Macrophage depletion in tissues of HO-1^−/− mice was associated with increased expression of Hp in the liver. (A) Quantitative RT-PCR showed 2.2-fold increased (P < .001) mRNA levels of Hp (Hb scavenger protein) probably in response to hemolysis of senescent RBCs. (B) Expression of the CD163, the receptor for Hb-Hp complexes, was markedly diminished in spleen, liver, and BM (P < .001) as determined by quantitative RT-PCR. In contrast, expression of CD163 in kidney was increased 3-fold (P < .01). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (n = 5 for each WT and HO-1^−/− mice). All mice were 12 months old. Error bars represent SD of the mean. (C) Immunohistochemistry for the pan-macrophage marker, F4/80, indicated that the resident macrophage populations of the spleen and liver were markedly reduced in HO-1^−/− animals. Arrowheads point to diaminobenzidine positively stained macrophages (brown staining). The increased number of F4/80⁺ cells in kidneys correlates with the elevated CD163 expression. Two different fields are shown for the tissue samples of the same HO-1^−/− mice. All animals were 12 months old.