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Molecular Immunology 70 (2016) 1–7

Contents lists available at ScienceDirect

Molecular Immunology

j ournal homepage: www.elsevier.com/locate/molimm

Functional analysis of membrane-bound complement regulatory

protein on T-cell immune response in ginbuna crucian carp

a,b a,c a a

Indriyani Nur , Nevien K. Abdelkhalek , Shiori Motobe , Ryota Nakamura ,

a a,∗ a

Masakazu Tsujikura , Tomonori Somamoto , Miki Nakao

Laboratory of Marine Biochemistry, Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu

University, Fukuoka 812-8581, Japan

Aquaculture Department, Fisheries and Marine Science Faculty, Halu Oleo University, Kendari 93232, Indonesia

Department of Internal Medicine, Infectious and Fish diseases, Faculty of Veterinary Medicine, El-Mansoura University, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Complements have long been considered to be a pivotal component in innate immunity. Recent

Received 9 August 2015

researches, however, highlight novel roles of complements in T-cell-mediated adaptive immunity.

Received in revised form

Membrane-bound complement regulatory protein CD46, a costimulatory protein for T cells, is a key

25 November 2015

molecule for T-cell immunomodulation. Teleost CD46-like molecule, termed Tecrem, has been newly

Accepted 29 November 2015

identiﬁed in common carp and shown to function as a complement regulator. However, it remains unclear

whether Tecrem is involved in T-cell immune response. We investigated Tecrem function related to T-cell

Keywords:

responses in ginbuna crucian carp. Ginbuna Tecrem (gTecrem) proteins were detected by immunoprecip-

Membrane-bound complement regulatory

protein itation using anti-common carp Tecrem monoclonal antibody (mAb) and were ubiquitously expressed

+ +

on blood cells including CD8␣ and CD4 lymphocytes. gTecrem expression on leucocyte surface was

CD46-like protein

T-cell enhanced after stimulation with the T-cell mitogen, phytohaemagglutinin (PHA). Coculture with the

Teleost anti-Tecrem mAb signiﬁcantly inhibited the proliferative activity of PHA-stimulated peripheral blood

Ginbuna crucian carp lymphocytes, suggesting that cross-linking of Tecrems on T-cells interferes with a signal transduction

PHA

pathway for T-cell activation. These ﬁndings indicate that Tecrem may act as a T-cell moderator and

imply that the complement system in teleost, as well as mammals, plays an important role for linking

adaptive and innate immunity.

1. Introduction T-cell immunity have been documented (Russell, 2004; Liszewski

et al., 2005; Heeger and Kemper, 2012; Kemper and Kohl, 2013;

Complements have long been considered vital only to innate Yamamoto et al., 2013). Binding of CD46 ligands triggers alterations

immunity, without any role in the adaptive immune responses, in cytokine, chemokine (Vaknin-Dembinsky et al., 2008; Cardone

until approximately 40 years ago. From the early 1970s, growing et al., 2010) and nitric oxide production in T-cells, proliferation of T-

evidence in mammals has suggested that complements may be cells when cross-linked with CD3 (Zaffran et al., 2001; Astier et al.,

involved in the instruction and regulation of adaptive immunity, 2006), isotype switching in cooperation with interleukin (IL)-4 and

especially in B-cell-mediated antibody responses (Carroll, 2004; alterations in T-cell morphology (Marie et al., 2002). Furthermore,

Carroll and Isenman, 2012). In addition, several researches have some recent studies have shown that CD46 is a potent costimulator

shown the involvement of the complement in the regulation of for the induction of interferon-␥ (IFN-␥)-secreting effector T helper

T-cell responses (Astier et al., 2000; Carroll, 2004; Longhi et al., type 1 (Th1) cells and their subsequent switch into IL-10-producing

2006; Morgan et al., 2005; Kemper and Atkinson, 2007; Heeger and regulatory T cells (Cope et al., 2011; Le Friec et al., 2012; Ghannam

Kemper, 2012; Clarke and Tenner, 2014). et al., 2014).

In recent years, multiple functions of CD46, a membrane-bound In teleost, a CD46-like molecule, termed Tecrem, has been

regulator of complement activation (RCA) protein in mammals, on recently identiﬁed in common carp and was shown to function as

a complement regulator (Tsujikura et al., 2015). However, it is still

unclear whether Tecrem is involved in T-cell activation or regula-

∗ tion. The ginbuna crucian carp has been shown to be an ideal model

Corresponding author. Fax: +81 92 642 2897.

ﬁsh for comparative immunology research, especially for studying

E-mail address: [email protected] (T. Somamoto).

http://dx.doi.org/10.1016/j.molimm.2015.11.010

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2 I. Nur et al. / Molecular Immunology 70 (2016) 1–7

T-cell-mediated immunity, because of the availability of clonal ﬁsh IgG (diluted 1/100, Abcam). The cells were then washed three times

strains and antibodies speciﬁc for T-cell subset markers (Nakanishi with medium. The expression of these cell surface proteins in the

et al., 2011; Somamoto et al., 2013, 2014a,b, 2015). In a previous lymphocyte fraction was analysed by ﬂow cytometry, as described

study, we found three isoforms of membrane-bound RCA protein earlier. It was conﬁrmed that these secondary antibodies did not

termed ginbuna Tecrem (gTecrem-1, gTecrem-2 and gTecrem-3) and cross-react with rat or mouse IgG.

characterised at the mRNA level in ginbuna crucian carp (Nur et al.,

2013). gTecrem-1, in particular, has a tyrosine phosphorylation site 2.4. Immunoprecipitation of Tecrem protein on cell surface

in its cytoplasmic tail and is expressed in T-cell subsets. These

evidences suggest that gTecrem-1 possesses functions modulating Erythrocytes were puriﬁed using a Histopaque gradient, as

T-cell activation or/and regulation. described earlier. The puriﬁed erythrocytes were washed twice

The monoclonal antibody (mAb) speciﬁc for common carp with phosphate-buffered saline (PBS) and then adjusted to a den-

Tecrem (cTecrem) has recently been established using cTecrem- sity of 10 cells/ml in PBS, to be used for biotinylation of cell surface

expressing CHO cells as an immunogen (Tsujikura et al., 2015). proteins with 10 mM sulfo-NHSBiotin (Pierce), according to the

Because common carp and ginbuna crucian carp are close species, manufacturer’s instructions. After washing three times with PBS,

the mAb-recognising cTecrem is expected to be cross-reactive to the biotinylated cells were lysed and sonicated using 1 ml of NP40

gTecrem. In this study, we ﬁrst conﬁrmed the cross-reactivity of lysis buffer (1% Nonidet P40, 10 mM Tris–HCl pH 7.4, 150 mM NaCl,

the mAb against gTecrem. Furthermore, to learn whether gTecrem is 10 mM NaF, 0.5 mM EDTA and 1 mM PMSF) to each millilitre of

involved in T-cell immunomodulation, the function of gTecrem pro- cell suspension. The cell lysates were centrifuged, and supernatants

teins after stimulation with a T-cell mitogen, phytohaemagglutinin were used in the immune precipitation assay using 1F12 (5 ␮g/ml of

(PHA), has been analysed using the mAb. the cell lysate) that was coupled to the swollen protein G Sepharose

4 Fast Flow beads (GE Life Sciences) according to the manufacturer’s

2. Materials and methods instructions. The beads were incubated with 1 SDS–PAGE sample

buffer and boiled for 3 min; then, the supernatant obtained was

2.1. Animals directly loaded on to 12% SDS-polyacrylamide gel under reducing

conditions. The proteins in the gel were electro-transferred onto

Clonal triploid ginbuna crucian carp (S3n strain), weighing nitrocellulose membranes, treated with avidin and peroxidase-

approximately 40–120 g, was obtained from the National Research conjugated biotin (Vectastain ABC kit; Vector Laboratories) and

Institute of Aquaculture and maintained in a circulating water tank then visualised using 3,3 -diaminobenzidine (DAB).

◦

at 24 C, fed with commercial diets at Kyushu University.

2.5. Proliferation assay of PBLs after PHA-stimulation

2.2. Blood cell separation

PBLs (3 × 10 cells) in 100 ␮l of RPMI 1640 medium supple-

Erythrocytes and leucocytes were isolated using a Histopaque mented with 2% ginbuna serum were seeded in each well of 96-well

(d = 1.083, Sigma–Aldrich) gradient method as previously described ﬂat-bottomed microtitre plates (Nunc, Denmark). These PBLs were

␮

(Nur et al., 2013). Cell suspensions were applied to Histopaque then stimulated by the addition of PHA 10 g/ml (Sigma–Aldrich

and centrifuged for 30 min at 380 × g to separate erythrocytes and Japan). Unstimulated PBLs served as the negative control. Then,

◦

leucocytes. Leucocytes on the Histopaque were collected, and ery- cells were incubated at 25 C in a 5% CO2 humidiﬁed incubator

throcytes below the Histopaque were harvested. Separated cells for 72 h. Thereafter, the cells were subjected to a colorimetric MTT

␮

were washed twice with RPMI 1640 (Nissui Pharmaceutical) and assay for cell proliferation assessment. Brieﬂy, 10- l MTT solution

adjusted to suitable concentration in each experiment. (5 mg/ml in PBS) was added directly to each well and incubated

◦

for 4 h in the dark at 37 C. The cells were then solubilised with

2.3. Detection of Tecrem protein on blood cells by ﬂow cytometry 100 ␮l of acidic isopropanol (0.1 N HCl in absolute isopropanol)

and incubated overnight at room temperature. The absorbance was

PBLs and erythrocytes (1–5 10 cells/mL) were incubated in measured at 590 nm using a microplate plate reader (ImmunoMini

the presence of 5% foetal bovine serum (FBS) on ice with 1/10 NJ-2300; Nunc Japan, Tokyo, Japan).

diluted hybridoma supernatant of anti-Tecrem mAb (1F12, isotype

IgG1) (Tsujikura et al., 2015) for 45 min, washed twice with RPMI 2.6. Detection of Tecrem protein on PHA-stimulated PBLs

1640 and incubated with ﬂuorescein isothiocyanate (FITC)-labelled

goat anti-mouse IgG (diluted 1/100, Sigma Japan) on ice for 1 h. The The PBL suspension in RPMI 1640 medium with 5% FBS was

cells were then washed twice with RPMI 1640. Cells without anti- adjusted to 1.5 × 10 cells/ml and then stimulated by adding

Tecrem mAb treatment were used as negative controls. Expressions 10 ␮g/ml PHA. Non-stimulated PBLs served as the control. The sus-

of Tecrem were analysed with a Beckman Coulter Epics XL Flow pension (0.5 ml) was seeded onto a 48-well plate and incubated

◦

Cytometer equipped with System II software (Beckman Coulter). for 5 h at 25 C in a humidiﬁed air containing 5% CO2. Cells were

The mean ﬂuorescent intensity of Tecrem was normalised by that then collected, washed twice with RPMI 1640 and treated with

of the negative control and expressed as the mean ﬂuorescence anti-Tecrem mAbs and FITC-labelled second antibody, as described

intensity rate (MFIR). earlier. The mean ﬂuorescent intensity of Tecrem was normalised

+ +

The expression of Tecrem protein on CD4 and CD8␣ T cells by that of the negative control and expressed as the MFIR.

was analysed by two-colour immunoﬂuorescence using anti-CD4

and anti-CD8␣ mAbs (mouse ascites: 6D1 and 2C3) (Toda et al., 2.7. Effects of anti-Tecrem mAb on proliferation triggered by PHA

2011). PBLs were ﬁrst incubated with 1F12 mAb for 30 min, washed

twice with RPMI 1640 and then incubated with Alexa Fluor 488- PBLs were cultured in RPMI 1640 medium in 96-well ﬂat-

labelled goat anti-mouse immunoglobulin G (IgG; diluted 1/500, bottomed microtitre plates (Nunc, Denmark) supplemented with

Life technology) on ice for 30 min. After washing twice, the cell 2% ginbuna serum and 10 ␮g/ml PHA, in the presence of 5, 10 or

suspension was equally divided, and the portions were incubated 15 ␮g/ml of puriﬁed anti-Tecrem IgG or 15 ␮g/ml of an isotype-

with 6D1 or 2C3 mAb for 30 min. Each labelled cell was washed matched irrelevant antibody (normal mouse IgG1). PHA stimulated

twice with medium and incubated with PE-labelled goat anti-rat with PBLs without anti-Tecrem was used as the control. Then, cells

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I. Nur et al. / Molecular Immunology 70 (2016) 1–7 3

Fig. 1. Tecrem expression on leucocyte and erythrocyte cell surfaces. Flow cytometry histogram (A) and mean ﬂuorescence intensity ratio (MFIR) (B) are shown. Leucocytes

and erythrocytes were incubated with or without anti-Tecrem mAb as the experiment and negative control, respectively. Grey or white peaks represent anti-Tecrem mAb-

treated or negative control samples, respectively. Three individual samples were independently analysed, and representative data are shown (A). Error bars indicate the

standard deviation (SD) (B).

◦

were incubated under standard culture conditions (25 C, 5% CO2 3. Results

and humidiﬁed air) for 72 h. Proliferation was measured by an MTT

assay, as described earlier. 3.1. Expression of Tecrem on surface of PBLs, erythrocytes and + +

CD4 and CD8˛ lymphocytes

2.8. Statistical analysis

Flow cytometric analysis showed that almost all cells in both

leucocytes and erythrocytes are Tecrem positive, indicating that

All experiments were repeated at least three times. Results were

the intrinsic cell surface complement regulator is present on blood

expressed as the mean ± standard deviation. Statistical differences

cells (Fig. 1A). The MFIR showed no signiﬁcant differences of Tecrem

were assessed for more than two groups with one-way analysis of

expression patterns between the erythrocyte and the leucocyte

variance (ANOVA) followed by Dunnett test or were analysed by

(Fig. 1B). Double staining with T-cell subset markers and Tecrem

paired or unpaired Student’s t -test for comparison between the + +

showed that more than 98% of CD8␣ and CD4 lymphocytes are

two groups. The signiﬁcance level was set at 0.05.

+ +

Fig. 2. Tecrem expression on CD8␣ and CD4 lymphocytes. Peripheral lymphocytes were gated on FS and SS dot plots, and they were analysed for two colours staining

with anti-Tecrem mAbs and anti-CD8␣ or anti-CD4 mAb. Three individual samples were independently analysed, and representative data are shown. Numbers indicate

percentages of positive cells within each region.

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4 I. Nur et al. / Molecular Immunology 70 (2016) 1–7

0.8 * 0.7 0.6 0 nm)

59 0.5 (A

ce 0.4 0.3 0.2 Absorban 0.1 0

Control PHA-stimulated PBL

Fig. 4. Proliferative response of ginbuna PBLs stimulated with PHA. Data are shown

as mean ± SD of four individual ﬁsh as the absorbance at 590 nm measured by an

MTT assay. The difference was considered signiﬁcant at P < 0.05.

3.3. Increase of gTecrem expression on PBLs after stimulation

with PHA

PHA-stimulated cell proliferation was examined using ginbuna

PBLs, which include T-cells. As shown in Fig. 4, absorbance of PHA-

stimulated PBLs is signiﬁcantly higher than that of non-stimulated

PBLs. This result indicated that T cells in the PBLs proliferated by

the activation trigger with the PHA.

Cell surface expression of Tecrem on PBLs was measured after

stimulation with PHA. Tecrem expression on PHA-stimulated PBLs

appeared stronger than that on non-stimulated PBL (Fig. 5A). The

MFIR of PHA-stimulated PBLs is signiﬁcantly higher (P < 0.05) than

that of non-stimulated PBLs (Fig. 5B). These results indicated that

increased Tecrem expression on PBLs is induced following stimu-

lation with PHA, a T-cell mitogen.

3.4. Effect of anti-Tecrem mAb on PBL activation triggered by PHA

To examine the functional relationship with the Tecrem expres-

sion and PHA-triggered activation of T-cells, the effect of the

anti-Tecrem mAb on PHA-stimulated T-cell proliferation was

tested using puriﬁed 1F12 mAb. As shown in Fig. 6, the forma-

tion of MTT-derived formazan dye on PHA-stimulated PBLs with

Fig. 3. Immunoprecipitation assay using anti-Tecrem mAb to ginbuna erythrocytes.

␮

Biotinylated cell-surface molecular binding with the mAb was detected using avidin, the highest dose of anti-Tecrem mAb (15 g/ml) is signiﬁcantly

peroxidase-conjugated biotin and 3,3 -diaminobenzidine. The molecular mass val- lower than that without the antibody, although the lower mAb dose

ues (kDa) are shown to the left. The arrows indicate detected proteins.

only affected dye formation marginally. The inhibition by the mAb

occurred in a dose-dependent manner. Isotype control of normal

IgG1 did not inhibit the formation of formazan dye. These results

indicate that the blocking of Tecrem molecules on T-cells inhibits

PHA-stimulated T-cell proliferation.

Tecrem positive (Fig. 2A and B). Thus, it was conﬁrmed that most

+ + Furthermore, the effect of 1F12 mAb on the formation of cell

CD8␣ and CD4 T cells of ginbuna carp constitutively express

Tecrem. aggregation during PBL culture in the presence of PHA was observed

under a microscope (Fig. 7). The appearance of larger cell aggregates

was noticed in the presence of anti-Tecrem mAb, whereas only

small cell aggregates were observed in PBLs cultured with control

3.2. Detection of gTecrem protein on erythrocytes by IgG1 and without antibody. immunoprecipitation

4. Discussion

An immunoprecipitation assay of erythrocytes with anti-

Tecrem mAb showed two bands, detected at approximately 50 and

Membrane-bound complement regulatory protein CD46 acts

70 kDa (Fig. 3). The theoretical molecular masses of three gTecrem

as a key sensor of immune activation and an important modula-

isoforms are 35.1 and 36.5 kDa (short-form isotype, gTecrem-2 and

tor of T-cell immunity in mammals (Heeger and Kemper, 2012).

gTecrem-3) and 56.7 kDa (long-form isotype, gTecrem-1) (Nur et al.,

Although a previous study has shown that the teleost CD46-like

2013). As reported for the cTecrem (Tsujikura et al., 2015), the differ-

molecule, Tecrem, functions as a complement regulator (Tsujikura

ence in the observed molecular mass and theoretical value suggests

et al., 2015), it remains unclear whether Tecrem plays a role in the

that gTecrem proteins are heavily O-glycosylated at the Ser/Thr/Pro

activation or regulation of T-cells. The present study focuses on the

(STP)-rich region region. Therefore, the result suggests that 1F12

function of Tecrem on T-cell response using anti-Tecrem mAb in

recognises both long and short forms of the gTecrem protein.

ginbuna crucian carp.

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I. Nur et al. / Molecular Immunology 70 (2016) 1–7 5

Fig. 5. Tecrem expression on PHA-stimulated PBLs. Flow cytometry histogram (A) and mean ﬂuorescence intensity ratio (MFIR) (B) are shown. Non-stimulated or PHA-

stimulated leucocytes were incubated with or without anti-Tecrem mAb as the study and negative control, respectively. Grey or white peaks represent anti-Tecrem mAb

treated or negative control samples, respectively. Three individual samples were independently analysed, and representative data are shown (A). Error bars indicate SD (B).

The difference was considered signiﬁcant at P < 0.05.

Three different sequences of Tecrem (gTecrem-1, gTecrem-2 0.7

and gTecrem-3) have been previously found in ginbuna crucian

carp (Nur et al., 2013). A notable difference among their primary 0.65

structure is the number of SCR modules, which are composed

of extracellular domains. Similar to Tecrem from the common 0.6 *

carp, gTecrem-2 and gTecem-3 comprise four SCRs. gTecrem-1 is

(A590 nm)

0.55

a unique isoform of the ginbuna carp and consists of seven SCRs. ce

As gTecrem-1 possesses a tyrosine phosphorylation site in its cyto-

+ 0.5

plasmic region and its mRNA was strongly expressed in CD4 and +

Absorban

CD8 T cells, it presumably has roles in T-cell moderation. 1F12

0.45

mAb recognises 50- and 70-kDa proteins of ginbuna erythrocytes,

suggesting that it detects both long (gTecrem-1) and short (gTecrem-

0.4

2 and gTecrem-3) forms of gTecrem . This ﬁnding is reasonable,

0 5 10 15 15

as cTecrem includes four SCR domains that are common with all

gTecrems and was used as an immunogen for the establishment of Anti -Tecrem Normal

1F12 (Tsujikura et al., 2015).

The present study showed that the gTecrem protein was Concentration of IgG ( g/mL)

+ +

expressed on almost all cells, including CD4 and CD8 T cells.

Fig. 6. Inhibitory effect of anti-Tecrem mAb on the proliferation of PBLs. PBLs were

Previous study has already shown that leucocytes and erythro-

stimulated with PHA and with added anti-Tecrem IgG1, normal mouse IgG1 or PHA

cytes express Tecrem mRNA in ginbuna crucian carp (Nur et al.,

alone. Data are shown as mean ± SD of six individual ﬁsh as the absorbance at 590 nm

2013). Thus, the ubiquitous expression of the gTecrem protein on

measured by MTT assay. The level of signiﬁcance between control (0 ␮g/ml) was set

blood cells is consistent with the results of expression analyses at at P < 0.01 (*).

the mRNA. It is interesting to note that gTecrem expressions were

clearly detected on both erythrocytes and leucocytes. This result

was in contrast to the expression pattern of human CD46, which

is yet to be determined which cell types elevate gTecrem expres-

is not expressed on erythrocytes (Liszewski and Atkinson, 1991;

sion in response to PHA stimulation; however, a large proportion

Johnstone et al., 1993). Roles of tyrosine phosphorylation motif in

of PBLs showed high levels of gTecrem expression. This ﬁnding indi-

gTecrem-1 on erythrocytes are still unclear, although the motif is

cates that Tecrem expression increases not only on T-cells but also

thought to induce cell activations. Ohashi et al. (2010) reported that

on other leucocytes and implies that the activated T-cells synergis-

teleost G6F-like molecule, which possesses an immunoreceptor

tically promote increased Tecrem expression on other leucocytes.

tyrosine-based activation motif (ITAM), is expressed on erythro-

In humans, polyclonal T-cell stimulation by anti-CD3 antibody and

cyte, and speculated that it may have an active role in hemostasis.

costimulation with CD46 leads a synergistic activation of extra-

Thus, participation in hemostatic cascade may be one of possi-

cellular signal-related kinase mitogen-activated protein kinase

ble role of gTecrem-1 on erythrocytes. It has been reported that

+ (Zoffran et al., 2002). The cross-linking by anti-CD3 and anti-CD46

engagement of CD46 by its ligand C3b on CD4 T cells is required

antibodies induces morphological changes and aggregations of T-

for IFN-␥ production (Cardone et al., 2010). FCM analysis showed

+ cells. In the present study, anti-Tecrem IgG inhibited proliferation

that almost all CD4 T-cells express Tecrem, suggesting that Tecrem

by PHA stimulation, implying that blocking of Tecrem interferes

has the potential to stimulate T cells like CD46 in mammals. To

with a pathway of the signal transduction of T cells. In addition,

further understand the role of complements in T-cell immunity,

large cell aggregates were observed in the culture with anti-Tecrem

studies should focus on the costimulatory function of gTecrem on

IgG and PHA, suggesting that the binding of the mAb to Tecrem

CD4+ cells.

enhances cell–cell contacts. Therefore, the binding of the mAb

As in mammals, PHA is known to be a T-cell mitogen in many ﬁsh

would trigger cross-linking of T-cells, but it does not function as

species (Scapigliati, 2013). gTecrem expression on PHA-stimulated

a costimulation of T cells. Although the cause of the inhibition of

PBLs was higher than that on PBLs without PHA stimulation, indi-

the mitogenic effect remains unclear, these ﬁndings provide strong

cating that gTecrem expression is upregulated by PBL activation. It

evidence that Tecrem is involved in T-cell immunity.

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6 I. Nur et al. / Molecular Immunology 70 (2016) 1–7

Fig. 7. Microscopic observation of PHA-stimulated PBLs in the presence of anti-Tecrem IgG. PBLs were cultured with PHA and various concentrations of anti-Tecrem IgG1

(A = 0; B = 5; C = 10; D = 15 ␮g/ml) and 15 ␮g/ml of normal mouse Tecrem IgG1 (E).

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