Characterization of Natural Killer Cells in the Liver of Young Mice

Mice

C57BL/6 (B6) mice were used at 2 (young) and 8 (adult) weeks of age. All of the mice were maintained under specific pathogen-free conditions in the animal facility of Harbin Medical University.

Cell preparation

Hepatic lymphocytes were isolated by a previously described method^[6]. Briefly, mice anaesthetized with ether,were sacrificed by exsanguination via a cardiac puncture. To obtain lymphocytes, the liver was removed, pressed through a 200-gauge stainless steel mesh, and then suspended in Eagle’s minimum essential medium supplemented with 5 mM HEPES and 2% heat-inactivated newborn calf serum. After being washed once with this medium, the cell pellet was resuspended in the same medium. Lymphocytes were isolated from the parenchymal hepatocytes, the nuclei of hepatocytes, and Kupffer cells by the Percoll-gradient method (35% Percoll containing 100 U/ml heparin).

Splenocytes were obtained by pressing the spleen through a 200-gauge stainless steel mesh; erythrocytes in the spleen were lysed with 0.83% NH₄Cl-Tris buffer (pH 7.6).

Immunofluorescence tests

Lymphocyte phenotypes were identified by two or three-color immunofluorescence^[6]. Fluorescein iso-thiocyanate (FITC), phycoerythin (PE), or biotin-conjugated mAbs were used and biotin-conjugated reagents were developed with TRI color-conjugated streptavidin (Caltag Lab., San Francisco, CA). The mAbs used included anti-CD3 (145-2C11), anti-NK1.1 (PK136), anti-CD69 (H1.2F3), anti-Ly49C/I (5E6), anti-CD94 (18d3), and anti-Mac-1 (M1/70) mAbs (PharMingen Co., San Diego, CA). Cells were analyzed by flow cytometry (Becton Dickinson Co.). To prevent non-specific binding of mAbs, CD 16/32 (2.4G2) (PharMingen) was added before staining with labeled mAb. Forward scatter, side scatter and propidium iodide gating excluded dead cells.

Cytotoxicity assay

Cytotoxicity assays were performed as previously described^[7].YAC-1, syngeneic thymocytes and EL-4 target cells were labeled with sodium [⁵¹Cr] chromate for 2 h and washed three times with RPMI 1640 medium. Effecter cells were serially diluted and mixed with [⁵¹Cr]-labeled target cells (1×10⁴ or 2×10⁴ cells) in 96-well U-bottomed micro culture plates, and incubated for 4 h at 37°C. At the end of the culture, 100 μl supernatant was counted in a gamma counter.

Detection of perforin mRNA by PT-PCR analysis

To assess the expression of mRNA for perforin,a cytotoxic RNA was reverse-transcribed (RT) using primers noted below and the cDNA then amplified by the PCR method^[8]. Briefly, total RNA was prepared from various MNC by an acid-guanidinium thiocyanate-phenol-chloroform method. cDNA was synthesized using 1 μg of RNA with a first-strand cDNA synthesis kit (Pharmacia Biotech, US).

The following primers were used: perforin sense 5’ -GCT CCT TCC CAG TGA ACA CA-3’, antisense 5’-GTA GTA AGC ATG CTC TGT GG-3’; G3PDH: sense 5’-ACC ACA GTC CAT GAA ATC AC-3’, antisense 5’-TCC ACC ACC CTG TTG CTG TA-3’.

Statistical analysis

All parameters were expressed as the mean±SD. The difference between the values was determined by the Student’s t-test. P<0.05 was considered to be statistically significant.