Mol Cell Biol 23(24): 9349-9360

Deletion of α4 Integrins from Adult Hematopoietic Cells Reveals Roles in Homeostasis, Regeneration, and Homing

Division of Hematology, University of Washington, Seattle, Washington 98195-7710

^{Corresponding author. Mailing address: Division of Hematology, University of Washington, Box 357710, 1705 NE Pacific St., Seattle, WA 98195-7710. Phone: (206) 543-5756. Fax: (206) 543-3050. E-mail:}ude.notgnihsaw.u@plaht.

^{Present address: University Department of Hæmatology, Cambridge Institute of Medical Research, Cambridge CB2 2XY, United Kingdom.}

Received 2003 Jun 24; Revised 2003 Aug 15; Accepted 2003 Sep 18.

Abstract

We have explored the functional implications of inducible α4 integrin deletion during adult hematopoiesis by generating a conditional-knockout mouse model, and we show that α4 integrin-deficient hematopoietic progenitor cells accumulate in the peripheral blood soon after interferon-induced gene deletion. Although their numbers gradually stabilize at a lower level, progenitor cell influx into the circulation continues at above-normal levels for more than 50 weeks. Concomitantly, a progressive accumulation of progenitors occurs within the spleen. In addition, the regeneration of erythroid and myeloid progenitor cells is delayed during stress hematopoiesis induced by phenylhydrazine or by 5-fluorouracil, suggesting impairment in early progenitor expansion in the absence of α4 integrin. Moreover, in transplantation studies, homing of α4^{cells to the bone marrow, but not to the spleen, is selectively impaired, and short-term engraftment is critically delayed in the early weeks after transplantation. Thus, conditional deletion of α4 integrin in adult mice is accompanied by a novel hematopoietic phenotype during both homeostasis and recovery from stress, a phenotype that is distinct from the ones previously described in α4 integrin-null chimeras and β1 integrin-conditional knockouts.}

Abstract

Development of hematopoietic stem cells and their differentiated descendants in selective anatomic sites during fetal or adult life relies on favorable interactions with a specialized microenvironment that provides mechanical support and facilitates the enhancement of hematopoietic stem cells' proliferation and differentiation. The relationship between hematopoietic cells and cells within the microenvironment is a highly dynamic one, such that in response to stimuli, their coordinated responses can accommodate acute demands in cell expansion and migration in or out of the hematopoietic compartment to meet physiologic needs. As the molecular demands of stem cells residing in each anatomical site are likely to be different from those of differentiated cells, specialized niches are envisioned to accommodate these requirements (61). Members of the integrin family of cytoadhesive molecules are widely expressed in the hematopoietic system (56, 63) and exercise decisive roles in the interactions between hematopoietic cells and their microenvironment. This specialized function is dependent on the ability of integrins to serve not only as adhesion receptors but also as bona fide bidirectional signaling molecules that transduce signals to downstream effectors (20, 55). Integration of signaling networks is of particular importance in hematopoiesis, permitting cross talk with additional integrin molecules or with growth factor receptors, metalloproteinases, and chemokines in order to influence motility and other cellular functions (22, 26, 47). Moreover, the specificity in the actions of integrins in different anatomic locations may rely on the fact that signaling after ligation to immobilized ligands is both topographically constrained and cell type dependent.

To uncover integrin functions in the process of hematopoiesis, early studies used antibodies that not only defined their expression patterns but also uncovered their function and responses to stimuli in vitro and in vivo. These studies found that the β1 integrins are expressed by a wide variety of cell types, whereas the β2 integrins are found exclusively in hematopoietic cells (56, 63). Expression of β1 and β2 integrins is also differentiation dependent, with mature cells exhibiting diminished or inactive expression of α4β1 integrin (41, 56, 63) in contrast to stem and progenitor cells, which express α4 integrin in a constitutively active state (21, 38). Cells from different stages of development also display differential patterns of integrin expression. For example, fetal cells express more α2β1 integrin than do adult cells, a factor which dictates functional differences in their ability to adhere to collagen IV (52). Similarly, yolk sac-derived primitive erythroid cells display little α4 or α5 integrin expression in contrast to high levels expressed by fetal liver (FL) erythroid and adult bone marrow (BM) cells (40, 41). Perturbation of α4 integrin function in long-term BM cultures by an anti-α4 integrin antibody blocked the in vitro development of progenitors into mature erythroid, lymphoid, and myeloid cells (36, 70). Similarly, anti-α4 integrin antibodies injected into pregnant mice resulted in inhibition in FL erythroid development, but with little effect on lymphoid or myeloid development (15). In vivo studies have revealed additional roles for α4β1 integrin and its ligand, vascular cell adhesion molecule 1 (VCAM-1), in the homing to and short-term engraftment in the BM (42, 75). In vivo studies also stressed the role of α4 integrin in influencing migration of hematopoietic progenitors from the BM into circulation (9, 23, 43, 62, 71).

Experiments in mice deficient in β1 or α4 integrins have provided direct evidence of the involvement of integrins in hematopoiesis. Deletion of either the α4 or β1 integrin gene caused embryonic lethality from nonhematological defects (10, 58, 72); deletion of VCAM-1 resulted in a phenotype identical to that of the α4 integrin knockout (13, 28). Early lethality consequently precluded significant analysis of the roles of these adhesion molecules in hematopoietic development, although yolk sac hematopoiesis did remain intact in the α4 integrin knockout (3). The transition from α4 integrin independence to dependence at the liver colonization stage might explain the preservation of primitive erythropoiesis in α4 knockout animals. Studies with chimeric mice showed that α4 integrin-deficient hematopoiesis was compromised in the FL and was extinguished shortly after birth (3, 4). Although FL progenitor cells differentiated normally in vitro, their terminal differentiation in the FL and BM was impaired in the absence of α4 integrin, resulting in decreased proliferation and differentiation. Furthermore, β1 integrin-deficient cells were not able to colonize the FL, nor were they able to colonize the adult BM, spleen, or thymus of chimeras (18). However, in contrast to the significant defects in hematopoiesis in α4 chimeric mice and the migratory failure of β1-null cells during development, recent analysis of adult mice with conditional deletion of β1 integrin demonstrated no perturbations in myelopoiesis in the absence of all β1 integrins (6). Moreover, it is noteworthy that both of these genetic studies appear to diverge from studies obtained in vivo with the use of α4 or β1 antifunctional antibodies, although the interpretation of data with monoclonal antibodies has been questioned because of unwanted side effects either by steric hindrance of other interactions or by partial activation of target cells and/or binding to irrelevant cells (6).

Because of the difficulties in assigning specific roles to α4 integrin in hematopoiesis, especially between fetal and adult stages, we have generated conditional-knockout mice with α4 integrin alleles designed to be disrupted upon treatment of adult animals with interferon. This approach not only bypasses the embryonic lethality observed in α4 integrin knockouts but also causes a cell-intrinsic deficiency that cannot be restored by a normal BM microenvironment. By studying myelopoiesis before and after induction of stress, a novel hemopoietic phenotype was unveiled in these mice that has certain similarities to that of α4 integrin chimeras but displays significant differences from both these mice and the β1 integrin-conditional knockouts. The present studies expand our understanding of the role of α4 integrin in adult hematopoiesis at both the cellular and molecular levels.

Acknowledgments

We acknowledge the technical assistance of Denise Farrar, Betty Nakamoto, Alex Rohde, and Vivian Zafiropoulos. We are grateful to Ken Peterson for his contribution in the early stages of this work. James Downing (St. Jude's Children's Research Hospital, Memphis, Tenn.) kindly provided the MSCV-cre-iresGFP plasmid.

This work was supported by National Institutes of Health grant numbers HL46557 and HL58734, which were awarded to T.P.

Acknowledgments

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