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STEM CELLS AND DEVELOPMENTVolume 18, Number 6, 2009© Mary Ann Liebert, Inc.DOI: 10.1089/scd.2008.0269

Blockade of α6-Integrin Reveals Diversity in Homing Patterns Among Human, Baboon, and Murine Cells

Halvard Bonig,1,2 Gregory V. Priestley,1 Martin Wohlfahrt,3 Hans-Peter Kiem,3 and Thalia Papayannopoulou1

Our understanding of the mechanisms by which intravenously transplanted hematopoietic stem/progenitor cells (HSPCs) home to and engraft the bone marrow (BM) remains incomplete, but participation of adhesion molecules has been documented. We here demonstrate that blockade of the α6-integrin enhanced BM homing of human and nonhuman primate BM-derived HSPCs by >60% in the xenogeneic transplant model and led to signifi cantly improved engraftment. The effect was limited to BM-derived HSPCs, as granulocyte-colony-stimulating factor mobilized peripheral blood or cord blood HSPCs express little or no α6 integrin. By contrast, despite high α6 integrin expression, no effect of α6 blockade on murine BM-HSPCs homing/engraftment was observed.

Introduction

Homing of intravenously (i.v.) transplanted hematopoi-etic stem/progenitor cells (HSPCs) to bone marrow (BM)

involves a series of well-coordinated interactions between HSPC adhesion receptors and stromal cell/extracellular matrix ligands [1,2]. Expression of a number of adhesion recep-tors on HSPCs has been reported [3], and functional roles for several α-partners of the β1-integrins have been documented. Thus blockade of α4β1 integrin negatively infl uences homing of human and murine HSPCs [4,5]. Divergent effects between human and murine HSPCs emanate from studies into the role of α5β1 integrin in HSPC homing/engraftment [5–8], and no effect on homing was associated with inhibition of α2β1 integrin in the mouse, presumably because α2-integrin is not expressed on the most primitive HSPCs [9].

Little is known about the functional role of the α6-integrin on HSPCs, even though its expression by most, if not all, BM–HSPCs has been unequivocally reported. α6-Integrin can heterodimerize with β1 and β4. Both dimers are laminin recep-tors. α6β4 binds laminin-5 and is thus required for epithelial integrity. Since hematopoietic cells do not appear to express β4-integrin [10], α6β1 (VLA6) is likely the sole hematopoietic α6-integrin heterodimer. On mature hematopoietic cells, α6 can function as an adhesion molecule and as a receptor for laminin-induced outside-in signals mediating haptotaxis.

Outside hematopoiesis, complex roles for α6-integrin have been described, including roles in reproduction, epithelial integrity, and organogenesis, all of which also appear to hinge on interactions with various laminin isoforms [11–13]. Mediated through α6β1, HSPCs bind to several laminin iso-forms, including, but likely not limited to, laminins 1, 8, and 10/11 [14,15]. However, observations about the functional role of α6β1-integrin in primitive hematopoiesis are thus far incomplete [16,17], and were therefore pursued here.

In this study, differential expression levels of α6-integrin on human BM- and granulocyte-colony-stimulating fac-tor mobilized peripheral blood- (MPB-) HSPCs were docu-mented, which are likely responsible for the differential functional consequences of α6-integrin blockade for homing and engraftment. In contrast to human cells, similar studies with murine HSPCs did not demonstrate α6-integrin depen-dent roles for homing/engraftment.

Materials and Methods

Mice

B6x129, nonobese diabetic/severe combined immunodefi -ciency (NOD/SCID)β2-microglobulin−/− or NOD/SCIDγc−/− (Jackson Laboratories, Bar Harbor, ME) were housed at the

1 Department of Medicine Division of Hematology, University of Washington, Seattle, Washington.

2 German Red Cross Blood Center and Institute for Transfusion Medicine and Immunohematology of the Johann-Wolfgang-Goethe

University, Frankfurt, Germany.3

Fred Hutchinson Cancer Research Center, Seattle, Washington.

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BONIG ET AL.840

Flow cytometry

Flow cytometry, using directly conjugated Abs (BD-Pharmingen), was performed according to standard protocols using the FACSCalibur (BD Immunocytometry Systems, San Jose, CA).

Statistics

Homing effi ciency was compared using the t-test. Probability of engraftment among groups was compared using the χ2 test. Descriptive statistics, t-tests and χ2 statis-tics were calculated using Excel (Microsoft, Redmond, WA).

Results

Expression of α6-integrin on human and baboon CD34+ cells

α6-Integrin is expressed on virtually all CD34+ cells from human and baboon BM (Fig. 1A and C). By compari-son, expression of α6-integrin was much lower on human MPB-CD34+ (Fig. 1B), and virtually absent from human cord blood (CB) CD34+ cells (Fig. 1B). Thus α6-integrin joins the list of adhesion molecules for which reduced expression on MPB- and CB-CD34+ cells was shown [23,24].

Effect of α6-blockade on homing and engraftment of human and baboon HSPCs

HSPCs were incubated in vitro with saturating concen-trations of α6-integrin-blocking Ab (anti-α6-Ab) (Fig. 1C) or with similar concentrations of isotype-matched control Abs prior to transplantation. This treatment had no effect on colony formation in vitro (Fig. 1D). Homing of anti-α6-Ab-treated CFU-C from human BM, assessed 20 h after injection into lethally irradiated NOD/SCIDβ2-microglobulin−/− mice, was increased by more than two-thirds compared to control-treated HSPCs (mean ± SEM from three indepen-dent experiments: control Ab 1.2 ± 0.13% vs. anti-α6-Ab 2.2 ± 0.17% of injected human CFU-C/2 femurs, Fig. 1E). Similar results were observed when baboon BM-HSPCs were used as transplants (Fig. 1G). At the same time, the number of HSPCs recovered from spleens was signifi cantly reduced in recipients of anti-α6-Ab treated HSPCs (Fig. 1E and G). Recovery from lungs was highly variable, but also trended toward lower values (Fig. 1E). Since enumeration of CFU-C homed to liver and lung could not be successfully achieved with our in vivo homing assays, a surrogate in vitro adhesion assay to sections from irradiated lung and liver was performed and demonstrated decreased binding of anti-α6-Ab-treated CD34+ cells compared to controls (Supplementary Fig. 1; Supplementary materials are avail-able online at http://www.liebertpub.com). In keeping with their superior homing, 8-week engraftment of human cells in sublethally conditioned NOD/SCIDγc−/− mice was signif-icantly increased after incubation with anti-α6-Ab (Fig. 1H). In contrast to BM-HSPCs, BM homing of human MPB-HSPC was not signifi cantly affected by anti-α6-Ab incubation (Fig. 1F). In agreement with these data, short-term engraftment of these cells was also no different between anti-α6-Ab and control-treated HSPCs (not shown).

specifi c pathogen-free facilities at University of Washington Medical Center or Fred Hutchinson Cancer Research Center. All procedures were approved by the institutional animal care and use committee.

Cells

Cryopreserved human CD34+ BM- or MPB cells were gifts from Shelley Heimfeld (Fred Hutchinson Cancer Research Center, Seattle, WA) and JoAnna Reems (Puget Sound Blood Center, Seattle, WA). CD34+ cord blood (CB) cells were a gift from Chris Miller (University of Washington, Seattle, WA). Baboon CD34+ BM cells were isolated as previously described [18]. Murine BM cells were fl ushed from long bones; where indicated, c-kit+ cells were purifi ed with immunomagnetic beads according to manu-facturer’s instructions (Miltenyi Biotech, Auburn, CA). Cells were incubated with the α6-function-blocking antibody (Ab) GoH3 (BD-Pharmingen, San Diego, CA) or isotype-matched control Ab (BD) (20 μg/mL, 4 C, 30 min) [19].

Transplantation

For homing experiments, hosts were irradiated with a single dose of 1,250 cGy total-body irradiation (TBI), fol-lowed by i.v. injection of HSPC suspensions as indicated. Progenitor cell (colony-forming cell culture, CFU-C) homing to BM as well as, where indicated, CFU-C content in blood, spleen, and lung were assessed by plating aliquots of cell sus-pensions from these organs, as described [20], 3 or 20 h after transplantation, using commercially available semi-solid complete colony assay media (Miltenyi Biotech for human CFU-C, StemCell Technologies, Vancouver, BC, Canada for murine CFU-C) [18]. For engraftment studies, recipients of human cells received 350 cGy TBI, recipients of mouse cells 1,250 cGy TBI. Engraftment was analyzed by quantifi ca-tion of donor-derived CD45+ cells in blood [21] or donor CFU-C contents in femurs [20]. For human engraftment, a threshold of 1% was considered indicative of engraftment. Transplanted cell doses per recipient were 0.4–0.6 × 106 for human or baboon CD34+ cells, 3–5 × 106 for murine c-kit+ cells, or 20–30 × 106 for total murine BM cells.

Adhesion assay

Adhesion to tissue sections was assessed as described [22]. Frozen tissue sections of liver (fresh) and lung (pre-fi xed) from lethally irradiated mice were prepared. A 1:1 mix of control and anti-α6-Ab-treated CD34+ cells, loaded with red (SNARF) or green (CFSE) cell dyes (Molecular Probes, Eugene, OR) according to manufacturer’s instructions, was allowed to adhere to the sections (1 h, room temperature). After washing by 10 times dipping into phosphate-buffered saline at room temperature, to remove nonadherent cells, slides were dried and mounted with Vectashield+DAPI. Adhering cells were counted by fl uorescence microscopy in all sections (Microscope: Leica DMIRE2, Wetzlar, Germany, with triple bandpass fi lter (420, 520, and 610 nm); camera: Spot RT Slider, Diagnostic Instruments, Sterling Heights, MI; Acquisition software: Openlab 3.1.7, Improvision, Lexington, MA). Red, green, and blue pseudocolored images were merged after adjustment of contrast and brightness.

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ROLE OF 𝛂6-INTEGRIN IN HOMING 841

Likewise, short-term engraftment was not affected by anti-α6-Ab incubation (Fig. 2F).

Discussion

Many earlier studies have addressed the effects of anti-functional Abs on homing and engraftment of HSPCs [5,6,9,25]. Although the interaction between HSPCs and Ab in vivo is likely transient, the interaction is suffi ciently long to affect homing, because the bulk of HSPCs homing to BM do so within 3 h of transplantation, and little change occurs thereafter [26]. Thus negative effects of antifunctional Abs on

Expression of α6-integrin and effects on homing and engraftment of murine BM-HSPCs

Similarly to human and baboon BM-CD34+ cells, virtu-ally all murine c-kit+ cells express α6, at a similar density as primate HSPCs (Fig. 2A). Nevertheless, homing of anti-α6-Ab-treated (saturating conditions, Fig. 2B) murine HSPCs in isogeneic (Fig. 2C and D) or in NOD/SCIDβ2-microglobulin−/− (Fig. 2E) hosts, tested 3 or 20 h after transplantation, was no different from control-treated HSPCs. This outcome was ob-served whether whole BM or c-kit-enriched BM-HSPCs were used, in normal as in splenectomized recipients (Fig. 2D).

160

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FIG. 1. Blockade of α6-integrin enhances marrow homing of human and baboon BM CFU-C. (A) Expression of α6-integrin on human BM (dark gray) or human MPB (light gray) CD34+ cells (unfi lled black line: isotype control). (B) Expression of α6-integrin on human CB (unfi lled black line) CD34+ cells (light gray: isotype control). (C) Saturation of α6-integrin-binding sites under the conditions used for α6-blockade. In contrast to control-Ab-incubated HSPCs, anti-α6-Ab-treated HSPCs (here, baboon CD34+ BM cells) did not bind PE-conjugated anti-α6-Ab (dark gray: anti-α6-Ab-incubated HSPCs; unfi lled black line: control incubated HSPCs; light gray: isotype control). (D) No effect of incubation of HSPCs (here, human CD34+ cells) with antifunctional α6-Ab on CFU-C formation, suggesting the absence of direct effects of anti-α6-Ab on prolifera-tion. (E) Marrow homing of human BM CFU-C in lethally irradiated (1,250 cGy) NOD/SCIDβ2-m−/− recipients, enumerated 20 h after transplantation, was >75% increased after α6-integrin blockade (three experiments, each with 3–5 mice/group, one representative experiment shown here). Concomitantly, CFU-C content in spleens was signifi cantly reduced. Average CFU-C content in lungs of recipients of anti-α6-Ab-treated HSPCs was also reduced >50%, but because of great variability this did not reach statistical signifi cance. (F) Marrow homing of human MPB CFU-C in lethally irradiated (1,250 cGy) NOD/SCIDβ2m−/− recipients, enumerated 20 h after transplantation, was neither signifi cantly increased nor was spleen homing different from controls (one experiment, 4 mice/group). (G) 20-h marrow homing of baboon BM CFU-C in lethally irradiated (1,250 cGy) NOD/SCIDβ2m−/− hosts was almost 70% increased, and spleen homing 50% reduced, after α6-blockade (two experiments, each with 4 recipients/group). (H) The probability of human engraftment, measured 8 weeks after transplanta-tion, was signifi cantly increased in sublethally (350 cGy) radioconditioned NOD/SCIDγc−/− recipients of anti-α6-Ab-treated human BM-CD34+ cells (7/9 mice) compared to recipients of control-treated cells (4/10 mice). Black diamonds indicate en-graftment (i.e., chimerism >1%), empty diamonds indicate nonengraftment (P = 0.027).

04-SCD-2008_0269.indd 841 10/8/2009 12:34:43 PM

BONIG ET AL.842

differential α6-integrin expression, although the differences between the cell types are complex, and additional factors are clearly involved [29].

Our data further describe a novel difference in the molec-ular mechanisms of BM homing between human and mouse HSPCs. Similar effects concern α5-integrin and CXCR4, which at least in some reports were considered essen-tial for human, but redundant for murine HSPC homing [5–7,21,30]. In this respect, earlier reports on negative effects of α6-blockade on murine BM-HSPCs homing without an effect on short-term engraftment [17] are inherently contra-dictory. Furthermore, they are diffi cult to reconcile with the absence of effects of α6-deletion on fetal liver HSPC homing [16] or with the data presented in this article, which were generated using very similar techniques, including use of

homing were regularly refl ected in short-term engraftment [7,21,25,27,28]. The observation that α6-blockade can enhance homing (and, consequently, engraftment) of human and baboon BM–derived HSPCs is notable in that it is the fi rst modifi cation of an integrin to have a positive effect. Although our homing data for human/baboon HSPCs were generated using xenogeneic recipients, it should be stressed that all pre-vious studies of human HSPCs homing were also performed in this model. Our data also demonstrate functional conse-quences of the differential expression of α6-integrin on BM- and MPB-HSPCs, by showing that homing of MPB-HSPCs, which express little α6, is not affected by α6-blockade. Based on these data we can speculate that the previously reported superior BM homing of human MPB-HSPCs compared with BM-HSPC may among other parameters be a refl ection of

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FIG. 2. Blockade of α6-integrin has no effect on marrow homing of murine CFU-C. (A) α6-Integrin is expressed on almost all c-kit+ murine BM cells. (B) The anti-α6-Ab concentration/incubation time was suffi cient to completely block binding of directly fl uorescence-conjugated anti-α6-Ab, indicating saturating conditions (solid black line histogram: anti-α6-Ab-treated c-kit+ cells; gray histogram: control-Ab-treated c-kit+ cells; fi ne gray line: isotype control). (C and D) Marrow hom-ing of murine BM CFU-C in lethally irradiated (1,250 cGy) isogeneic hosts, measured at 3 h (C), or 20 h (D) was not affected by α6-blockade. This outcome was observed irrespectively of whether whole BM cells (C and D, left) or c-kit-enriched BM cells (D, middle, right) were used, in normal (C; D, left, middle) or in splenectomized (D, right) recipients (one experiment each, with fi ve recipients/group). The number of circulating CFU-C, a sensitive measure of altered marrow homing, also did not differ between recipients of anti-α6-treated and control cells (C). Spleen homing was also not different between the groups in nonsplenectomized recipients of whole BM or c-kit+ BM cells (not shown). (E) The effi ciency of marrow and spleen hom-ing of murine BM CFU-C in lethally irradiated NOD/SCIDβ2m−/− recipients instead of isogeneic ones also did not differ be-tween anti-α6-Ab-incubated and control incubated CFU-C (one experiment, three mice/group). (F) Short-term engraftment of CFU-C 8 days after transplantation did not differ between recipients of control treated and anti-α6-Ab-treated cells.

04-SCD-2008_0269.indd 842 10/8/2009 12:34:44 PM

ROLE OF 𝛂6-INTEGRIN IN HOMING 843

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the same Ab clone and concentration. Earlier studies have also demonstrated the absence of an effect of anti-α6-Ab on human CB-HSPC homing [28], an observation that is consis-tent with our data reporting the absence of α6 on CB-CD34+ cells reported here.

With respect to mechanisms involved in the observed enhancement of homing in the absence of α6-integrin, we can only speculate at present. Laminin 1, a major constit-uent of the perivascular extracellular matrix (ECM) and of basal membranes, is highly abundant and ubiquitously expressed. It would likely be the fi rst laminin encountered by HSPCs, even more so after lethal irradiation, when endo-thelial surfaces have been denuded. We thus speculate that retention of HSPC on laminin 1, but also on other ECM con-stituents, such as fi bronectin and other laminins, is a rele-vant “trap” for transplanted HSPCs, retention of which in nonhematopoietic organs is well documented. If such trap-ping is reduced (such as by integrin blockade), the number of HSPCs circulating in blood would be increased, similar to what has been described for blocking of liver uptake of HSPCs by asialoglycoprotein receptor inhibition [31]. Thus the number of HSPCs available for homing through canon-ical homing receptors (α4, LFA-1, CXCL12/CXCR4, etc.) would be increased. Our fi nding of reduced HSPC recovery from spleens is consistent with this interpretation. Given the small total number of HSPCs retained in spleen, however, the increased marrow homing of anti-α6-treated HSPCs cannot be attributed to reduced retention in spleen alone. Instead, we propose that the modestly reduced retention of anti-α6-Ab-treated HSPCs in all extramedullary organs, also demonstrated by us, is in aggregate responsible for the observed phenotype.

The theoretical possibility that outside-in signals through α6 are elicited by the antifunctional Ab cannot be ruled out. For instance, laminin-induced haptotaxis, mediated through α6-integrin, has been reported [32]. Such effects could con-tribute to the observed phenotype.

In summary, α6-integrin blockade improves homing and engraftment of human HSPCs in mice, although this effect is limited to BM-derived cells, and if verifi ed in a large animal model, it presents a strategy to enhance engraftment. Our studies furthermore describe different expression levels of α6-integrin between human CD34+ HSPCs from BM, MPB, and CB, and identify a novel differential homing mecha-nism between human and murine HSPCs.

Acknowledgments

The study was supported by a CCEMH pilot grant from the Fred Hutchinson Cancer Research Center (FHCRC) (to H.B.), by National Institutes of Health (NIH) grant HL58734 (T.P.), and by NIH grant P01 HL53750 (to H.P.K.). H.B. con-ceived of the studies, planned and performed experiments, analyzed data, and wrote the article. G.V.P. and M.W. per-formed experiments. H.P.K. provided vital reagents and advised about experiments. T.P. planned experiments and wrote the article.

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Address correspondence to:Dr. Halvard Bonig

German Red Cross Blood Center andInstitute for Transfusion Medicine and Immunohematology

of the Johann-Wolfgang-Goethe UniversitySandhofstr. 1

Frankfurt 60528Germany

E-mail: hbonig@u.washington.edu; h.boenig@blutspende.de

Received for publication September 8, 2008Accepted after revision October 7, 2008

Prepublished on Liebert Instant Online October 8, 2008

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