Adult female inbred Lewis (LEW, RT1l) and Lewis × Brown Norway F1 hybrid (LBNF1, RTl1/n) rats weighing 250 to 350 grams were purchased from Kyudo Co. (Saga, Japan). The animal studies were conducted in accordance with protocols approved by the Animal Care and Use Committee of Fukuoka Dental College.
Induction of GVHD
Spleens removed from LEW rats were dissected in Hanks’ solution, forced through a stainless steel sieve, and filtered through a nylon mesh (Cell Strainers; BD Biosciences, CA, USA). The cells were washed three times in Hanks’ solution and resuspended at 108/ml in RPMI-1640 medium with 10% fetal calf serum. Cell viability was determined by trypan blue exclusion analysis. GVHD was induced by a 3-ml intraperitoneal injection of 3 × 108 cells into LBNF1 rats. Untreated LBNF1 rats and LBNF1 rats injected with an equal number of syngeneic LBNF1 splenocytes were used as controls. All rats were weighed daily and carefully observed for clinical signs of disease.
Assessment of GVHD
Clinical assessment of GVHD was determined by weight loss and the development of cutaneous or mucosal erythema, especially on the ears, nasal mucosa, foot-pads, and lips. Both clinical conditions appeared on day 10 after injection and became severe thereafter. A spleen weight assay was performed at autopsy to confirm the immunological assessment of GVHD. After day 10, the experimental rats showed remarkable splenomegaly as an overexpression of GVHD-related immune response
. All control animals survived and appeared healthy.
The whole tongue was excised 1–21 days after injection from five animals in each treatment group. Half of the tongue specimens were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS). Paraffin sections (4 μm) were then stained with hematoxylin and eosin (H&E) to examine histopathological changes. The other half was immediately frozen in liquid nitrogen, and serial frozen sections were used for immunostaining, lectin histochemistry, and in vitro adhesion assays.
LCA, Ulex europaeus I (UEA-1), and Archis hypogaea (peanut: PNA) were used for detection of α-D-Man, α-L-fucose, and galactose β1,3galactosamine, respectively (Vector Laboratories, Inc., Burlingame, CA, USA). LCA binding was detected using the Alexa Fluor 568 - labeled streptavidin biotin method. Acetone-fixed frozen sections were incubated with biotinylated LCA (25 μg/ml; Vector Laboratories) for 30 min and Alexa Fluor 568-labeled streptavidin (1:400; Molecular Probes, Eugene, OR, USA) for 30 min at room temperature. Histochemical controls substituted unconjugated LCA for the LCA-biotin conjugate and reaction with biotinylated LCA inactivated by its specific sugar inhibitor (D-mannose, 2 mM; EY Laboratories, Inc., San Mateo, CA, USA).
Abs used in this study were as follows: (a) a rabbit polyclonal Ab against ALG11, reactive with mannosyltransferase (1:300; Novus Biologicals, Littleton, CO, USA), (b) a rabbit polyclonal Ab against LMAN2, reactive with MBP 2 (1:100; Aviva Systems Biology, San Diego, CA, USA), (c) a mouse monoclonal Ab against 1A29, reactive with ICAM-1 (1:100; Cederlane Lab., Ontario, Canada), and (d) a mouse monoclonal Ab against OX-8, reactive with CD8 (1:100; Cederlane Lab.). Acetone-fixed frozen sections were first incubated with normal rabbit serum to decrease nonspecific binding and then reacted with one of the Abs, overnight at 4°C. Sections were then incubated with alkaline phosphatase-conjugated anti-rabbit Ab or anti-mouse Ab (1:150 dilution; DakoCytomation, Tokyo, Japan) for 45 min at room temperature. Immunohistochemical reactions were visualized using 5-bromo-4-chloro-3-indolyl phosphate/ nitro blue tetrazolium chloride solution (BCIP/NBT solution; DakoCytomation). As a control, sections were treated with normal rabbit IgG instead of the first set of Abs.
Total protein was extracted from spleen and tongue specimens in control or GVHD rats using ice-cold cell lysis buffer (20 m M Tris–HCl, pH 7.5; 150 mMNaCl; 1 mM ethylenediaminetetraacetic acid [EDTA]; 1 mM Na2EDTA; 1 mM ethylene glycol tetraacetic acid; 1% [v/v] Triton-X 100; 2.5 mM sodium pyrophosphate; 1 mMβ-glycerophosphate; 1 mM Na3VO4; and 1 μg/ml leupeptin and phenylmethylsulfonyl fluoride). Equal amounts of protein (20 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 12% separating gel). After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Tokyo, Japan). The blots were blocked with 1% casein in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. Primary Abs against LMAN2 (described earlier) and β-actin (Sigma-Aldrich) were used. Membranes were washed in TBS-T and incubated with secondary horseradish peroxidase-labeled Ab for 1 h at room temperature. Bound Ab complexes were detected by enhanced chemiluminescence (Bio-RadLaboratories).
Isolation of CD8+ cells from the spleens of GVHD-mediated rats
CD8+ cells from GVHD rats were used in transwell migration assays and Stamper-Woodruff binding assays. Lymphocytes were teased from spleen, exhibiting splenomegaly, of GVHD rats and suspended in RPMI-1640 medium at 4°C. The cells were dispersed by rapid, in-out pipetting, and the cell clumps were removed by passage through a nylon mesh (Cell Strainers; BD Biosciences). The resultant single-cell suspension was washed three times in the same medium and resuspended at a concentration of 3 × 107mononuclear cells /ml. CD8+ cells from the suspension were isolated by magnetic bead purification using Miltenyi CD8a microbeads according to the manufacturer’s protocol (Miltenyi Biotec, Tokyo, Japan).
Transwell migration assay for effector cells
Migration of CD8+ cells from the GVHD-spleen were evaluated in a transwell® insert system in which the top and bottom wells were separated by polycarbonate membrane (3-μm pore size; Corning, NY, USA). CD8+ cells, pre-incubated or not with anti-MBP Ab (50 μg/ml), were seeded at a density of 5 × 104 cells /well onto 3-μm transwell inserts. The lower chamber was filled with 500 μl RPMI medium only or medium containing Man (2 mM), a mixture of Man and LCA (50 μg/ml), or galactose (Gal, 2 mM; EY Laboratories). The cells were incubated for 4 h at 37°C (5% CO2) and then stained with Diff-Quik (Sysmex Corp., Hyogo, Japan). Migration activity was evaluated as the percent migration of cells from the upper chamber of the transwell insert to the lower chamber in three high power fields (100x) per well. The experiment was performed in triplicate.
Stamper-Woodruff binding assay (SWBA)
Stamper-Woodruff-type frozen-section assays were performed as described previously
. Briefly, aliquots containing 2.5 × 105 isolated CD8+ cells in 200 μl of RPMI-1640 medium were added to freshly cut (8 μm) frozen sections of the tongue obtained from rats in the control and GVHD groups. The sections were agitated on a rotary shaker (60 rpm) for 60 min at room temperature. Next, the cell suspension was carefully decanted, and cells adhering to sections were fixed with 2.5% glutaraldehyde in PBS for 5 min. Slides were then washed in PBS and stained with 0.5% toluidine blue. The number of adherent CD8+ cells was determined by light microscopy examination of fields at 200 × magnification (Each high-power field represented approximately 400 μm of oral epithelium). The number of CD8+ cells attached directly over KCs (but not in the cornified layer) was counted. Several blocking experiments were performed as follow: (a) frozen sections of tongue from GVHD rats were incubated with 25 μg/ml of LCA, PNA or UEA-I for 30 min at room temperature following a brief wash with PBS, and then added to lymphocytes; (b) CD8+cells were preincubated with 50 μg/ml of MBP Ab for 30 min at room temperature, before the entire reaction mixture was transferred onto frozen sections of the GVHD-tongues; (c) the two approaches were used simultaneously, i.e., CD8+ cells were treated with MBP Ab and the tissue sections were treated with MBP. Results are calculated as the percentage of binding relative to control lymphocytes and tissue sections exposed to buffer alone.
Statistical analysis was performed with one-way analysis of variance (ANOVA) and Scheffe’s multiple comparison tests to determine statistical differences among the samples. Data are presented as the mean ± standard deviation (SD) and P values of < 0.05 were considered statistically significant.