Animals
All experimental procedures were conducted in compliance with prevailing animal welfare regulations. Heterozygous male or female hPLAP-tg F344 rats were mated with wild-type F344 rats, and the resulting wild-type and heterozygous transgenic offspring were genotyped by enzyme histochemistry using a drop of tail blood as described [4]. The rats were housed in pairs at 24°C and a 12 h/12 h light/dark cycle with free access to tap water and commercial rat diets (Altromin 1324 for maintenance and 1314 for breeding, Altromin, Lage, Germany).
Histology and hPLAP detection
Because hPLAP is sensitive to fixation with formalin [2], tissues and bones were harvested and were fixed in 40% ethanol at 4°C for 48 h. Subsequently, the tissue specimens were dehydrated and embedded in paraffin or in a modified MMA embedding mixture that preserves enzyme activities and can also be used for immunohistochemistry [3]. Five-μm-thick paraffin and MMA sections were cut with a HM360 microtome (Microm, Walldorf, Germany), and were mounted on slides pre-treated with 3-aminopropyltriethoxy-silane (APES, Sigma-Aldrich, Deisenhofen, Germany).
Paraffin sections were deparaffinated using xylene, whereas MMA sections were deplasticized using 2-methoxyethylacetate as described [3]. Deparaffinated and deplasticized sections were rehydrated and heated at 65°C for 30 min in deionized water to block endogenous alkaline phosphatase activity. Sections were then incubated in TRIS buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, 5 mM MgCl2) containing 0.17 mg/ml of the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) at room temperature overnight. Subsequently, sections were counterstained with nuclear fast red (Sigma), dehydrated, and mounted using Vectamount (Vector, Burlingame, CA, USA).
Flow cytometry
Peripheral whole blood was taken from a tail vein. BMC were harvested from the tibia or femur by short centrifugation of the bone, and were dispersed in PBS by repeated pipetting. For labeling of cell surface hPLAP on peripheral blood and BMC, cell suspensions (approximately 106 cells each) were incubated with a monoclonal mouse anti-hPLAP antibody (supernatant of clone 8B6, Dako) diluted 1:20 for 30 min on ice. Non-immune mouse IgG1 (MOPC-21, Sigma) and cell suspensions from wild-type rats were used as negative controls. After washing twice, the cells were incubated with rat-adsorbed, FITC-labeled goat anti-mouse IgG antibody (Sigma) for 30 min on ice. Prior to FACS analysis, erythrocytes were hemolyzed in blood samples using FACS lysing solution (Becton Dickinson, Heidelberg, Germany). The analyses were performed on a FACScan flow cytometer using CellQuest Pro software (Becton Dickinson, Heidelberg, Germany).
To examine the presence of circulating anti-hPLAP antibodies in serum of wild-type rats that had previously received cells from hPLAP-tg rats, wild-type and transgenic peripheral blood cells were incubated for 30 min on ice with 10 μl undiluted serum from naïve wild-type rats or from wild-type rats that had received transgenic PBL earlier. Using FITC-labeled goat anti-rat IgG antibody (Serotec, Harwell, UK) as secondary antibody, the samples were processed and analyzed as described above.
Transplantation of transgenic blood and bone marrow cells into wild-type recipients
In order to determine the fate of genetically labeled donor cells in wild-type recipients, we intravenously injected 36 wild-type F344 rats with PBL or BMC (n = 18 each) isolated from hPLAP-tg F344 rats. Transgenic rats were killed by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. PBL were harvested using a density gradient centrifugation kit (NycoPrep 1.077A, Axis-Shield, Oslo, Norway), washed several times and resuspended in PBS. The percentage of viable cells after the isolation procedure was > 90% (trypan blue exclusion). At the same time, BMC were harvested and washed as described above. After isolation, all cells were resuspended in PBS, and were kept on ice until use. Isolated genetically labeled PBL or BMC were injected intravenously at a dose of 25 × 106 or 5 × 106 cells per animal, respectively, into sex-matched wild-type recipients via a lateral tail vein. All recipients received daily injections of genetically labeled cells over 7 consecutive days (days 1 – 7). The recipient rats were killed on days 8, 15, 22, or 28 (n = 4 – 6 each for PBL and BMC recipients) by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. From all recipient animals a variety of soft tissues and bones were harvested, fixed in 40% ethanol at 4°C for 48 hours, and processed as described above.
Humoral and cellular immune response to transgenic cells
To examine the development of antibody production against transgenic cells, we repeatedly exposed wild-type rats to PBL from hPLAP-tg donors. The PBL were harvested from hPLAP-tg rats as described above, and resuspended in PBS. Subsequently, 1 × 106 PBL were subcutaneously or intraperitoneally injected three times into wild-type F344 rats (1 rat each) with an 11-day interval between the injections. At baseline, and 10 days after each injection, blood was drawn from a tail vein of the wild-type recipients to obtain serum. For mixed lymphocyte reaction, spleen cells were isolated by density gradient centrifugation (NycoPrep 1.077A). The spleen cells from naïve wild-type rats or from wild-type rats previously exposed to transgenic cells (3 intraperitoneal injections at 10-day intervals) were co-cultured with transgenic spleen cells for 5 days at a total density of 5 × 105 cells per well (2.5 × 105 wild-type and transgenic cells each). During the last 18 hours of culture, 3H thymidine (2 Ci/mmol) was added to quantify cell proliferation. ConA (10 μg/ml) was used as a positive control.
Lethal irradiation and bone marrow transplantation
To transplant bone marrow from hPLAP-tg rats into wild-type F344 rats, wild-type animals (n = 6) were lethally irradiated with a dosage of 8.5 Gy at 0.9473 Gy/min using a cobalt-60 irradiator (Eldorado, Atomic Energy of Canada, Ottawa, Canada). Four hours after the irradiation, the rats were intravenously injected with 3 – 4 million BMC isolated from hPLAP-tg rats as described above. To rule out unsuccessful engraftments, the same injection of freshly prepared transgenic BMC was repeated 24 hours after the irradiation.
Intraarticular injection of genetically labeled cells
Next, we wanted to explore whether intraarticularly injected BMC from hPLAP-tg rats would survive, proliferate, and differentiate in the knee joint of irradiated WT (hPLAP-BMT) rats. For this experiment 6 male and female wild-type F344 rats at the age of 3 months were lethally irradiated and transplanted with BMC from hPLAP-tg F344 rats. Two weeks after the bone marrow transplantation, 5 × 106 BMC isolated from hPLAP-tg rats were injected once into the cavity of the left or right knee joint under medetomidine/midazolam/fentanyl anesthesia. The rats were killed 7 days or 28 days (n = 3 each) after the intraarticular injection by exsanguination from the abdominal aorta under ketamine/xylazine anesthesia. The intact knee joints were harvested, trimmed, fixed in 40% ethanol at 4°C for 48 hours, and embedded in MMA. Frontal 5-μm-thick sections were cut as described above.
Induction of tolerance by neonatal exposure to transgenic cells
To induce tolerance to the marker gene by an alternative method, 6 neonatal wild-type F344 rats pups, within 4 hours after birth, were subcutaneously injected once with 20 μl of whole blood from hPLAP-tg F344 donors. One control litter mate did not receive transgenic cells. Four months later, all 7 rats received skin grafts (approx. 1 cm2) from sex-matched hPLAP-tg donors at the back under anesthesia with medetomidine/midazolam/fentanyl. The grafts were controlled daily. Four weeks post-transplantation, biopsies were taken under anesthesia from the margin of the skin grafts, fixed in 40% ethanol at 4°C for 48 hours, embedded in paraffin, sectioned at 5 μm thickness, and stained for hPLAP enzyme activity as described above.
Tracking of transgenic cells in neonatally tolerized rats
Tolerance to hPLAP in F344 WT hosts was induced and confirmed by skin grafts as described above. At the age of 4 months, 4 neonatally tolerized WT rats were intravenously injected once with 108 BMC from hPLAP-tg donors, and were killed 2 months post-injection. Various tissues were harvested, fixed in 40% ethanol at 4°C for 48 hours, embedded in paraffin or MMA, sectioned at 5 μm thickness, and stained for hPLAP enzyme activity as described above. Neonatally tolerized control rats were killed without prior injection of labeled cells to rule out a background of labeled cells in the marker tolerant recipients.
Statistical analysis
Statistics were computed using SPSS for Windows 14.0 (SPSS, Chicago, IL). The data of the mixed lymphocytes reaction experiments were analyzed using one-way analysis of variance (ANOVA). When the analysis of variance performed over all groups indicated a significant (p < 0.05) difference among the groups, statistical differences between two groups were subsequently evaluated with Fisher's least significant difference test. P values of less than 0.05 were considered significant. The data in Figure 2D are given as means ± SEM.
