TSG-6 protein expression in the pancreatic islets of NOD mice
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- Kvezereli, M., Michie, S.A., Yu, T. et al. J Mol Hist (2008) 39: 585. doi:10.1007/s10735-008-9199-5
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The histologic hallmark of the development of type 1 diabetes (T1D) is insulitis, characterized by leukocytic infiltration of the pancreatic islets. The molecules controlling the early influx of leukocytes into the islets are poorly understood. Tumor necrosis factor α (TNFα)-stimulated gene 6 (TSG-6) is involved in inflammation, extracellular matrix formation, cell migration, and development. In the present study, we examined the expression and cellular localization of TSG-6 protein in islets of female non-obese diabetic (NOD) mice using frozen section immunofluorescence staining. Pancreata from nondiabetic (8 and 25 weeks old), prediabetic (230–280 mg/dl blood glucose) and diabetic (>300 mg/dl blood glucose) NOD mice were stained for TSG-6, insulin, CD3, CD11c, Mac3 and CD31. TSG-6 protein was detected in 67% of islets of prediabetic mice, 27% of islets of 25-week old nondiabetic mice, and less than 7% of islets of diabetic mice and 8-week old nondiabetic mice. Lastly, islet-derived TSG-6 protein was localized to the infiltrating CD3 and CD11c positive leukocytes.
KeywordsType 1 diabetesInsulitisInflammationTSG-6Dendritic cellsT lymphocytes
T1D is a T cell-mediated autoimmune disease that results in the destruction of insulin-producing β cells in the pancreatic islets (Roep et al. 1995). In the NOD mouse model of T1D, a nondestructive accumulation of T lymphocytes, B lymphocytes, macrophages and dendritic cells around islets (peri-insulitis) is first seen at about 4 weeks of age (Charlton et al. 1988). At 6–8 weeks of age, the lymphocytes begin to invade the islets, resulting in destructive insulitis. Between 12 and 16 weeks of age, 60–80% of female NOD mice develop T1D, characterized by uncontrolled hyperglycemia (Pozzilli et al. 1993; Andre et al. 1996). The signaling events leading to the development of insulitis and the subsequent destruction of the β cells are poorly understood.
Cytokines and cytokine-induced proteins generated by the inflammatory cells in the islet milieu play an important role in the pathogenesis of insulitis and T1D (von Herrath and Oldstone 1997; DiCosmo et al. 1994). We propose to investigate the importance of the multifunctional protein TSG-6 in type 1 diabetes. TSG-6 is known to play a role in developmental processes, extracellular matrix (ECM) formation and inflammatory cell migration (Lee et al. 1992; Fülöp et al. 1997; Maier et al. 1996; Wisniewski and Vilcek 1997; Day and Prestwich 2002). TSG-6 has a potent anti-inflammatory effect in murine models of inflammatory arthritis (Mindrescu et al. 2000; Bárdos et al. 2001; Mindrescu et al. 2002). The link module of TSG-6 binds to hyaluronan (HA), which is a major component of the ECM (Parkar and Day 1997; Parkar et al. 1998; Day and Prestwich 2002). HA also binds to CD44, which is expressed on the cell surface of leukocytes; this interaction supports T cell adhesion and transmigration into the islets, which results in insulitis (DeGrendele et al. 1997). Lesley et al. suggested that the binding of TSG-6 to HA might facilitate CD44-mediated recruitment of inflammatory cells or inhibit the adhesion of leukocytes to endothelia, depending on the concentration of TSG-6 and HA at the inflammatory site (Lesley et al. 2004). Our laboratory is investigating the potential role of TSG-6 protein in the development of T1D.
In the present study, we evaluated the expression of TSG-6 in islets of nondiabetic, prediabetic and diabetic female NOD mice. First, we found that islet expression of TSG-6 protein was most common in prediabetic and 25-week old nondiabetic NOD mice. Second, we localized TSG-6 to CD3 and CD11c positive leukocytes in the islets of the prediabetic mice.
Materials and methods
Female NOD and C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) and housed in the Stanford Blood Center animal facility. The Stanford University Institutional Animal Care and Use Committee (IACUC) approved all procedures. Blood glucose concentrations were checked once a week, starting at 10 weeks of age, using a glucose test strip reader (Roche Diagnostics, Indianapolis, IN).
Goat polyclonal IgG antibody to mouse TSG-6 was purchased from R&D Systems (Minneapolis, MN); chicken polyclonal IgG antibody to insulin was obtained from Chemicon (Temecula, CA); rat monoclonal antibody (mAb) to CD3 (clone 17A2) was purchased from Biolegend (San Diego, CA); rat mAbs to Mac3 (clone M3/84) and CD31 (PECAM-1; clone MEC 13.3) were purchased from BD Pharmingen (San Jose, CA); and rat mAb to CD11c (clone 223H7) was purchased from MBL International (Woburn, MA). Alexa Fluor 488-anti-goat IgG, Alexa Fluor 594-anti-chicken IgG and Alexa Fluor 350-anti-rat IgG were purchased from Invitrogen (Carlsbad, CA). Cy3-anti-chicken IgG, peroxidase-anti-goat IgG and tetramethyl rhodamine isothiocyanate (TRITC)-anti-goat IgG were purchased from Chemicon.
HeLa cells and 293T cells were purchased from ATCC (Manassas, VA). Top 10 chemically competent E. coli cells were purchased from Invitrogen. Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (Ipswich, MA). The lentiviral vector pHR’tripCMV-IRES-eGFP-sin backbone was obtained from Dr. K. Breckpot (Vrije Universiteit Brussels, Belgium) and modified in Dr. C.G. Fathman`s laboratory (Stanford University, Stanford, CA). Recombinant TSG-6 protein was obtained from ProSci (Poway, CA).
TSG-6 cloning and lentiviral vector production
The TSG-6 gene was amplified from mouse cDNA library made of various tissues. The primers were designed from the cDNA sequence with an accession number NM_009398/NCBI: 5′-GCAACTGCAGAGATGGTCGTCGTCCTCCTTT-3′ forward, 5′-AAAACCGCGGTTTATAGATGGCTAAACCTTCCAGG-3′ reverse.
Amplified fragment covered the entire coding sequence for murine TSG-6, which includes the signal sequence of the TSG-6 protein. The TSG-6 gene (851 bp insert) (Supplementary Figure 1) was cloned into the lentiviral vector pHR’tripCMV-IRES-eGFP-sin (9.695 kb) within the multiple cloning sites available between the CMV promoter and the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV). Therefore, TSG-6 and GFP (downstream of the IRES) will be co-expressed on a bicistronic mRNA and translated independently into protein.
The vector and insert were digested by restriction enzymes Sac II and Pst I for 3 h at room temperature. Digested DNAs were purified and then ligated using T4 DNA ligase at 25°C for 5 min. The ligation product was transformed into chemically competent Top 10 E. coli cells using heat-shock for 30 s at 42°C. DNA was isolated using Genelute HP endotoxin free plasmid maxiprep kit (Sigma). DNA was sequenced at Stanford University sequencing facility. Lentiviral vector particles were generated by transient transfection of 293T cells with a packaging plasmid, an envelope plasmid and pHR’tripCMV-TSG-6-IRES-eGFP-sin or pHR’tripCMV-IRES-eGFP-sin using a modified calcium phosphate method, as described previously (Breckpot et al. 2003). The viral preparations were frozen at −80°C.
293T cell transduction and immunoblotting
293T cells were harvested after transduction with TSG-6 GFP lentivirus or GFP lentivirus at a MOI of 10 and 15. Untransduced cells were harvested as controls. About 106 trypsinized cells were washed, lysed, and boiled in loading buffer at 94°C for 5 min. The samples and recombinant TSG-6 protein were electrophoresed on 4–15% polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA) and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk in Tris buffered saline containing 0.05% Tween-20 (TBST) and probed with goat anti-mouse TSG-6 antibody (1:500) overnight. Membranes were washed four times in TBST and incubated with peroxidase-anti-goat IgG antibody. Protein bands were detected using the Enhanced Chemiluminescence (ECL) Western Blotting Detection System (Amersham Biosciences, Piscataway, NJ) and visualized using a FUJI Imager LAS3000 (Valencia, CA).
HeLa cell transduction, immunofluorescence staining and fluorescence microscopy
HeLa cells were cultured on coverslips in six well-plates at 0.4 × 106 cells per well and transduced with pHR’tripCMV-TSG-6-IRES-eGFP-sin expressing-lentivirus (hereafter referred to as TSG-6 GFP lentivirus) or pHR’tripCMV-IRES-eGFP-sin expressing-lentivirus (hereafter referred to as GFP lentivirus) at a multiplicity of infection (MOI) of 10 and 15 at 37°C for 72 h in the presence of protamine sulfate. GFP expression was confirmed by fluorescence microscopy. Untransduced cells were kept in the same conditions and served as controls. The coverslips with cultured HeLa cells were carefully removed from the six well-plates, mounted on glass slides, and fixed in 4% paraformaldehyde. Slides were sequentially incubated at room temperature with 2% bovine serum albumin (BSA) for 1 h, goat anti-mouse TSG-6 antibody (1:1000) overnight, and TRITC-anti-goat IgG (1:1000) for 45 min. There were three washes with phosphate buffered saline with 0.05% Tween-20 (PBST) after each incubation. Slides were coverslipped with Vectashield anti-fading media with DAPI (Vector Labs, Burlingame, CA). Images were taken with a CCD camera (Applied Biosystems, Foster City, CA) mounted on an Olympus microscope (Center Valley, PA) and analyzed with Open Lab software (Improvision, Waltham, MA).
Pancreas immunofluorescence staining and fluorescence microscopy
There were four experimental groups of NOD mice: (A) 8-week old nondiabetic mice with blood glucose levels less than 120 mg/dL (n = 6); (B) 25-week old nondiabetic mice with blood glucose levels less than 120 mg/dL (n = 6); (C) Prediabetic mice (11–18 weeks old) with blood glucose levels of 230–280 mg/dL (n = 6); (D) Diabetic mice (11–20 weeks old) with blood glucose levels greater than 300 mg/dL (n = 6). Prediabetic and diabetic mice were sacrificed on the day they presented with glucose concentrations of 230–280 mg/dL and >300 mg/dL, respectively. C57BL/6 mice served as controls (n = 3).
Pancreata were harvested, snap frozen in OCT compound, and serially cryosectioned (5 micron). Slides were air-dried and fixed in ice-cold acetone for 10 min. Five slides, from various levels of the tissue, were stained with hematoxylin and eosin (H&E) for the evaluation of islets and inflammation. Adjacent slides were sequentially incubated with 1% BSA and 10% goat serum in PBST for 1 h at room temperature; goat anti-mouse TSG–6 (1:500) overnight at 4°C; Alexa Fluor 488-anti-goat IgG (1:2000) for 45 min at room temperature; chicken anti-mouse insulin (1:500 dilution) and a rat mAb to a lineage marker (anti-CD3 (1:100), anti-CD11c (1:100), anti-Mac3 (1:100), or anti-CD31 (1:100)) for 2 h at room temperature; and Alexa Fluor 594-anti-chicken IgG (1:1000) or Cy-3-anti-chicken IgG (1:500) and Alexa Fluor 350-anti-rat IgG (1:100) for 45 min at room temperature. There were three washes with PBST after each step. For slides stained for TSG-6 and insulin only, incubations with the rat mAb to a lineage marker and Alexa Fluor 350-anti-rat IgG were omitted. Negative control slides were incubated without primary antibodies to identify nonspecific binding.
Slides stained for TSG-6 and insulin only were coverslipped with Vectashield with DAPI. All other slides were coverslipped with Vectashield. Images were captured with a CCD camera mounted on an Olympus microscope and analyzed with CytoVision 3.92 (Applied Biosystems) and Open Lab software.
Evaluation of TSG-6 expression and TSG-6 co-localization
To determine the prevalence of TSG-6-expressing islets, we examined the pancreas sections that were stained with antibodies to TSG-6 and insulin. Four to six islets per mouse were counted for a total of 30 islets in each group. Results are given as the percentage of islets positive for TSG-6 staining.
To identify the specific cell types that express TSG-6, we examined sections of prediabetic mouse pancreata that were stained for TSG-6 protein, insulin, and one of the following lineage markers: CD3, CD11c, Mac3 or CD31. Islets with staining for TSG-6 and CD3, for TSG-6 and CD11c, for TSG-6 and Mac3, and for TSG-6 and CD31, respectively, were analyzed for co-localization of TSG-6 with the lineage marker. Twenty-five such islets were counted for each lineage marker. Results are expressed as percent of islets showing TSG-6 co-localization with the lineage marker.
Data are presented as mean ± standard deviation (SD). Results were assessed by the Fisher’s exact test for multiple comparisons. The value P < 0.05 was considered statistically significant.
TSG-6 protein expression in transduced 293T cells
In order to test for TSG-6 protein expression in mammalian cells, 293T cells transduced with TSG-6 GFP lentivirus or GFP lentivirus and untransduced 293T cells were processed for immunoblotting. TSG-6 recombinant protein was used as a positive control (Supplementary Figure 2, Lane 1). TSG-6 protein (35 kDa) was not detected in untransduced or GFP lentivirus transduced cells (Supplementary Figure 2, Lanes 2, 3), but was detected in cells transduced with TSG-6 GFP lentivirus at a MOI of 10 and 15 (Supplementary Figure 2, Lanes 4 and 5).
Specificity of the anti-TSG-6 polyclonal antibody
TSG-6 expression in islets
TSG-6 co-localization studies
To determine which cells express TSG-6 protein, we stained frozen sections of pancreata from prediabetic NOD mice for TSG-6 protein, insulin (to locate islets), and one of the following lineage markers: CD3 for T cells, CD11c for dendritic cells, Mac3 for macrophages, and CD31 for endothelial cells. We captured single color images of islets that stained for TSG-6 and a lineage marker. The single color images were overlaid and co-localization of TSG-6 and the lineage marker was evaluated (n = 25 islets for each lineage marker).
TSG-6 co-localization in islets of prediabetic NOD mice
Islets (n = 25) with TSG-6 co-localization (%)
Although TSG-6 is involved in the development of several inflammatory diseases, its role in the context of T1D has not yet been described (Wisniewski et al. 1993; Bayliss et al. 2001; Milner and Day 2003). The aim of the present study was to evaluate the expression of TSG-6 protein in pancreata of NOD mice in different developmental stages of T1D. We found that TSG-6 is highly expressed in islets of prediabetic NOD mice and 25-week old nondiabetic mice. Very few islets in diabetic NOD mice and 8-week old nondiabetic NOD mice expressed TSG-6. We did not detect expression of TSG-6 in islets of non-autoimmune prone C57BL/6 mice or in the exocrine portion of the pancreas in any of the mice. These data correlate with published results on the detection of TSG-6 protein, which is expressed during inflammatory processes and rarely in normal physiological conditions (Wisniewski et al. 1993, Bayliss et al. 2001).
Since TSG-6 production is linked to inflammation, we identified the cellular source of the protein among the major cell types involved in islet inflammation, including CD3+ T lymphocytes, CD11c+ dendritic cells, Mac3+ macrophages and CD31+ endothelia. Macrophages are scavenger cells that phagocytose, process antigens, and modulate the immune response through antigen presentation and activation of other immune cells (Ihm and Yoon 1990; Gordon 1998). Endothelia adhesion and activating molecules are critical for recruitment of leukocytes from blood vessels into islets. Staining for TSG-6 co-localized with staining for Mac3 or CD31 in few islets of prediabetic NOD mice, suggesting that these cells might produce small amounts of TSG-6 and/or secrete TSG-6 rapidly after production.
Dendritic cells and macrophages are thought to be important in the development of peri-insulitis and early insulitis. CD11c+ myeloid dendritic cells take up islet antigens and present them to CD4+ T helper cells in the draining lymph nodes in the early stages of diabetes in NOD mice (Turley et al. 2003). After autoantigen recognition, CD4+ and CD8+ T cells attack and destroy the β cells (Eisenbarth 1986; Haskins et al. 1988; Serreze and Leiter 1994; Tisch and McDevitt 1996). We found that CD11c+ and CD3+ cells are major cellular sources of TSG-6 in NOD islets. Although CD11c is a major cell surface marker for dendritic cells, it may also be expressed on activated CD3+ T cells during the mounting of some immune responses such as the one reported by Huleatt et al. during graft versus host disease or by Lin et al. during an infection with lymphocytic choriomeningitis virus (Huleatt and Lefrancois 1995; Lin et al. 2003). We conclude that CD3+ T cells and CD11c+ dendritic cells and/or activated T cells represent cellular sources of TSG-6 in islets of prediabetic NOD mice.
TNFα is an inflammatory cytokine that can be produced by CD11c+ cells and/or CD3+ T lymphocytes during diabetogenesis. TNFα plays an important role in the initiation of T1D in the NOD mouse by regulating the maturation of DCs and by initiating cross-presentation of islet antigens to CD8+ T cells (Lee et al. 2005; Green et al. 2000). TNFα may also induce expression of TSG-6; further studies are needed to determine if TSG-6 protects the islets from invading lymphocytes or facilitates lymphocyte migration into the islets. Lymphocyte migration is a multistep process with at least four steps: (1) tethering and rolling of the lymphocyte on the endothelia surface; (2) lymphocyte activation; (3) firm adhesion of the lymphocyte to the endothelia surface; and (4) diapedesis of the lymphocyte through the vessel wall into the tissues (Muller 2002). Steps 1 and 4 can be mediated by CD44 on the lymphocyte surface binding to HA on endothelia and ECM, respectively (DeGrendele et al. 1996). In an endothelial cell culture transmigration assay, TSG-6 has been shown to inhibit transmigration of leukocytes by affecting leukocyte rolling (Cao et al 2004). On the other hand, TSG-6 has also been shown to modulate the interaction between HA and CD44 at sites of inflammation (Lesley et al. 2004). CD44 is a cell surface molecule by which lymphocytes bind to HA on endothelia and ECM during migration. TSG-6 can form complexes with HA, induce the binding of HA to CD44 and allow the cells to roll on the substrates comprising these complexes (Lesley et al. 2004). Further mechanistic studies need to be performed to determine the role of TSG-6 during lymphocyte migration into the pancreatic islets during T1D development.
In summary, we show that TSG-6 is highly expressed in islets of prediabetic NOD mice and 25-week old nondiabetic NOD islets. We have localized islet-derived TSG-6 to the infiltrating CD3+ and CD11c+ leukocytes in islets of prediabetic mice. Further studies are needed to determine the exact role of TSG-6 in the development and progression of insulitis in the NOD mouse model of T1D.
We would like to acknowledge Dr. C. Garrison Fathman for providing us with lentiviral plasmids, Anet James for assistance with the artwork, and Claudia Benike and Donna Jones for critical reviews of the manuscript. We would like to thank Dr. Edgar Engleman for making all the necessary laboratory equipment accessible.
This work was supported by:
Dean’s Postdoctoral Fellowship, Stanford University (to M.K.)
Department of Pathology Research Fund at Stanford (to M.J.F.)
Juvenile Diabetes Research Foundation Grant 1-2001-56 (to S.A.M.)
National Institutes of Health Grant R01 DK67592 (to S.A.M.)
Juvenile Diabetes Research Foundation Postdoctoral Fellowship (to R.J.C.)