Critical role for P-glycoprotein expression in hematopoietic cells in the FVB.Mdr1a(-/-) model of colitis

Abstract

Objective: P-glycoprotein (P-gp), the functional product of the multidrug resistance gene (MDR), is a transmembrane protein that extrudes substrates from the intracellular environment. P-gp is expressed on the apical surface of epithelial cells and on cells from the hematopoietic lineage. Human MDR polymorphisms have been associated with the increased risk of inflammatory bowel disease, and FVB/N animals deficient in mdr1a expression develop spontaneous colitis. Previous studies using adult bone marrow chimeras indicated that colitis development in this animal model was contingent on P-gp deficiency in radiation-resistant epithelial cells; however, the use of adult animals may mask the role of hematopoietic immune cells in colitis initiation, due to preexisting epithelial abnormalities.

Subjects and methods: To assess the importance of P-gp expression in intestinal epithelial and hematopoietic-derived cells on colitis induction in FVB.mdr1a(-/-) animals, we developed a neonatal model of bone marrow reconstitution. FVB/N and FVB.mdr1a(-/-) adult and neonatal animals were lethally irradiated and reconstituted with bone marrow from FVB/N or FVB.mdr1a(-/-) donors. Animals were observed for 20 weeks.

Results: Adult FVB/N animals deficient in P-gp expression in hematopoietically derived immune cells developed colitis similar to adult animals deficient in P-gp expression in radiation-resistant epithelial/stromal cells. Neonatal animals deficient in P-gp expression in hematopoietically derived immune cells developed a more histologically significant colitis than those deficient in P-gp expression in epithelial tissue.

Conclusions: The use of a neonatal model of bone marrow reconstitution has revealed a critical role for P-gp expression in hematopoietically derived immune cells in colitis development in the FVB.mdr1a(-/-) model.

Figure 1

Weight Changes and Mortality in Adult Bone Marrow Chimeras. Mice were 2–4 months of age at reconstitution. Reconstituted animals were monitored for weight loss (A) or increased mortality (animals were sacrificed following loss of approximately 20% of highest body weight) (B). Animals were sacrificed 20 weeks following reconstitution. † indicates an animal died or was sacrificed at this time point. Group 4A was significantly different (P<0.05) from the other groups at or after week 7 (weight loss) and week 15 (mortality).

Figure 2

Histologic Analysis of Adult Reconstituted Bone Marrow Chimeras. Mice were 2–4 months of age at reconstitution. Representative photomicrographs of H&E stained distal colonic tissue from Group 1A, WT ⇒ WT (A), Group 2A, KO ⇒ WT (B), Group 3A, WT ⇒ KO (C), and Group 4A, KO ⇒ KO (D). Histological scoring data from cecal and colonic tissue from reconstituted animals (E &F). * indicates significance of P<0.05. Reference bar =1μm.

Figure 3

Serum Levels of FITC-Dextran following Irradiation in Neonatal and Adult Animals. Neonatal (day 7 from birth) and 6–8 week adult FVB/N mice were exposed to 900 RADSγ-radiation. 3 days post irradiation animal were orally gavaged with 60mg/100gm of body weight with FITC-Dextran. Total blood volume was collected at sacrifice and serum was evaluated for FITC-Dextran content. N.D. indicates not detected. * indicates significance of P<0.05 vs. the FVB Adult.

Figure 4

Weight Changes and Mortality in Neonatally Reconstituted Bone Marrow Chimeras. Mice were 7 days of age at reconstitution. FVB.mdr1a^−/− mice were lethally irradiated and reconstituted with bone marrow from FVB, or FVB.mdr1a^−/− donors, and monitored for weight loss (A) or increased mortality (B). Reconstituted FVB.mdr1a^−/− and FVB/N animals were sacrificed upon reaching 5 month of age, following the loss of 20% maximum weight gain, or demonstration of fecal blood. † indicates an animal died at this time point. Weight loss and mortality in Group 4N was significantly different (P<0.05) from the other groups at or after week 14.

Figure 5

Histologic Analysis of Neonatally Reconstituted Bone Marrow Chimeras. Mice were 7 days of age at reconstitution. Representative photomicrographs of H&E stained distal colonic tissue from Group 1N, WT ⇒ WT (A), Group 2N, KO ⇒ WT (B), Group 3N, WT ⇒ KO (C), and Group 4N, KO ⇒ KO (D). Histological scoring data from cecal and colonic tissue from reconstituted animals (E & F). * indicates significance of P<0.05. Reference bar =1μm

Figure 6

RNA expression of Inflammatory Cytokines in Neonatally Reconstituted FVB/N and FVB.mdr1a^−/−animals. Colonic tissue for RNA isolation was harvested from animals at sacrifice. Target gene expression was normalized to expression of the 18S housekeeping gene, and to the average fold change in gene expression in Group 1N, WT ⇒ WT. Data is shown on a log 2 scale. All values demonstrating a 2 fold alteration in gene expression from control values are considered physiologically altered from control values. N=4 in each data set. * indicates significance of P<0.05 vs. Group 1N.