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. 2016 Sep 29;128(13):1756-65.
doi: 10.1182/blood-2016-05-714527. Epub 2016 Aug 19.

The macrophage contribution to stress erythropoiesis: when less is enough

Tatiana Ulyanova  1 , Susan R Phelps  1 , Thalia Papayannopoulou  1
Affiliations

The macrophage contribution to stress erythropoiesis: when less is enough

Tatiana Ulyanova et al. Blood. .

Abstract

Although the importance of native bone marrow and spleen macrophages in enhancing baseline and stress erythropoiesis has been emphasized over several decades, their kinetic and phenotypic changes during a variety of stress responses have been unclear. Furthermore, whether monocyte-derived recruited macrophages can functionally substitute for inadequate or functionally impaired native macrophages has been controversial and seem to be not only tissue- but also stress-type dependent. To provide further insight into these issues, we made detailed observations at baseline and post-erythroid stress (E-stress) in 2 mouse models with genetically depressed macrophage numbers and compared them to their controls. We documented that, irrespective of the stress-induced (hemolytic or post-erythropoietin [Epo]) treatment, only native CD11b(lo) splenic macrophages expand dramatically post-stress in normal mice without significant changes in the monocyte-derived CD11b(hi) subset. The latter remained a minority and did not change post-stress in 2 genetic models lacking either Spi-C or VCAM-1 with impaired native macrophage proliferative expansion. Although CD11b(lo) macrophages in these mice were one-fifth of normal at their peak response, surprisingly, their erythroid response was not compromised and was similar to controls. Thus, despite the prior emphasis on numerical macrophage reliance to provide functional rescue from E-stress, our data highlight the importance of previously described non-macrophage-dependent pathways activated under certain stress conditions to compensate for low macrophage numbers.

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Figures

Figure 1
Figure 1
Macrophage subsets in WT mice at steady state and during response to stress. (A) Morphology and expression of F4/80, CD11b, and VCAM-1 was analyzed in EI preparations isolated from spleens of PHZ-treated or Epo-treated mice on day 4; Hema3 stain: note the different maturation stages of Ebs that adhere to themselves and to the central macrophage engorged in damaged red cells after PHZ treatment (i); and DAPI-counterstained EIs (ii-v). Light green stain for F4/80 and magenta nuclear stain (assigned color) for surrounding Ebs (ii); light green CD11b+ cells (iii); dotted circles indicate central macrophages. Light green F4/80+ cells and DAPI-stained Ebs (iv); and green VCAM-1 positivity on central macrophages (v). CD169 expression on F4/80+ cells was analyzed by FACS after EI disaggregation (bottom right panel). (B) Percentage of F4/80+ cells in the BM and spleen that are CD11bhi or CD11blo at steady state (CD11bhi: red; CD11blo: blue). VCAM-1 expression in the 2 subsets (right panel). Note that only CD11blo express VCAM-1 in the spleen. (C) Quantitative kinetic changes in the 2 F4/80+ subsets after PHZ or Epo challenge. (D) Quantitative changes in the 2 F4/80+ subsets in transplanted mice (WT→WT) treated with PHZ 9 weeks after transplantation (left). Cycling status (Ki-67 antibodies; see “Methods”) of the 2 subsets before and after stress (right). Note differences only in the CD11blo subset. The high Ki-67 positivity in this subset changed from 9.63 ± 1.27% to 45.9 ± 6.15% and finally to 76.15 ± 1.25% on days 0 (light blue), 4 (peach) and 6 (red), respectively. No such population was present in CD11bhi subset (arrow). Number of mice: day 0, n = 6; PHZ-induced stress, day 4, n = 4; day 6, n = 6; and Epo-induced stress, n = 3. Images in (Ai,iv-v) were taken with a Leica DMLB camera (objective N PLAN 40×/0.65, eye piece HC PLAN 10×/22) and 20× objective for (Aii-iii). d, day; DAPI, 4,6 diamidino-2-phenylindole; Spl, spleen.
Figure 2
Figure 2
Total and CD11blo macrophage subsets are greatly reduced before and during E-stress response in Spi-C–deficient and VCAM-1–deficient mice. Numbers of CD11blo (lighter shades) and CD11bhi (darker shades) macrophages, as well as VCAM-1 expression in F4/80+ cells in the BM and spleen were determined at several points after PHZ challenge. (A) Spi-C–deficient mice (blue bars and lines, n = 3 for each time point). (B) VCAM-1 knockouts (peach bars and lines, n = 3 to 5 mice for each time point). (C) Normal irradiated recipients transplanted (Tx) with either normal donor cells (red hatched bars) or VCAM-1−/− donor cells (peach hatched bars, n = 5 for each time point). Red bars and lines represent the respective controls (n = 6 to 12 mice for each time point). Significant difference from control; *P < .05. Note that total F4/80+ cells are significantly reduced in mutant mice compared with their controls before and especially after PHZ challenge, mainly in the spleen. Far right panels in (A-B) indicate levels of VCAM-1 expression in total macrophages. Note that less VCAM-1 expression (lower MFI) is seen in Spi-C–deficient and virtually no expression in VCAM-1–deficient macrophages (low MFI VCAM-1 was seen in <10% of F4/80+ cells). Ctrl, control; d, day; MFI, mean fluorescence intensity.
Figure 3
Figure 3
Erythroid responses to PHZ challenge in Spi-C–deficient and VCAM-1–deficient mice. (A) Cellularity and total number of Ebs in the femur (left panels) and spleen (right panels) of respective controls for each group of mice (red bars), Spi-C–deficient mice (blue bars, upper panels), VCAM-1–deficient mice (peach bars, middle panels), and control mice irradiated and transplanted with either control donor cells (red striped bars) or VCAM-1–deficient donor cells (peach striped bars, lower panel). (B) Erythroid maturation profiles in femur and spleen of Spi-C–deficient mice (blue lines, left panels) VCAM-1–deficient mice (peach lines, middle panels) and control mice irradiated and transplanted (Tx) with either control donor cells or VCAM-1–deficient donor cells (peach lines, right panels) and their respective controls (red lines) at day 4 (solid lines) and at day 6 post-PHZ (broken blue, peach, and red lines, respectively). Blue bars and lines, n = 3 for each time point; and peach bars and lines, n = 3 to 5 mice for each time point; red bars and lines, n = 6 to 12 mice for each time point. Significant difference over controls; *P < .05 d, day; Baso, basophilic Eb; Poly, polychromatophilic Eb; Ortho, orthochromatic Eb; Pro, proerythroblast.
Figure 4
Figure 4
Dynamics of macrophage subsets CD11blo (lighter shades) and CD11bhi (darker shades) in response to aCD24 antibody treatment. WT (red bars) and VCAM-1−/− blue bars) mice were treated with aCD24 antibody or control immunoglobulin G (see “Methods”). Cellularity (left panels), macrophage (middle panels), and Eb (right panels) responses were followed in the BM (A) and spleen (B). Note the major changes after treatment in the spleen only. These data are consistent with the fact that the spleen and not the BM is the major source of SCF produced after CD24 engagement. SCF, stem cell factor.
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