The macrophage contribution to stress erythropoiesis: when less is enough.

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.

Combinatorial and distinct roles of α5 and α4 integrins in stress erythropoiesis in mice.

To delineate the role of specific members of ß1 integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of ß1-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in ß1(Δ/Δ) reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to ß1-deficient mice, α4(Δ/Δ) mice showed only a kinetic delay in recovery and similar to ß1(Δ/Δ), terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1(Δ/Δ) mice. Convergence of information from these comparative studies lends new insight to the distinct in vivo roles of α4 and α5 integrins in erythroid stress, suggesting that the presence of mainly α5ß1 integrin in all hematopoietic progenitor cells interacting with splenic microenvironmental ligands/cells is instrumental for their survival and accumulation during hemolytic stress, whereas presence of α4 or of both α5 and α4, is important for completion of terminal maturation steps.

Is the post-transplantation treatment with AMD beneficial?

Recent data have suggested novel ways to enhance donor cell engraftment by treating transplanted recipients with CXCR4/CXCL12 inhibitors, thereby expanding the biologic potential of these molecules primarily used for mobilization purposes. We tested whether repeated pulse inhibitions of CXCR4/CXCL12 signaling using AMD, an inhibitor of CXCR4/CXCL12 signaling, would enhance engraftment in non-myeloablated murine recipients, similar to data published in a myeloablative setting. We documented an increased proportion of circulating neutrophils (both donor- and host-derived) in the AMD-treated group, but this increase was not kinetically influenced by AMD treatment and multilineage engraftment was not enhanced. Although our results with neutrophils are similar to recent clinical data in neutropenic patients chronically treated with AMD, the absence of multilineage engraftment diverges from data in myeloablated recipients. We conclude that pulses of mobilization by AMD post transplantation do not enhance multilineage engraftment.

On the adaptation of endosteal stem cell niche function in response to stress.

Although the influence of microenvironmental "niche" on the function of a variety of stem cells is undisputed, the details of hematopoietic stem cell/niche interactions at the cellular and molecular level have sparked a continuous debate. We studied the microanatomic partitioning of transplanted normal and alpha4 integrin-deficient Lin-kit+ cells in trabecular and compact bone before and after irradiation and present robust quantitative data on both. We found that (1) the microanatomic distribution of normal highly enriched progenitor cells is random in nonirradiated recipients based on area distribution analyses, (2) in contrast, in irradiated hosts normal cells distribute preferentially near the endosteum, (3) the overall cell seeding efficiency was higher in trabecular versus compact bone both before and after irradiation, and (4) alpha4 integrin-deficient cells not only lodge with reduced overall efficiency confirming previous data, but fail to preferentially partition themselves into endosteal regions in irradiated hosts, as normal cells do. A similar phenotype was observed with cells rendered G(i)-protein signaling incompetent by pertussis toxin treatment, supporting an active stromal-derived factor 1 (SDF-1) gradient near endosteum after irradiation.

Diverse hematopoietic potentials of five human embryonic stem cell lines.

Despite a growing body of literature concerning the hematopoietic differentiation of human embryonic stem cells (hESCs), the full hematopoietic potential of the majority of existing hESC lines remains unknown. In this study, the hematopoietic response of five NIH-approved hESC lines (H1, hSF6, BG01, BG02, and BG03) was compared. Our data show that despite expressing similar hESC markers under self-renewing conditions and initiating mesodermal differentiation under spontaneous differentiation conditions, marked differences in subsequent hematopoietic differentiation potential among these lines existed. A high degree of hematopoietic differentiation was attained only by H1 and BG02, whereas this process appeared to be abortive in nature for hSF6, BG01, and BG03. This difference in hematopoietic differentiation predisposition was readily apparent during spontaneous differentiation, and further augmented under hematopoietic-inducing conditions. This predisposition appeared to be intrinsic to the specific hESC line and independent of passage number or gender karyotype. Interestingly, H1 and BG02 displayed remarkable similarities in their kinetics of hematopoietic marker expression, hematopoietic colony formation, erythroid differentiation, and globin expression, suggesting that a similar, predetermined differentiation sequence is followed. The identification of intrinsic and extrinsic factors governing the hematopoietic differentiation potential of hESCs will be of great importance for the putative clinical utility of hESC lines.

VCAM-1 ablation in nonhematopoietic cells in MxCre+ VCAM-1f/f mice is variable and dictates their phenotype.

OBJECTIVE: The goal of the present study was to assess the extent of vascular cell adhesion molecule-1 (VCAM-1) gene deletion in hematopoietic vs nonhematopoietic cells in the bone marrow (BM) of MxCre(+)VCAM-1(f/f) mice and its impact on the phenotypic features of these mice. METHODS: VCAM-1 ablation was evaluated at the genomic level by polymerase chain reaction (PCR), at the mRNA level by real-time PCR, and at the protein level by fluorescein-activated cell sorting and immunohistochemistry. The homing or mobilization of colony-forming unit cultures was assessed by standard assays. RESULTS: A previously accepted interferon-induction scheme yielded efficient VCAM-1 ablation in hematopoietic cells but variable ablation in BM fibroblasts and endothelial cells. The level of ablation in the latter populations correlated with alterations in the hematopoietic phenotype. CONCLUSIONS: Poly(I:C)-induced MxCre-mediated gene ablation is highly efficient in hematopoietic cells but variable and partial in nonhematopoietic cells in BM. Ablation of VCAM-1 in hematopoietic cells does not contribute to their mobilization, nor does it impair their homing. The latter is dependent on VCAM-1 ablation in nonhematopoietic cells of BM.

Unique and redundant roles of alpha4 and beta2 integrins in kinetics of recruitment of lymphoid vs myeloid cell subsets to the inflamed peritoneum revealed by studies of genetically deficient mice.

OBJECTIVE: Leukocyte recruitment to inflammatory sites is a prominent feature of acute and chronic inflammation. Instrumental in this process is the coordinated upregulation of leukocyte integrins (among which alpha4beta1 and beta2 integrins are major players) and their cognate receptors in inflamed tissues. To avoid the ambiguity of previous short-term antibody-based studies and to allow for long-term observation, we used genetically deficient mice to compare roles of alpha4 and beta2 integrins in leukocyte trafficking. MATERIALS AND METHODS: Aseptic peritonitis was induced in alpha4 or beta2 integrin-deficient (conditional and conventional knockouts, respectively) and control mice, and recruitment of major leukocyte subsets to the inflamed peritoneum was followed for up to 4 days. RESULTS: Despite normal chemokine levels in the peritoneum and adequate numbers, optimal recruitment of myeloid cells was impaired in both alpha4- and beta2-deficient mice. Furthermore, clearance of recruited neutrophils and macrophages was delayed in these mice. Lymphocyte migration to the peritoneum in the absence of alpha4 integrins was drastically decreased, both at steady state and during inflammation, a finding consistent with impaired lymphocyte in vitro adhesion and signaling. By contrast, in the absence of beta2 integrins, defects in lymphocyte recruitment were only evident when peritonitis was established. CONCLUSIONS: Our data with concurrent use of genetic models of integrin deficiency reveal nonredundant functions of alpha4 integrins in lymphocyte migration to the peritoneum and further refine specific roles of alpha4 and beta2 integrins concerning trafficking and clearance of other leukocyte subsets at homeostasis and during inflammation.

Sustained alterations in biodistribution of stem/progenitor cells in Tie2Cre+ alpha4(f/f) mice are hematopoietic cell autonomous.

We have generated Tie2Cre+alpha4(f/f) mice with documented alpha4-integrin ablation in hematopoietic and endothelial cells. A prominent feature in this model is a sustained, significant increase in circulating progenitors at levels higher than the levels seen with Tie2Cre+VCAM-1(f/f) mice. To test whether phenotypic differences are due to contributions by ligands other than VCAM-1 in bone marrow, or to alpha4-deficient endothelial cells or pericytes, we carried out transplantation experiments using these mice as donors or as recipients. Changes in progenitor biodistribution after transplantation were seen only with alpha4-deficient donor cells, suggesting that these cells were necessary and sufficient to reproduce the phenotype with no discernible contribution by alpha4-deficient nonhematopoietic cells. Because several similarities are seen after transplantation between our results and those with CXCR4-/- donor cells, the data suggest that VLA4/VCAM-1 and CXCR4/CXCL12 pathways contribute to a nonredundant, ongoing signaling required for bone marrow retention of progenitor cells during homeostasis.

Lack of alpha4 integrin expression in stem cells restricts competitive function and self-renewal activity.

Alpha4 integrin or VLA4 (CD49d/CD29) is a multitask molecule with wide expression within and outside the hematopoietic system. Because targeted ablation of alpha4 integrin leads to embryonic lethality, to study its effects on adult hematopoiesis, we used animals with conditional excision of alpha4 integrin (alpha4Delta/Delta) in hematopoietic cells. In such animals, we previously documented weakened bone marrow retention of progenitor cells during homeostasis and impaired homing and short-term engraftment after transplantation. In the present study we show that long-term repopulating cells lacking alpha4 integrins display a competitive disadvantage in hematopoietic reconstitution compared to normal competitors. Although initial dominance of alpha4+ competitors is due to their better homing and proliferative expansion early after transplantation, a progressive decline in contribution of alpha4Delta/Delta hematopoiesis is compatible with neither normal homing nor normal function of alpha4Delta/Delta hematopoietic stem cells (HSCs) in post-homing hematopoiesis. In the absence of alpha4+ competitor cells, alpha4Delta/Delta HSCs can establish long-term hematopoiesis in primary recipients, however, some resurgence of host hematopoiesis is evident, and it becomes dominant in secondary transplants, so that no survivors with exclusively alpha4Delta/Delta cells are seen in tertiary transplants. Collectively, our data provide compelling evidence that under regenerative stress alpha4 integrin assumes a greater importance than for maintenance of steady-state hematopoiesis.

VCAM-1 expression in adult hematopoietic and nonhematopoietic cells is controlled by tissue-inductive signals and reflects their developmental origin.

Although expression of vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells and its functional implications have been previously appreciated, VCAM-1 expression in other than endothelial cells, especially hematopoietic cells, has been recently recognized and has not been explored in detail. Using normal mice and mice with a conditional ablation of VCAM-1 through a Tie2-driven cre transgene, we have studied the biodistribution and the pattern of VCAM-1 expression in circulating versus tissue-residing cells before and after their enforced mobilization. In the normal mouse, both at basal hematopoiesis or following mobilization, VCAM-1 expression is confined to myeloid cells residing in hematopoietic tissues, whereas free cells in circulation or in body cavities are devoid of VCAM-1 messenger RNA (mRNA) and protein. However, following culture, proliferating myeloid cells, but not lymphoid cells, express VCAM-1. In the VCAM-1-ablated mouse, there is an increase in circulating progenitors as a consequence of their ongoing release from bone marrow, a process enhanced by splenectomy. We postulate that the main mechanism leading to their release is the ablation of VCAM-1 by fibroblastic and by endothelial cells. Ablation of VCAM-1 in fibroblasts by Tie2-driven cre is a novel finding and likely denotes their developmental ancestry by Tie2-expressing (mesenchymal?) progenitor cells during development.