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(Journal of Leukocyte Biology. 2002;72:486-491.)
© 2002 by Society for Leukocyte Biology

Interferon-{gamma} stimulates the expression of galectin-9 in cultured human endothelial cells

Tadaatsu Imaizumi*, Mika Kumagai*, Naoko Sasaki*, Hidekachi Kurotaki{dagger}, Fumiaki Mori{ddagger}, Masako Seki§, Nozomu Nishi||, Koji Fujimoto*, Kunikazu Tanji{ddagger}, Takeo Shibata*, Wakako Tamo*, Tomoh Matsumiya#, Hidemi Yoshida*, Xue-Fan Cui*, Shingo Takanashi**, Katsumi Hanada{dagger}{dagger}, Ken Okumura**, Soroku Yagihashi{dagger}, Koichi Wakabayashi{ddagger}, Takanori Nakamura||, Mitsuomi Hirashima§ and Kei Satoh*

Departments of
* Vascular Biology,
{ddagger} Molecular Biology, Institute of Brain Science,
{dagger} Pathology,
# Dentistry and Oral Surgery,
** the Second Department of Internal Medicine, and
{dagger}{dagger} Dermatology, Hirosaki University School of Medicine, Japan; and Departments of
§ Immunology and Immunopathology and
|| Endocrinology, Kagawa Medical School, Japan

Correspondence: Tadaatsu Imaizumi, M.D., Department of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. E-mail: timaizum{at}cc.hirosaki-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Galectin-9 is a member of the galectin family and has been identified as an eosinophil chemoattractant produced by activated T lymphocytes. Vascular endothelial cells play an important role in the initial step of eosinophil recruitment and activation in immune and inflammatory responses. We have addressed the stimulation of galectin-9 expression in endothelial cells. Galectin-9 was detected in membrane and cytosolic fractions of human umbilical vein endothelial cells stimulated with interferon-{gamma} (IFN-{gamma}). IFN-{gamma} also enhanced the adhesion of human eosinophilic leukemia-1 cells to endothelial monolayers, and it was inhibited by the presence of lactose. Interleukin-4, which induces eotaxin expression, did not affect the expression of galectin-9. The in situ endothelium from patients with inflammatory diseases was found to express galectin-9. IFN-{gamma}-induced production of galectin-9 by endothelial cells may play an important role in immune responses by regulating interactions between the vascular wall and eosinophils.

Key Words: eosinophils • adhesion • cytokine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Galectins are a family of animal lectins that have affinity for ß-galactosides and have a conserved, specific sequence motif of carbohydrate-binding domains [1 ]. Each member of the family has a specific function, and galectin-9 was isolated as a potent chemoattractant for eosinophils from activated T cells [2 3 4 ]. Therefore, galectin-9 is considered to constitute part of the molecular control on eosinophil traffic and play a key role in immune responses and allergic reactions. It has two distinct N- and C-terminal carbohydrate-binding domains connected by a link peptide, and both domains are essential for eosinophil-chemoattractant activity [5 ]. There are three isoforms of galectin-9: short type of 311 amino acids, medium type of 323 amino acids, and long type of 355 amino acids [4 ].

Galectin-9 was isolated from an activated T cell line [2 ], mouse embryonic kidney [6 ], and the tissue affected by Hodgkin’s disease [7 ]. Monocytes/macrophages, Jurkat, THP-1, and RPMI-8866 cells are also known to produce this eosinophil chemoattractant [4 ]. In mouse, galectin-9 was found to widely distribute in the liver, small intestine, thymus, kidney, spleen, lung, etc. [6 , 8 ], and Wada et al. [9 ] showed that galectin-9 immunoreactivity was detected in blood vessels of rat kidney. However, there is no information about the mechanism of regulation and functional significance of its expression in the vascular wall.

Interferon-{gamma} (IFN-{gamma}) is one of the most pivotal cytokines that regulates immune responses [10 ]. It activates various functions of endothelial cells and is known to up-regulate the expressions of chemokines such as IFN-inducible protein-10 (IP-10) [11 ] and fractalkine [12 ]. This study was undertaken to examine the expression of galectin-9 in cultured endothelial cells stimulated with IFN-{gamma}. It is known that endothelial cells produce eotaxin [13 , 14 ], another agonist for eosinophils, in response to interleukin-4 (IL-4), and a possible effect on eotaxin expression was also studied. We also addressed in vivo expression of galectin-9 by examining biopsied human tissues for the immunoreactivity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Cell culture medium Humedia EB-2 and its supplements were from Kurabo (Osaka, Japan). Fetal bovine serum (FBS), Medium 199 (M199), dibutyryl-cyclic adenosine monophosphate (cAMP), primer oligo(dT)12–18, and M-Mulv reverse transcriptase (RT) were from Gibco-BRL (Gaithersburg, MD). An RNeasy total RNA isolation kit and Taq DNA polymerase were from Qiagen (Hilden, Germany). Recombinant human [r(h)]IFN-{gamma} was from Roche Boehringer Mannheim (Germany). r(h)IL-4 was from R&D Systems (Minneapolis, MN). Antigalectin-9 immunoglobulin G (IgG) was generated in a rabbit by immunization with a C-terminal peptide and was affinity-purified [2 ]. A chemiluminescent substrate for Western blotting (SuperSignal West Pico) was from Pierce (Rockford, IL). Oligonucleotide primers for polymerase chain reaction (PCR) were synthesized by Greiner Japan (Atsugi).

Cell culture
Human umbilical vein endothelial cells (HUVEC) were isolated using collagenase and were cultured in gelatin-coated plates as described [15 16 17 ] with slight modifications. HUVEC were cultured in Humedia EB-2 supplemented with 2% FBS, 10 ng/ml r(h) epidermal growth factor, 1 µg/ml hydrocortisone, 5 ng/ml r(h) basic fibroblast growth factor, and 10 µg/ml heparin. When the cells reached about 80% confluence, the medium was replaced with Humedia EB-2 containing only 20% human serum (Humedia-HS). The tightly confluent monolayers of first to fifth passage were used for the experiments. The primary cultures contained <1% CD45+ cells, but no CD45+ cells were found after first passage. HUVEC were stimulated for the indicated time periods by incubating in Humedia-HS containing IFN-{gamma} or IL-4.

Human eosinophilic leukemia (EoL)-1 cells were cultured using RPMI 1640 supplemented with 10% FBS.

RNA extraction, RT-PCR
Total RNA was extracted from HUVEC using an RNeasy total RNA isolation kit. Single-strand cDNA for a PCR template was synthesized from 1 µg total RNA using a primer oligo(dT)12–18 and M-Mulv RT. Specific primers were designed from cDNA sequences for galectin-9, eotaxin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and each cDNA was amplified by PCR using Taq DNA polymerase. The sequences of the primers were as follows:

galectin-9-F (5'-GAGATGGCCTTCAGCAGTTCC-3'),
galectin-9-R (5'-CGCCTATGTCTGCACATGGGT-3'),
eotaxin-F1 (5'-TCACGCCAAAGCTCACACCT-3'),
eotaxin-F2 (5'-CCCAACCACCTGCTGCTTTAACCTG-3'),
eotaxin-R1 (5'-TTATGGCTTTGGAGTTGGAGAT-3'),
eotaxin-R2 (5'-TGGCTTTGGAGTTGGAGATTTTTGG-3'),
GAPDH-F (5'-CCACCCATGGCAAATTCCATGGCA-3'),
and GAPDH-R (5'-TCTAGACGGCAGGTCAGGTCCACC-3').

The reaction condition for galectin-9 was 1x (94°C, 1 min), 26x (94°C, 1 min; 62°C, 1 min; 72°C, 1 min), and 1x (72°C, 10 min). As the expression of eotaxin mRNA was low, eotaxin cDNA was amplified by nested PCR. The first-round PCR was performed using the primers eotaxin-F1 and eotaxin-R1, and subsequently 5 µl 1/20 dilution of the first-round product was subjected to the second-round amplification using the primer set of eotaxin-F2 and eotaxin-R2. The condition for the first-round amplification was 1x (94°C, 1 min) and 30x (94°C, 1 min; 55°C, 1 min; 72°C, 1 min), and the second-round amplification was 15x (94°C, 1 min; 59°C, 1 min; 72°C, 1 min) and 1x (72°C, 10 min). The condition for GAPDH was 1x (94°C, 1 min), 30x (94°C, 1 min; 55°C, 1 min; 72°C, 1 min), and 1x (72°C, 10 min).

The products were analyzed by electrophoresis on an agarose gel (1% for galectin-9 and GAPDH or 2% for eotaxin) containing ethidium bromide. There are three isoforms of galectin-9 according to the length of the linker peptide [4 ]: The short-, the medium-, and the long-sized galectin-9 have 311, 323, and 355 amino acids, respectively. The primers for galectin-9 were designed to identify the mRNA for these isoforms. The expected sizes for the PCR products for short, medium, and long isoforms of galectin-9, eotaxin, and GAPDH were 942 bp, 978 bp, 1074 bp, 208 bp, and 696 bp, respectively. As all of these primer pairs were designed from different exons, the products with the expected size were amplified from single-strand cDNA but not from contaminating genomic DNA. The PCR products were confirmed to be specific for each cDNA by sequencing.

Western blot
Cells were washed twice with cold 20 mM phosphate-buffered saline (PBS), pH 7.4, and lysed with Laemmli’s reducing sample buffer. The lysate was subjected to electrophoresis on a 4–20% polyacrylamide gel, and the proteins were transferred to a polyvinylidene difluoride membrane. The membrane was incubated with rabbit antigalectin-9 IgG (0.2 µg/ml) and then with anti-rabbit IgG labeled with horseradish peroxidase. Immunodetection was performed using a chemiluminescent substrate.

To determine the subcellular localization of gelectin-9, cells were homogenized and subfractionated. Cells were scraped in PBS containing 0.01% protease-inhibitor cocktail and were sonicated. The homogenates were centrifuged at 105,000 g for 60 min, and the supernatant was designated as the cytosolic fraction. The pellet, the membrane fraction, was lysed with Laemmli’s reducing buffer. Both fractions were subjected to Western blotting as described above.

Adhesion assay
Eosinophil adhesion assay was performed as described previously [12 , 18 , 19 ] with slight modifications. EoL-1 cells were differentiated to eosinophils by the treatment with 0.1 mM dibutyryl-cAMP for 3 days before the experiments. Then the cells were suspended in M199 containing 5% FBS (M199-FBS). Monolayers of HUVEC grown to confluence in a six-well plate were stimulated with 10 ng/ml IFN-{gamma} for 24 h and washed twice. Then the HUVEC were incubated at 37°C for 1 h with 50 µg/ml antigalectin-9 antibody or a control antibody in M199-FBS. The medium was removed, and HUVEC were incubated for 15 min with suspension of differentiated EoL-1 cells (2x106 cells/600 µl/well). Nonadherent cells were removed, and the cells were gently washed twice with M199. Adherent cells in seven random fields were counted. The effects of lactose and sucrose on the adherence were also examined, and HUVEC were incubated with the EoL-1 suspension in the presence of 20 mM lactose or sucrose. Statistical analyses involving multiple comparisons were performed using an ANOVA followed by a Fisher’s Protected Least Significant Difference test.

Immunohistochemistry
Cellular distribution of galectin-9 in human tissues was examined by immunohistochemical staining of a nasal polyp excised from a patient with allergic rhinitis and a skin biopsy specimen from a patient with Sjögren syndrome. Tissue sections mounted on aminopropyltriethoxysilane-coated glass slides were deparafinized, rehydrated, and boiled by microwave irradiation in 0.01 M citrate buffer (pH 6.0) for antigen retrieval. The slides were immersed for 30 min in methanol containing 0.3% hydrogen peroxide to block endogenous peroxidase activity. The slides were incubated with 1% goat serum for 30 min at room temperature and then with an antigalectin-9 antibody (1:1000) or a nonimmune control antibody at 4°C in a moist chamber. After overnight incubation, samples were incubated with biotinylated anti-rabbit IgG and peroxidase-conjugated streptavidin. The peroxidase reaction was performed using a 3'-diaminobenzidine tetrahydrochloride/H2O2 solution. The nuclei of the nasal polyp tissue were counterstained with 1% methyl green solution buffered with veronal acetate.

We next performed the immunofluorescent staining of galectin-9 in HUVEC. The cells were stimulated with 10 ng/ml IFN-{gamma} for 24 h, and were fixed with 10% formaldehyde. The cells were incubated with normal goat serum/Superblock (1:1) and then with 10 µg/ml antigalectin-9 antibody or a control antibody. After washing with PBS, the cells were incubated with anti-rabbit IgG antibody labeled with biotin, followed by incubation with streptavidin-fluorescein isothiocyanate (FITC). The cells were examined by a laser confocal microscope (LSM 410; Carl Zeiss, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{gamma} enhances galectin-9 expression in HUVEC
RT-PCR analysis of galectin-9 mRNA in HUVEC is shown in Figure 1 A . A faint band of 978-bp cDNA, which corresponds to the medium type, was detected in unstimulated cells. IFN-{gamma} enhanced the expression of the medium and long-type galectin-9 in a concentration-dependent manner, and the band for the medium type was always more intense than that for the long type. The cDNA for the short-type galectin-9 was not detected.



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  		Figure 1. Expression of galectin-9 in HUVEC stimulated with IFN-{gamma}. (A) Concentration-dependence of the galectin-9 mRNA expression in HUVEC stimulated with IFN-{gamma}. HUVEC were stimulated with IFN-{gamma} for 24 h, and the cells were subjected to total RNA extraction. Single-strand cDNA was synthesized from 1 µg total RNA, and the specific cDNAs for galectin-9 and GAPDH were amplified by PCR. (B) Concentration-dependent expression of galectin-9 protein in HUVEC stimulated with IFN-{gamma}. After the stimulation, the cells were washed twice with cold PBS and lysed. Western blot analysis for galectin-9 protein was performed.

 
Western blot analysis of galectin-9 expression is shown in Figure 1B . IFN-{gamma} also increased galectin-9 protein expression in a concentration-dependent manner. The size of the band was 36K and consistent with the medium-type galectin-9.

Time-dependent expression of galectin-9 mRNA is shown in Figure 2 A . Galectin-9 mRNA reached a maximal level 24 h after the stimulation with 10 ng/ml IFN-{gamma} and decreased thereafter. Figure 2B shows the time-dependent production of galectin-9 protein, and it agreed with the time course of mRNA expression.



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  		Figure 2. Time course of the expression of galectin-9 in HUVEC stimulated with IFN-{gamma} (A) HUVEC were treated with 10 ng/ml IFN-{gamma} for up to 72 h, and total RNA was extracted. RT-PCR analysis was performed as described in Figure 1 . (B) Western blot analysis for galectin-9 was performed as described in Figure 1 .

 
Subcellular distribution of galectin-9 protein is shown in Figure 3 . Galectin-9 was not detected in extracellular medium even in a 50-fold concentrate, and it was found in the membrane and cytosolic fractions.



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  		Figure 3. Expression of galectin-9 in cytosolic and membrane fractions of HUVEC stimulated with IFN-{gamma}. HUVEC were incubated with 10 ng/ml IFN-{gamma} for 24 h. The cells were homogenized by sonication, and cytosolic and membrane fractions were separated by cetrifugation (105,000 g). Western blot analysis for galectin-9 was performed as described in Figure 1 .

 
Effect of IL-4 on galectin-9 and eotaxin expression in HUVEC
IL-4 did not induce the expression of galectin-9 nor did it affect the up-regulation of galectin-9 in response to IFN-{gamma} (Fig. 4 ). Figure 4 also shows that IFN-{gamma} by itself did not stimulate eotaxin expression but inhibited the IL-4-induced expression almost completely.



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  		Figure 4. Effect of IFN-{gamma} and IL-4 on the expression of galectin-9 and eotaxin. HUVEC were treated with 10 ng/ml IFN-{gamma} and/or 10 ng/ml IL-4 for 24 h. RT-PCR analysis of mRNAs for galectin-9 and eotaxin was performed as described in Figure 1 .

 
Eosinophil adhesion to HUVEC stimulated with IFN-{gamma}
Stimulation of HUVEC with IFN-{gamma} increased the adhesion of EoL-1 cells, which had been differentiated with dibutyryl-cAMP treatment (Fig. 5 ). The numbers of adherent cells to the control and IFN-{gamma}-stimulated HUVEC monolayers were 20 ± 9 and 96 ± 23 cells/field, respectively (mean±SD; n=7, P<0.01). Antigalectin-9 antibody reduced the number of adherent cells to 50 ± 17 cells/field (P<0.01 vs. HUVEC stimulated with IFN-{gamma}). The presence of 20 mM lactose reduced this adhesion (P<0.01), but 20 mM sucrose failed to inhibit the adhesion.



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  		Figure 5. Adhesion of EoL-1 cells to HUVEC. HUVEC were incubated with 10 ng/ml IFN-{gamma} for 24 h. After an additional 1-h incubation with antigalectin-9 antibody or a control antibody, the medium was removed and EoL-1 cells pretreated with dibutyryl-cAMP were added to a rinsed monolayer (2x106 cells/600 µl/well). After the incubation at 37°C for 15 min, nonadherent cells were removed and the cells were gently washed twice. The effects of lactose and sucrose, at the concentration of 20 mM, on the adherence of EoL-1 cells to the HUVEC monolayer were also examined. (A) The cells were photographed; original bar = 30 µm. (B) Adherent cells in seven random, low power fields were counted under a microscope. The value represents mean ± SD obtained from seven different fields. *, P < 0.01.

 
In vivo expression of galectin-9 in endothelial cells
Immunohistochemical identification of galectin-9 in tissues with inflammatory diseases is shown in Figure 6 . Vascular endothelial cells in a nasal polyp from a patient with allergic rhinitis were stained positively (Fig. 6A) , and only counterstained nuclei were identifiable in the control staining (Fig. 6B) . Endothelial cells showed intense immunoreactivity in a cutaneous tissue from a patient with Sjögren’s syndrome as well (Fig. 6C) . Galectin-9 immunoreactivity was also found in perivascular inflammatory cells (Fig. 6A and 6C) .



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  		Figure 6. Immunohistochemical detection of galectin-9 in vascular endothelial cells. Original bar = 30 µm. A nasal polyp excised from a patient with allergic rhinitis (A) and a biopsy specimen of skin from a patient with Sjögren’s syndrome (C) were stained with an antibody against galectin-9. (B and D) Controls for (A) and (C) stained using a nonimmune antibody instead of antigalectin-9 antibody. Nuclear counterstaining was performed in (A) and (B). Note that vascular linings were stained positively in (A) but not in (B).

 
Immunofluorescent staining for galectin-9 in HUVEC
Results of immunofluorescent staining for galectin-9 are shown in Figure 7 . HUVEC stimulated with IFN-{gamma} were positively stained for galectin-9, and the immunoreactivity was observed in cytoplasm and cell surface with regional punctate intensities.



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  		Figure 7. Morphological localization of galectin-9 in HUVEC. HUVEC were stimulated with 10 ng/ml IFN-{gamma} for 24 h and were subjected to immunofluorescent staining. Cells were fixed with 10% formaldehyde and incubated with a control antibody or an antigalectin-9 antibody, followed by the incubation with biotin-labeled anti-rabbit IgG antibody and FITC-streptavidin. Cells were observed using a laser confocal microscope. Original bar = 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Vascular endothelial cells play an important role in inflammatory and immune responses [20 , 21 ]. When endothelial cells are stimulated with IL-1 or bacterial lipopolysaccharide, blood cell tethering molecules such as E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 are expressed [22 ], and this is associated with simultaneous expression of various chemokines including IL-8 [21 ], neutrophil-activating peptide-78 [23 ], monocyte chemoattractant protein-1 [24 ], and eotaxin [13 ].

In the present study, we found that IFN-{gamma} stimulates the expression of galectin-9 in HUVEC. Immunohistochemical staining also revealed the expression of galectin-9 in vascular endothelial cells in human inflammatory lesions. IFN-{gamma} mediates a wide range of functions including antiviral, antiproliferative, anti-tumor, and immunomodulatory activities [10 ]. IFN-{gamma} is mainly produced by T-helper cell type 1 (Th1) lymphocytes, and vascular endothelial cells are one of the targets of this cytokine. It up-regulates the expression of IP-10 [11 ] and fractalkine [12 ] in HUVEC and may regulate the interactions between endothelial cells and leukocytes [12 ]. Galectin-9 may be regarded as one of such molecules mediating the interactions of eosinophils with the endothelium.

The two subtypes of helper T cells, Th1 and Th2, produce distinct profiles of cytokines [25 ]. Th1 lymphocytes are thought to be involved in host defense, and IFN-{gamma} is one of the major cytokines produced by this subtype. Th2 cells produce cytokines such as IL-4 and IL-5 and are involved in allergic reactions. It has been shown that Th2-type cytokine IL-4 induces eotaxin production, and Th1-derived cytokine IFN-{gamma} inhibits it [26 27 28 ]. The present study demonstrated the induction of galectin-9 by IFN-{gamma} and IL-4 was completely inactive in this aspect. Thus, the expression of these two eosinophil chemoattractants is differentially regulated, and the accumulation of eosinophils in tissue may be controlled by the balance between galectin-9 and eotaxin as well as the balance between Th1- and Th2-derived cytokines.

Galectin-9 in HUVEC stimulated with IFN-{gamma} was found in membrane and cytosolic fractions. We tried to detect galectin-9 in HUVEC-conditioned medium by Western blot analysis, but no immunoreactivity was found even after a 50-fold concentration. Eosinophil-chemotactic activity was negligible in the conditioned medium from IFN-{gamma}-stimulated HUVEC (data not shown). Activated T lymphocytes secrete galectin-9, a high level of cell-surface galectin-9 is detected on Jurkat T cells, and the majority is found in cytoplasm in THP-1 and RPMI-8866 cells [3 , 4 ]. The secretion and intracellular distribution of galectin-9 is regulated differentially according to cell types, and galectin-9 may play some physiological role on the membrane surface in vascular endothelial cells.

Galectin-9 has two carbohydrate-binding domains and is thus functionally bivalent [3 , 4 ]. Also, the two carbohydrate domains are essential for eosinophil chemotactic activity [5 ]. Treatment of HUVEC with IFN-{gamma} enhanced the adhesion of eosinophilic EoL-1 cells, and this was inhibited by an antigalectin-9 antibody. The presence of lactose also inhibited the adhesion of EoL-1 cells to HUVEC, but sucrose, used as a control, failed to inhibit the adhesion. Galectin-9 has binding affinity for lactose, and adhesion of eosinophils may be, at least in part, mediated through binding of galectin-9 with specific galactosyl groups on an eosinophil cell surface.

We conclude that galectin-9 is produced by endothelial cells stimulated with IFN-{gamma} and is expressed in the endothelium of human inflammatory lesions. Galectin-9 may mediate, in part, immune responses in endothelial cells elicited by IFN-{gamma}.


   ACKNOWLEDGEMENTS
 
A part of this study was supported by the Karoji Memorial Fund for Medical Research and a grant from Aomori Bank. The authors thank the staff of the Fujimori Clinic and Kumiko Munakata for their help.

Received April 5, 2001; revised February 7, 2002; accepted May 9, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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