Multiple apoptotic defects in hematopoietic cells from mice lacking lipocalin 24p3

Abstract

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.

FIGURE 1.

Generation of 24p3^−/− null mice. A, targeting strategy. Schematic diagram of the WT 24p3 gene (top), the targeting construct (middle), and the 24p3 KO allele (bottom). After homologous recombination, the targeting vector replaces exons 1–5 (indicated by gray boxes) with the phosphoglycerate kinase-neomycin resistance gene (PGK-neo) cassette. The external 5′-flanking probe is indicated, as are the positions of PCR primers for detecting WT (P1 and P2) and mutant KO (P1 and P3) alleles. The position of the external 5′-flanking probe used for Southern blot analysis is indicated, as are the BamHI sites (denoted by the letter B). B, multiplex PCR analysis for genotype identification of 24p3^+/+, 24p3^+/−, and 24p3^−/− mice by multiplex PCR analysis for WT and mutant alleles. Genomic DNA was prepared from tail snips of 24p3^+/+, 24p3^+/−, and 24p3^−/− mice and amplified with P1, P2, and P3 primer pairs (P1 and P2 or P1 and P3) as described in A (P1 and P2 are for the WT allele, and P1 and P3 are for the mutant allele). A PCR product of 264 bp represents the WT allele, whereas a product of 164 represents the targeted KO allele. C, immunoblot analysis of proteins extracted from monitoring the presence of 24p3 in concentrated urine samples of from 24p3^+/+, 24p3^+/−, and 24p3^−/− mice. Immunoblot analysis was performed using specific antisera directed against 24p3 (Santa Cruz Biotechnology, Inc.). An immunoreactive band migrating at the size expected for 24p3 was detected in urine samples from 24p3^+/+ and 24p3^+/− mice but not in the urine sample from 24p3^−/− mice. An asterisk indicates a contaminating band that serves as a loading control.

FIGURE 2.

Control experiments confirming the success of bone marrow transplantation. A, schematic representation of the experimental strategy for bone marrow transplantation experiments. B, PCR analysis confirming the success of transplantation. Bone marrow cells isolated from recipient mice were analyzed by PCR for the presence of a Y chromosome-specific gene, Smcy (represented by the 312 bp band). The primers also amplify a homologous gene on the X chromosome, Smcx, due to intronic differences, (represented by the 341-bp band; ref. 8). Both donor and recipient mice were on the 129/SVE genetic background.

FIGURE 3.

Role of the 24p3/24p3R pathway in stress hematopoiesis. A, bone marrow cells were harvested from both hind limbs of either 24p3^−/− or control WT mice (both on C57BL/6 background CD45.2 +) and mixed with competitor Ly-5.1 BoyJ bone marrow cells (CD45.2⁺) at a ratio of 1:1. The mixed bone marrow cells were injected into lethally irradiated (11 Gy (1100 rads)) Ly-5.1 BoyJ recipient mice at a ratio of 1 donor equivalent to five recipients. The percentage of donor-derived CD45.2 chimerism in the peripheral blood of each recipient mouse was determined by flow cytometry. The donor engraftment was analyzed 9 weeks or later after the transplantation. Results shown are mean ± S.D. (error bars) from two independent transplantations with a total of 10 mice in each group. B, percentage of donor-derived chimerism in Gr-1- and B220-positive populations in the peripheral blood of each recipient mouse. The donor engraftment was analyzed 6 months post-transplantation. Results shown are mean ± S.D. from 24p3^+/+ (n = 7) and 24p3^−/− (n = 8). C and D, 5-FU was injected intraperitoneally (200 mg/kg), and at 0 days (before injection) and 7 days (after injection), mice were euthanized, and bone marrow from hind limbs (femur and tibia) was collected. The bone marrow counts are shown for total white blood cells (C) and for the Sca-1⁺Lin⁻ fraction (D).

FIGURE 4.

24p3/24p3R proapoptotic pathway regulates neutrophil apoptosis. A, total number of cells in bone marrow cultures derived from 24p3^+/+ and 24p3^−/− mice. B, percentage of Gr-1⁺/Mac-1⁺ cells in bone marrow cultures derived from 24p3^+/+ and 24p3^−/− mice. C, apoptosis assays. Cultured Gr-1⁺ bone marrow cells from 24p3^+/+ and 24p3^−/− mice were analyzed by annexin V-FITC staining. D, cultured Gr-1⁺ bone marrow cells from 24p3^+/+ and 24p3^−/− mice were analyzed for apoptosis by annexin V-FITC staining following the removal of G-CSF. E, colony formation assay in bone marrow cell cultures from 24p3^+/+ and 24p3^−/− mice. CFU-GM, colony-forming units, granulocyte and macrophage; CFU-GEMM, colony-forming units, granulocyte, erythroid, macrophage, and megakaryocyte. Error bars, S.D.

FIGURE 5.

The 24p3/24p3R proapoptotic pathway is critical for apoptosis induction in IL-3-dependent mast cells. A, apoptosis assays. BMMCs were cultured in the presence or absence of IL-3 or in the absence of IL-3 with the addition of FeCl₃ and analyzed for apoptosis by annexin V-FITC staining. B, naive BMMCs were treated with CM from BMMCs cultured in the presence or absence of IL-3, or in the absence of IL-3 with the addition of FeCl₃. As a control, BMMCs were treated with CM from IL-3-deprived FL5.12 cells. Error bars, S.D.

FIGURE 6.

The 24p3/24p3R proapoptotic pathway is required for glucocorticoid-mediated apoptosis in thymocytes. A, analysis of spontaneous apoptosis in primary thymocytes from 24p3^+/+ and 24p3^−/− mice. B, analysis of spontaneous and Dex-induced apoptosis in cultured thymocytes from 24p3^+/+ and 24p3^−/− mice. C, total thymic cellularity in 24p3^+/+ and 24p3^−/− mice following systemic administration of Dex or, as a control, saline. D, analysis of apoptosis in thymocytes in 24p3^+/+ and 24p3^−/− mice following systemic administration of Dex. E, FACS analysis monitoring the percentage of CD4⁺/CD8⁺ thymocytes in 24p3^+/+ and 24p3^−/− mice. F, analysis of splenic lymphocyte number in 24p3^+/+ and 24p3^−/− mice following systemic administration of Dex. G, analysis of B220⁺ bone marrow-derived pre-B cells in 24p3^+/+ and 24p3^−/− mice following systemic administration of Dex. Error bars, S.D.

FIGURE 7.

The 24p3/24p3R proapoptotic pathway regulates apoptosis in erythroid cells. A, analysis of Ter-119⁺ cells in peripheral blood (left), bone marrow (middle), and spleen (right) from 24p3^+/+ and 24p3^−/− mice. B, apoptosis assays in bone marrow and spleen from 24p3^+/+ and 24p3^−/− mice. C, analysis of apoptosis in Ter-119⁺ cells in bone marrow and spleen from 24p3^+/+ and 24p3^−/− mice. D, in vitro colony formation assay monitoring the number of BFU-E colonies from peripheral blood, bone marrow, and spleen from 24p3^+/+ and 24p3^−/− mice. Error bars, S.D.