Erythroblast apoptosis and microenvironmental iron restriction trigger anemia in the VK*MYC model of multiple myeloma

Abstract

Multiple myeloma is a malignant disorder characterized by bone marrow proliferation of plasma cells and by overproduction of monoclonal immunoglobulin detectable in the sera (M-spike). Anemia is a common complication of multiple myeloma, but the underlying pathophysiological mechanisms have not been completely elucidated. We aimed to identify the different determinants of anemia using the Vk*MYC mouse, which spontaneously develops an indolent bone marrow localized disease with aging. Affected Vk*MYC mice develop a mild normochromic normocytic anemia. We excluded the possibility that anemia results from defective erythropoietin production, inflammation or increased hepcidin expression. Mature erythroid precursors are reduced in Vk*MYC bone marrow compared with wild-type. Malignant plasma cells express the apoptogenic receptor Fas ligand and, accordingly, active caspase 8 is detected in maturing erythroblasts. Systemic iron homeostasis is not compromised in Vk*MYC animals, but high expression of the iron importer CD71 by bone marrow plasma cells and iron accumulation in bone marrow macrophages suggest that iron competition takes place in the local multiple myeloma microenvironment, which might contribute to anemia. In conclusion, the mild anemia of the Vk*MYC model is mainly related to the local effect of the bone marrow malignant clone in the absence of an overt inflammatory status. We suggest that this reproduces the initial events triggering anemia in patients.

Figure 1.

The Vk*MYC disease model. Analysis of serum monoclonal gammopathy in samples from 20-, 35-, 50- and 80-week old Vk*MYC mice and wild-type littermates. (A) Incidence of monoclonal component (M-spike). (B) Percentage of M-spike on total serum proteins in affected Vk*MYC and wild-type mice. (C) Hemoglobin (Hb) and red blood cells (RBC) count in 50-week old Vk*MYC affected mice, wild-type and non-affected Vk*MYC littermates. All the analyses were performed in both sexes. The number of mice for each group was described in the Methods section. *P<0.05. ns: not statistically significant.

Figure 2.

Body iron distribution. (A) Hepcidin (Hamp) expression measured by qRT-PCR. (B) Spleen and (C) Liver Iron Content (LIC). (D) Serum transferrin saturation. (E) Correlation between liver Hamp expression and LIC in 80-week old wild-type mice. (F) Correlation between liver Hamp expression and LIC in 80-week old Vk*MYC mice. All the analyses were performed on male Vk*MYC affected mice and wild-type littermates. The number of mice of each group is indicated by the symbols in the figures. ns: not statistically significant.

Figure 3.

Flow cytometry analysis of erythropoiesis. (A) Identification of clusters of erythroid precursors at different maturation stages. Left panels: recognition of bone marrow (BM) TER119⁺ cells (Erythroid precursors, gate 2) inside the Side Scatter (SSC)^low CD11b⁻ B220⁻ cells. Right panels: density plot of CD44 versus Forward Scatter (FSC) of BM erythroid precursors (Gate 2) showing naturally occurring clusters (fractions I–V). (B) Representative plots of BM erythroid precursors of Vk*MYC and wild-type littermates with the gates on erythroid fractions I–V. Right histogram: quantification of BM erythroid fractions I–V with respect of total BM cells. (C) Representative caspase 8 activation assay by IETD-FMK stain in erythroblast fraction III–V of wild-type (black line) and Vk*MYC mice (gray filled curve) with gates on IETD-FMK⁺ cells. Dotted line: negative control (Vk*MYC samples lacking IETD-FMK stain). Right histogram: quantification of the percentage of erythroid cells showing caspase 8 activation with respect of total erythroid cells of each fraction. (D) Representative plots of spleen erythroid precursors of Vk*MYC and wild-type littermates with the gates on erythroid fractions I–IV. Bottom histogram: quantification of spleen erythroid fractions I–IV with respect of total spleen cells. Quantifications were performed on 50-week old male mice on samples from 4 Vk*MYC affected mice compared with 4 wild-type littermates. Error bars indicate standard error. * P<0.05.

Figure 4.

Flow cytometry analysis of iron transporters in non-erythroid cells. (A) Representative plots of CD138 versus B220 with the gating on CD138⁺ B220⁻ cells. (B) Representative plots of F4/80 versus CD11b with the gating on F4/80⁺ and on F4/80⁻ CD11b⁺ cells. (C) Left and central panels: representative CD71 expression in F4/80⁺ and CD138⁺ B220⁻ cells. Right panel: quantification of CD71 expression. (D) Representative ferroportin expression in F4/80⁺ and CD138⁺ B220⁻ cells. Black line: wild-type. Gray filled curve: Vk*MYC. Dotted line: negative control (representative samples lacking CD71 or ferroportin stain). (E) Representative Perl’s staining shows evident iron accumulation in the residual hematopoietic marrow of Vk*MYC affected mice in comparison with age-matched wild-type animals. Higher magnification reveals that most iron is contained in macrophage cytoplasm. All the analyses were performed on 50-week old male mice on samples from 4 Vk*MYC affected mice compared with 4 wild-type littermates. Error bars indicate standard error. *P<0.05.

Figure 5.

Bone marrow (BM) markers of inflammation and apoptosis. (A) Gene expression analysis by qRT-PCR in CD11b⁺ cells separated from BM and in residual cell fraction. (B) Left panels: representative density plots of CD138 versus B220 with the gating on CD138⁺ B220⁻, on CD138⁺ B220⁺ and on CD138⁻ B220⁺. Right panels: representative Fas-L expression in cell populations gated in A. Black line: wild-type. Gray filled curve: Vk*MYC. Dotted line: negative control (Vk*MYC samples lacking Fas-L stain). All the analyses were performed on 50-week-old male mice on samples from 4 Vk*MYC affected mice compared with 4 wild-type littermates. Error bars indicate standard error. *P<0.05.