages produce hCCL18 [27]. These reports also
supposed that hCCL18 acted as a profibrotic factor andthat the hCCL18–CCR8 axis may be a therapeutic target
for IgG4-RD.
The role of chemokines in directing T cell polarizationin addition to their role in chemoattraction has beenshown in several reports; for example, CXCL10 for T
helper type-1 cells [28, 29], CXCL11 and CXCL12 for T
regulatory-1 cells [30, 31], and CCL1 for FOXp3+ Treg
cells [20] have been reported to be involved in thepolarization and differentiation of CD4+ T cells. Analysisof the effect of the mCCL8 (hCCL18)–CCR8 axis on
CD4-positive T cell differentiation needs to be further
investigated. Moreover, we hope that our research findings to lead to the development of treatments targetingthe hCCL18–CCR8 pathway as a rescue therapy for intractable IgG4-RD cases such as steroid-resistant cases
and recurrent cases.

Conclusions
In conclusion, we have demonstrated that upregulationof the mCCL8 (analog of hCCL18)–CCR8 axis in LAT
mice could play a crucial role in fibrosis via ERK1/2
pathway-dependent type-I collagen production, as wellas in cell infiltration in sialadenitis. In fact, anti-CCL8
Ab improved both the inflammation and the fibrosis of
SGs in LAT mice, suggesting that inhibition of thehCCL18 (mCCL8)–CCR8 axis should shed light on new
therapies for IgG4-RD.
Abbreviations
IgG4-RD: Immunoglobulin G4-related disease; LAT: The linker for activation of
T cells; LAT mice: LAT Y136F knockin mice; SGs: Salivary glands; anti-CCL8

Page 11 of 12

Ab: Anti-CCL8 neutralizing antibody; rmCCL8: Recombinant mouse CCL8;
LSGs: Labial salivary glands; mCCL8: Murine CCL8; hCCL18: Human CCL18;
Th2: Type-2 T helper; DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal
bovine serum; H&E: Hematoxylin and eosin; M&T: Masson’s trichrome;
PBS: Phosphate-buffered saline; qRT-PCR: Quantitative real-time reversetranscription polymerase chain reaction; cLNs: Cervical lymph nodes;
RBC: Red blood cell; cDNA: Complementary DNA; Gapdh: Glyceraldehyde-3phosphate dehydrogenase; ELISA: Enzyme-linked immunosorbent assay;
FACS: Fluorescence-activated cell sorting; PMA: Phorbol myristate acetate;
ERK: Extracellular signal-regulated kinase; MFI: Mean fluorescent intensity;
control Ig: Control immunoglobulin; MAPK: Mitogen-activated protein kinase;
CD4+CTL: CD4+ T cells with cytotoxic activity

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13075-021-02597-6.
Additional file 1: Figure S1. Flow cytometric analysis of spleen andSGs for cytokine production. (A) Gating strategy for flow cytometricanalysis of CD4+ T cells. Images are representative results of splenocytefrom littermate mice. (B) FACS plot for production of IFN-γ, IL-17, and IL-4
by CD4+ cells derived from 6-week-old littermates and LAT mice after
stimulation with phorbol myristate acetate and ionomycin for 4 hours.
CD4+ gated cells were shown in FACS plot. FACS plot is a representativeof analysis of six mice (littermate, n=3; LAT, n=3).
Additional file 2: Figure S2. CD25 and Foxp3 expression in
splenocytes from littermate and LAT mice. (A) FACS analysis of CD4,
CD25, and Foxp3 in splenocyte from 6-week-old littermates and LAT
mice. Dead cells were gated out by using fixable viability dye. FACS plot
is a representative of analysis of six mice (littermate, n=3; LAT, n=3). (B)The proportion of CD4+CD25+Foxp3+ cells were compared between littermates and LAT mice. *p<0.05, Unpaired t test. The data are presented
as means ± SEMs.
Acknowledgements
We thank Dr. F Miyamasu, Medical English Communication Center, Universityof Tsukuba, for critical reading of the manuscript.
Authors’ contributions
FH; conceptualization, investigation, formal analysis, visualization and writing
the original draft. H Tsuboi; conceptualization, supervision, and visualization
and writing the original draft. YO; conceptualization, SA; conceptualization, H
Takahashi, conceptualization and supervision, KI; conceptualization andresources, KY; conceptualization and resources, MK; conceptualization andresources, YK; conceptualization and supervision, KA; methodology andresources, MT; methodology and resources, MM; conceptualization andresources, BM; conceptualization and resources, IM; conceptualization andsupervision, TS; conceptualization and supervision. All authors contributed
and approved to the review and editing of the manuscripts.
Funding
This work was supported in part by a grant from the Practical Research
Project for Rare/Intractable Diseases (IgG4-related disease) of the Japan
Agency for Medical Research and Development (AMED).
Availability of data and materials
The data that support the findings of this study are available from thecorresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
All the animal protocols were approved by the Institutional Animal
Experiment Committee of the University of Tsukuba (Permit No.20-189), andall the experiments were performed in accordance with relevant guidelines
and regulations.
Consent for publication
All authors gave their consent to publication of this manuscript.

Honda et al. Arthritis Research & Therapy(2021) 23:214

Page 12 of 12

Competing interests
The authors declare that they have no competing interests.
18.
Author details
1
Departments of Internal Medicine, Faculty of Medicine, University ofTsukuba, 1-1-1 Tennodai, Tsukuba-city, Ibaraki 305-8575, Japan. 2Departmentof Rheumatology, Graduate School of Medical Science, Kanazawa University,
13-1 Takara-machi, Kanazawa-city, Ishikawa 920-8640, Japan. 3Laboratory of
Immune Regulation, School of Life Science, Tokyo University of Pharmacy
and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. 4Centred’Immunologie de Marseille-Luminy, Aix Marseille Université, INSERM, CNRS,
13288 Marseille, France.

19.

20.

21.
Received: 13 April 2021 Accepted: 2 August 2021
22.
References
1. Stone JH, Zen Y, Deshpande V. IgG4-related disease. N Engl J Med. 2012;
366(6):539–51. https://doi.org/10.1056/NEJMra1104650.
2. Umehara H, Okazaki K, Masaki Y, Kawano M, Yamamoto M, Saeki T,
et al. Comprehensive diagnostic criteria for IgG4-related disease (IgG4RD), 2011. Mod Rheumatol. 2012;22(1):21–30. https://doi.org/10.3109/s1
0165-011-0571-z.
3. Tanaka A, Moriyama M, Nakashima H, Miyake K, Hayashida JN, Maehara T,
et al. Th2 and regulatory immune reactions contribute to IgG4 production
and the initiation of Mikulicz disease. Arthritis Rheum. 2012;64(1):254–63.
https://doi.org/10.1002/art.33320.
4. Yamamoto M, Takahashi H, Shinomura Y. Mechanisms and assessment of
IgG4-related disease: lessons for the rheumatologist. Nat Rev Rheumatol.
2014;10(3):148–59. https://doi.org/10.1038/nrrheum.2013.183.
5. Tsuboi H, Nakai Y, Iizuka M, Asashima H, Hagiya C, Tsuzuki S, et al. DNA
microarray analysis of labial salivary glands in IgG4-related disease:
comparison with Sjögren’s syndrome. Arthritis Rheumatol. 2014;66(10):2892–
9. https://doi.org/10.1002/art.38748.
6. Puxeddu I, Capecchi R, Carta F, Tavoni AG, Migliorini P, Puxeddu R. Salivary
gland pathology in IgG4-related disease: a comprehensive review. J
Immunol Res. 2018;2018:6936727.
7. Chenivesse C, Tsicopoulos A. CCL18 – beyond chemotaxis. Cytokine. 2018;
109:52–6. https://doi.org/10.1016/j.cyto.2018.01.023.
8. Atamas SP, Luzina IG, Choi J, Tsymbalyuk N, Carbonetti NH, Singh IS, et al.
Pulmonary and activation-regulated chemokine stimulates collagenproduction in lung fibroblasts. Am J Respir Cell Mol Biol. 2003;29(6):743–9.
https://doi.org/10.1165/rcmb.2003-0078OC.
9. Tsuboi H, Iizuka-Koga M, Asashima H, Takahashi H, Kudo H, Ono Y, et al.
Upregulation and pathogenic roles of CCL18-CCR8 axis in IgG4-relateddisease. Mod Rheumatol. 2020;30(4):729–37. https://doi.org/10.1080/143
97595.2019.1632061.
10. Akiyama M, Yasuoka H, Yoshimoto K, Takeuchi T. CC-chemokine ligand 18 isa useful biomarker associated with disease activity in IgG4-related disease.
Ann Rheum Dis. 2018;77(9):1386–7. https://doi.org/10.1136/annrheumdis-2
017-212110.
11. Aguado E, Richelme S, Nuñez-Cruz S, Miazek A, Mura AM, Richelme M, et al.
Induction of T helper type 2 immunity by a point mutation in the LAT
adaptor. Science. 2002;296:2036–40.
12. Yamada K, Zuka M, Ito K, Mizuguchi K, Kakuchi Y, Onoe T, et al.
LatY136F knock-in mouse model for human IgG4-related disease. PLoS
One. 2018;13:1–14.
13. Cui Y, Masaki K, Zhang X, Yamasaki R, Fujii T, Ogata H, et al. A novel modelfor treatment of hypertrophic pachymeningitis. Ann Clin Transl Neurol.
2019;6(3):431–44. https://doi.org/10.1002/acn3.715.
14. Islam SA, Ling MF, Leung J, Shreffler WG, Luster AD. Identification of human
CCR8 as a CCL18 receptor. J Exp Med. 2013;210(10):1889–98. https://doi.
org/10.1084/jem.20130240.
15. Greenspan JS, Daniels TE, Talal N, Sylvester RA. The histopathology of
Sjögren’s syndrome in labial salivary gland biopsies. Oral Surg Oral Med Oral
Pathol. 1974;37(2):217–29. https://doi.org/10.1016/0030-4220(74)90417-4.
16. Asano K, Takahashi N, Ushiki M, Monya M, Aihara F, Kuboki E, et al. Intestinal
CD169 + macrophages initiate mucosal inflammation by secreting CCL8
that recruits inflammatory monocytes. Nat Commun. 2015;6:1–14.
17. Wallace ZS, Naden RP, Chari S, Choi H, Della-Torre E, Dicaire JF, et al. The
2019 American College of Rheumatology/European League Against

23.

24.

25.

26.

27.

28.

29.

30.

31.

Rheumatism Classification Criteria for IgG4-Related Disease. Arthritis
Rheumatol. 2020;72(1):7–19. https://doi.org/10.1002/art.41120.
Koonpaew S, Shen S, Flowers L, Zhang W. LAT-mediated signaling in CD4+
CD25+regulatory T cell development. J Exp Med. 2006;203(1):119–29.
https://doi.org/10.1084/jem.20050903.
Islam SA, Chang DS, Colvin RA, Byrne MH, McCully ML, Moser B, et al.
Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+
TH2 cells. Nat Immunol. 2011;12(2):167–77. https://doi.org/10.1038/ni.1984.
Barsheshet Y, Wildbaum G, Levy E, Vitenshtein A, Akinseye C, Griggs J, et al.
CCR8+FOXp3+ Treg cells as master drivers of immune regulation. Proc Natl
Acad Sci U S A. 2017;114(23):6086–91. https://doi.org/10.1073/pnas.16212
80114.
Spinetti G. The chemokine receptor CCR8 mediates rescue fromdexamethasone-induced apoptosis via an ERK-dependent pathway. J
Leukoc Biol. 2003;73(1):201–7. https://doi.org/10.1189/jlb.0302105.
Segar R, Krebs EG. The MAPK signaling cascade. FASEB J. 1995;9(9):726–35.
https://doi.org/10.1096/fasebj.9.9.7601337.
Ihn H, Tamaki K. Oncostatin M stimulates the growth of dermal fibroblasts
via a mitogen-activated protein kinase-dependent pathway. J Immunol.
2000;165(4):2149–55. https://doi.org/10.4049/jimmunol.165.4.2149.
Jinnin M, Ihn H, Yamane K, Tamaki K. Interleukin-13 stimulates thetranscription of the human α2(I) collagen gene in human dermal
fibroblasts. J Biol Chem. 2004;279(40):41783–91. https://doi.org/10.1074/
jbc.M406951200.
Mattoo H, Mahajan VS, Maehara T, Deshpande V, et al. Clonal expansion of
CD4(+) cytotoxic T lymphocytes in patients with IgG4-related disease.
J Allergy Clin Immunol. 2016;138(3):825–38. https://doi.org/10.1016/j.jaci.201
5.12.1330.
Maehara T, Mattoo H, Ohta M, Mahajan VS, Moriyama M, Yamauchi M, et al.
Lesional CD4+IFN-γ+ cytotoxic T lymphocytes in IgG4-related
dacryoadenitis and sialoadenitis. Ann Rheum Dis. 2017;76(2):377–85. https://doi.org/10.1136/annrheumdis-2016-209139.
Furukawa S, Moriyama M, Tanaka A, Maehara T, Tsuboi H, Iizuka M, et al.
Preferential M2 macrophages contribute to fibrosis in IgG4-related
dacryoadenitis and sialoadenitis, so-called Mikulicz’s disease. Clin Immunol.
2015;156(1):9–18. https://doi.org/10.1016/j.clim.2014.10.008.
Salomon I, Netzer N, Wildbaum G, Schif-Zuck S, Maor G, Karin N. Targetingthe function of IFN-γ-inducible protein 10 suppresses ongoing adjuvant
arthritis. J Immunol. 2002;169(5):2685–93. https://doi.org/10.4049/jimmunol.1
69.5.2685.
Wildbaum G, Netzer N, Karin N. Plasmid DNA encoding IFN-γ-inducible
protein 10 redirects antigen-specific T cell polarization and suppresses
experimental autoimmune encephalomyelitis. J Immunol. 2002;168(11):5885–92. https://doi.org/10.4049/jimmunol.168.11.5885.
Zohar Y, Wildbaum G, Novak R, Salzman AL, Thelen M, Alon R, et al.
CXCL11-dependent induction of FOXP3-negative regulatory T cells
suppresses autoimmune encephalomyelitis. J Clin Invest. 2017;127(10):3913.
https://doi.org/10.1172/JCI97015.
Meiron M, Zohar Y, Anunu R, Wildbaum G, Karin N. CXCL12 (SDF-1α)
suppresses ongoing experimental autoimmune encephalomyelitis byselecting antigen-specific regulatory T cells. J Exp Med. 2008;205(11):2643–
55. https://doi.org/10.1084/jem.20080730.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.