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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 826-837
HCA, an Immunoglobulin-Like Adhesion Molecule Present on the
Earliest Human Hematopoietic Precursor Cells, Is Also Expressed by
Stromal Cells in Blood-Forming Tissues
By
Fernando Cortés,
Frédéric Deschaseaux,
Nobuko Uchida,
Marie-Claude Labastie,
Annabelle M. Friera,
Dongping He,
Pierre Charbord, and
Bruno Péault
From the Institut d'Embryologie Cellulaire et Moléculaire,
CNRS UPR 9064, Nogent-sur-Marne, France; the Établissement de
Transfusion Sanguine, Besançon, France; and SyStemix Inc, Palo
Alto, CA.
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ABSTRACT |
We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a
member of the immunoglobulin family that mediates both homophilic and
heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at
the surface of all of the most primitive CD38 /lo,
Thy-1+, rho123lo, CD34+
hematopoietic cells in human fetal liver and fetal and adult bone
marrow. In the present report we show that HCA is also expressed by
subsets of stromal cells in the primary hematopoietic sites that
sequentially develop in the human embryo and fetus, ie, the paraaortic
mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal
cells established in vitro, including those derived from
Stro-1+ progenitors and cells from immortalized cell
lines, express HCA. In contrast, no HCA expression could be detected in
peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA
membrane molecules purified from marrow stromal cells interact with
intact marrow stromal cells, CD34+ CD38
hematopoietic precursors, and CD3+ CD6+
peripheral blood lymphocytes. Finally, low but significant levels of
CD6 are here for the first time detected at the surface of CD34+ rho123med/lo progenitors in the bone
marrow and in mobilized blood from healthy individuals. Altogether,
these results indicate that the HCA/ALCAM surface molecule is involved
in homophilic or heterophilic (with CD6) adhesive interactions between
early hematopoietic progenitors and associated stromal cells in primary
blood-forming organs.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HEMATOPOIESIS OCCURS IN higher
vertebrates in various tissues of different embryonic derivations.
Whereas T-cell differentiation requires the contact of stem cells with
the epithelio-mesenchymal framework of the thymus, myeloerythroid
development can occur in diverse, histologically distinct environments
such as those provided by yolk sac hemangioblastic clusters, embryonic
and fetal hepatocytes, or bone marrow (BM) heterogeneous stroma. In
pathological conditions such as myeloproliferative disorders and
chronic anemias, blood cells can also develop in atypical locations
such as adult liver and spleen.
Hematopoiesis is tightly controlled by interactions between blood cells
and their surrounding stromal compartments1; yet, the
nature of that communication remains largely obscure. Notably, the
influence exerted by blood-forming tissues on hematopoietic stem and
precursor cells is undefined, although some clues have recently been
given on the molecules involved in the contact of the latter with BM
stroma. For example, the  4 1 integrin VLA-4 is expressed by
immature hematopoietic cells and by their more differentiated progeny
in the BM, whereas its ligand VCAM-1 is present at the surface of cells
in the adherent layer of BM cultures.2-6 In vivo expression
of VCAM-1 by reticular and endothelial cells in the murine BM stroma
has also been documented.7 Antibodies against both VLA-4
and VCAM-1 have been shown to inhibit or partially affect
lymphomyelopoiesis in vitro and in vivo. Treatment of BM cells with an
antibody to VLA-4 significantly decreases their ability to colonize the
marrow of irradiated mice on intravenous injection, as does the
administration of anti-VCAM-1 antibodies to the mouse
hosts.8 Furthermore, knocking out the gene encoding the
4 integrin chain in embryonic stem (ES) cells
profoundly disturbs their postnatal lymphoid progeny in chimeric mice
by dysregulating T- and B-cell precursor development in the BM and by
impeding T-cell homing to Peyer's patches.9 The
inactivation of the 1 integrin gene results in the inability of
hematopoietic stem cells to colonize the embryonic liver.10
Heparan sulfate, a glycosaminoglycan of the stromal cell glycocalix,
also mediates binding of mouse hematopoietic progenitors via the Mac-1
and CD45 surface molecules11 and that of CD34+
human progenitors through CD31 (PECAM-1), a cell surface molecule also
involved in homophilic adhesion between hematopoietic cells and their
endothelium-like stromal environment.12 This nonexhaustive list of receptor/ligand pairs underlines the existence of multiple, complementary adhesion mechanisms between stromal and hematopoietic cells. These mechanisms are likely to be involved at all stages of
hematopoiesis. These include stem cell retention, possibly via
chemotactic factors,13 in various niches inside primary blood-forming tissues fetal liver and BMfor renewal, expansion, and
differentiation,14 but also emigration into the blood flow through sinusal walls and directed homing to other hematopoietic tissues or back to the tissue of origin.
We have presented in a previous work15 the characterization
and molecular cloning of HCA, the human member of a family of homophilic adhesion proteins that includes BEN/SC1/DM-GRASP in the
chicken,16-18 neurolin in the fish,19
KG-CAM/F84.1 in the rat,20,21 and mALCAM in the
mouse.22 Like its animal homologs, HCA is expressed
transiently by developing neurons in the embryonic and early fetal
central and peripheral nervous systems,15,23 as well as by
subsets of hematopoietic progenitor cells. The HCA protein is present
at the surface of 40% to 70% of CD34+ cells in fetal
liver and in fetal and adult BM (ABM), and on virtually all
CD34+ cells in peripheral blood after precursor cell
mobilization.15 Importantly, all of the most primitive
CD34+ cells from these sources, defined by their
CD38/lo, Thy-1+, rho123lo
phenotype,24-27 express HCA.15 Hematopoietic
precursor cell activity is confined to the subset of CD34+
cells that coexpress HCA, as measured by long-term in vitro culture and
severe combined immunodeficiency-hu bone reconstitution
assays.15 Glycoproteins of the BEN family exhibit typical
structural features of adhesion molecules, and their homophilic
adhesive properties have been documented in vitro in animal
studies.17,18,28 That HCA plays a similar role at the
surface of human cells has been suggested by the demonstration that HCA
cDNA expression confers strong self-adhesive properties to Chinese
hamster ovary (CHO) cells, which can be competed by soluble recombinant
HCA polypeptides.15 In addition, homotypic interactions
mediated by MEMD, a molecule identical to HCA, are essential for
cell-cell adhesion in several human melanoma cell lines.29
The constitutive expression of a novel homophilic adhesion protein by
human hematopoietic stem cells has prompted us to search for its
conjoint presence at the surface of stromal cells in blood-forming tissues. We have thus studied, by in situ hybridization of an HCA
riboprobe and antibody staining of tissue sections, the distribution of
the HCA message and protein in the following different anatomical sites
in which hematopoiesis occurs in the human embryo and fetus: yolk sac,
embryonic aorta, liver, thymus, BM, spleen, and lymph nodes. HCA
expression has been also examined by flow cytometry on stromal cells
from long-term cultured ABM and immortalized lines. This study shows
that HCA is present on nonhematopoietic stromal cells in all primary
hematopoietic tissues, but is absent from secondary ones, ie, spleen
and lymph nodes.
These data suggest that HCA is involved in adhesive interactions
between hematopoietic stem and progenitor cells and their supporting
stromas. However, HCA is also known to be identical to ALCAM (activated
leukocyte cell adhesion molecule), a glycoprotein expressed by
mesenchymal stem cells,30 and involved in the binding of
thymocytes to thymic epithelial cells through their CD6 surface antigen.31 We also report here that CD6 is expressed at the surface of a subset of CD34+ cells, and hypothesize that
both HCA/HCA and HCA/CD6 interactions may be involved in the adherence
of human primitive hematopoietic progenitors to surrounding stromal
cells in primary blood-forming tissues. In support of this assumption
we show that HCA molecules purified from marrow stromal cells can bind
intact marrow stromal cells, CD3+CD6+
peripheral blood lymphocytes (PBL), and
CD34+CD38 hematopoietic cell precursors.
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MATERIALS AND METHODS |
Cells and tissues.
Human embryonic and fetal tissues were obtained from voluntary or
therapeutic abortions performed in compliance with the French legislation. Developmental stages, indicated in weeks postconception, were estimated from menstrual history, and confirmed on anatomic criteria. Yolk sac, embryonic and fetal bone, liver and thymus, and
fetal spleen and mesenteric lymph nodes were obtained and processed as
described. In addition, liver pieces were biopsied at autopsy on two
stillborn infants.
ABM was aspirated from the posterior iliac crest of healthy adult
volunteers after informed consent was obtained according to the
criteria established by the Institutional Review Board at Stanford
Medical Center (Stanford, CA), or, with informed consent, from patients
undergoing cardiac surgery at Minjoz Hospital (Besançon, France).
Alternatively, we used peripheral blood apheresed from healthy
volunteers treated with granulocyte colony-stimulating factor (G-CSF;
10 to 15 mg/kg) at either Stanford Medical Center, Fred Hutchinson
Cancer Research Center (Seattle, WA), or MD Anderson Center (Houston, TX).
K562 erythroleukemia cells were obtained from the American Tissue
Collection Center (Rockville, MD). The stromal cell lines HS27A and
HS2332 were obtained from B. Torok-Storb (Fred Hutchinson Cancer Research Center), L87/4 and L88/533 from P. Dörmer (Forschungszentrum für Umwelt und
Gesundheit, Munich, Germany), PU-3434 from
D.A. Williams (Indianapolis, IN), KM10235 from J. Greenberger (Pittsburgh, PA), and L2Ori- from A. Keating (Toronto
General Hospital, Canada).
Tissue processing and section immunostaining.
Freshly dissected tissues were fixed in phosphate-buffered saline
(PBS), 4% paraformaldehyde, and then impregnated and included in
gelatin/sucrose medium before freezing in isopentane vapors as
described previously.36 Cryostat sections, 6 to 10 µm,
were incubated for 1 hour at room temperature in a humid chamber with the F84.1 antibody diluted in TBST (Tris-buffered saline, 0.25% Triton
X-100 [Sigma, St Louis, MO]) with 2% fetal calf serum
(FCS), followed by three washing steps in TBST. Slides were then
incubated for 30 minutes with appropriately diluted rabbit anti-mouse
IgG (Dako, Glostrup, Denmark), rinsed again with TBST, and incubated 30 minutes with diluted mouse anti-alkaline phosphatase coupled to
alkaline phosphatase (Dako). Immune reaction was revealed using the
Dako Fast Red substrate system according to manufacturer's instructions. Endogenous alkaline phosphatase activity was inhibited by
adding levamisole to the substrate solution at a final concentration of
1 mmol/L. Sections were counterstained with Gill's hematoxylin before
mounting in aqueous medium.
In situ hybridization.
Digoxygenin (DIG)-labeled sense and antisense HCA riboprobes were
synthesized by transcription from flanking promoters of a linearized
pGEM-T vector (Promega, Madison, WI) containing a coding sequence
fragment localized between positions 1381 and 2506 of the HCA cDNA. In
situ hybridization was performed on frozen tissue sections as described
by Henrique et al,37 with minor modifications. Briefly,
sections were dried for 16 hours at room temperature and hybridized
with the digoxygenin-labeled HCA probes overnight at 65°C in a
humid chamber. Slides were then rinsed three times in 50% formamide
1× SSC (saline-sodium citrate buffer) at 65°C,
followed by two 30-minute washes in MABT (100 mmol/L maleic acid, 150 mmol/L NaCl, 0.1% Tween-20, pH 7.5) at room temperature. Sections were
blocked for 1 hour with MABT containing 2% Boehringer's blocking
reagent (Boehringer, Mannheim, Germany) and 20% FCS, and incubated
overnight at room temperature with appropriately diluted alkaline
phosphatase-coupled anti-digoxygenin antibody (Boehringer). After five
20-minute washes in MABT, and rinsing twice for 10 minutes in staining
buffer (100 mmol/L NaCl, 50 mmol/L MgCl2, 0.1% Tween-20,
100 mmol/L Tris, pH 9.5), the staining reaction was performed by
incubating slides in nitroblue tetrazolium
salt/5-bromo-4-chloro-3-indolyl-phosphate solution with 5 mmol/L
levamisole for 3 to 5 hours at 37°C in the dark. The reaction was
stopped by several PBS washes. Subsequent staining with the anti-CD34
HPCA-1 monoclonal antibody (MoAb) (Becton Dickinson, San Jose, CA) was
performed as described above.
Cultures.
Primary stromal layers for flow cytometry analysis were established by
plating 2 × 106 ABM cells/mL in the presence of 50 ng/mL leukemia-inhibitory factor (LIF), 10 ng/mL
interleukin-6 (IL-6), 30 mg/mL endothelial cell growth supplement
(ECGS; Collaborative Research, Franklin Lakes, NJ) and
106 mol/L hydrocortisone (Sigma).
Cultures were fed weekly by replacing 50% of the medium consisting of
50% Iscove's modified Dulbecco's medium (JRH Bioscience, Lenexa,
KS), 50% RPMI with 10% FCS (Hyclone Laboratories, Logan, UT), 10 mmol/L HEPES, 4 × 105 mol/L
2-mercaptoethanol, 106 mol/L hydrocortisone, 100 U/mL penicillin-streptomycin, 4 mol/L glutamine (JRH Bioscience), 50 ng/mL LIF, 10 ng/mL human IL-6, and 10 mg/mL ECGS.
Primary ABM stromal cells used for secondary colony-derived cell line
(CDCL) cultures (see below) were established in vitro by plating
2 × 106 cells/mL in long-term culture
medium as described previously.38 The culture medium
consisted of McCoy's 5A medium supplemented with amino acids,
vitamins, antibiotics, 12.5% heat-inactivated FCS (GIBCO, Paisley,
UK), 12.5% heat-inactivated prescreened horse serum (GIBCO), and
106 mol/L hydrocortisone. Cultures were incubated at
37°C and 4% CO2 in a fully humidified atmosphere.
Monolayers of vascular smooth muscle-like stromal cells were obtained
either from isolated Stro-1+ cells or from stromal colonies
grown in methylcellulose, as previously described.39,40
Briefly, after 7 to 10 days of culture, primary layers from ABM were
trypsinized, washed twice, and incubated with M450 beads (Dynal, Oslo,
Norway) coated with Stro-1 MoAb41 (three beads per cell).
The cell suspension and beads were incubated under gentle agitation for
30 minutes. Cells bound to beads were then recovered using a magnetic
particle concentrator (MPC-1; Dynal). Unbound cells were removed by
three washes in PBS, 0.1% bovine serum albumin (BSA). Selected cells
were grown in long-term culture medium containing 2 ng/mL fibroblast
growth factor-2 (FGF-2). Confluent cell layers were sacrificed for flow
cytometry, immunofluorescence, and interaction studies. Alternatively,
primary layers were grown for 3 to 4 weeks, and then dissociated with
trypsin/EDTA and grown in methylcellulose in the presence of 20 U/mL
IL-1 and 200 U/mL tumor necrosis factor-. The cell concentration
was 1 to 2 × 104 cells/mL. After 2 weeks stromal
colonies were obtained using fine pipettes. CDCLs were then grown in
long-term culture medium with 10 ng/mL FGF-2. Confluent layers were
dissociated and analyzed by flow cytometry.
Immortalized stromal cell lines were cultured in long-term culture
medium as described above. K562 cells were cultured in RPMI 1640 (Biowhittaker, Walkersville, MD) and 10% FCS.
CD34+ cell purification.
On a Hypaque-Ficoll density gradient (Pharmacia, St Quentin, France)
(density = 1.077), 3 to 4 × 108
nucleated marrow cells were centrifuged and mononuclear cells at the
interface were collected. CD34+ cells were isolated using a
magnetic separation column according to the manufacturer's
instructions (mini-Macs; Myltenyi Biotec, Bergisch Gladbach, Germany).
The average final purity of the CD34+ cell fraction was, in
10 distinct experiments, 98% ± 1%.
Flow cytometry.
Primary antibodies used for the analysis of primary ABM cultures were
11-59 (anti-KG-CAM), obtained from Dr E. Geisert Jr (University of
Tennessee, Memphis, TN); and phycoerythrin (PE)-conjugated anti-Thy-1,
CD6, and CD49d, obtained from Pharmingen (San Diego, CA). The CD34
antigen was detected by staining cells with sulforhodamine-conjugated anti-CD34 F(ab')2 (SyStemix, Palo Alto, CA). hHCA was detected with the F84.1 MoAb (IgG1), obtained from Dr W. Stallcup (La Jolla Cancer Foundation, La Jolla, CA), and PE-conjugated goat anti-mouse IgG1 (Caltag, South San Francisco, CA). Isotype-matched negative control antibodies were used to delineate gated populations. Cells were
incubated for 20 minutes on ice for each step. After the final wash,
cells were resuspended in PBS supplemented with 2% FCS containing 1 mg/mL propidium iodide (PI). Labeled cells were analyzed and sorted
with a Vantage dual-laser fluorescence-activated cell sorter (FACS)
(Becton Dickinson Immunocytometry Systems, San Jose, CA)
at the SyStemix FACS facility. Dead cells were excluded from analysis
by their PI staining characteristics.
Primary antibodies used to analyze stromal cultures were F84.1, Stro-1
(IgM), purchased from the Developmental Studies Hybridoma Bank
(University of Iowa, Iowa City, IA); 6-19 (IgG1), provided by Dr C. Abboud42; 1B10 (IgM; Sigma)43;
anti-CD45 (IgG1; Dako); anti-CD68 (IgG1; Dako); and irrelevant IgG1 and
IgM controls (Dako). For 30 minutes at 4°C in the dark, 50,000 to
100,000 cells were labeled with the primary antibody and then washed
three times with PBS. Fluorescein isothyocyanate (FITC)-conjugated
F(ab')2 goat anti-mouse IgG + IgA + IgM (Cappel,
Malvern, PA) was added for 30 minutes at 4°C in the dark. Cells
were washed three times with PBS and analyzed on a FACScan flow
cytometer equipped with the Lysis II software (Becton Dickinson).
Following the model described by Andreoni et al,44 a region
with intermediate to high forward scatter and autofluorescence (FL2
channel) was defined and used in antigen distribution studies.
Primary antibodies used to analyze mononuclear cells from ABM and
mobilized peripheral blood progenitors (MPB) were the same as those
used for primary ABM cultures except for the 11-59 antibody. The
staining procedure for rhodamine 123 (rho123) was described previously15,45 as follows: a 1-mg/mL stock solution of
rho123 (Molecular Probes, Eugene, OR) was prepared in ethanol, stored at  20°C in the dark, and thawed just before use. Mononuclear cells or MPB cells were resuspended at 106/mL or less in
PBS containing 2% FCS, and incubated with 0.1 mg/mL of rho123 dye for
30 minutes at 37°C. The cells were washed and incubated at 37°C
for 40 minutes to allow efflux of the dye, washed again, and stained
with antibodies. Rho123 fluorescence was analyzed in the FITC channel
after setting compensation between the FITC and PE channels.
Purified BM CD34+ cells and lymphocytes were stained with
the following IgG1 MoAbs: anti-CD34 (HPCA-2) conjugated to PE (Becton Dickinson), anti-CD38 conjugated to FITC (Coulter, Miami, FL), anti-CD6
provided by Diaclone Research (Besançon, France), anti-CD45 RB
(Dako), and irrelevant controls.
T-lymphocyte preparation.
An aliquot of peripheral blood was centrifuged on a Hypaque-Ficoll
density gradient. Interface mononuclear cells were collected and
cultured in RPMI 1640 (Biowhittaker) with L-glutamin, penicillin, streptomycin, 10% allogeneic (AB group) serum, 500 U/mL
IL-2 (Chiron, Amsterdam, The Netherlands), and 10 ng/mL OKT3 (Cilag,
Levallois-Perret, France). After 10 days of culture, the cellular
suspension consisting of 95% CD3+ lymphocytes was
collected for adhesion studies.
Studies on HCA-mediated interactions between stromal cells and
CD34+ BM cells or PBL.
Marrow stromal cells obtained from Stro-1+ cell-derived
layers were incubated for 30 minutes at room temperature with the F84.1 antibody or an irrelevant mouse IgG1 antibody (Dako), and then washed
three times with PBS/BSA 0.4% (wt/vol). Cells were then incubated for
15 minutes at room temperature under gentle agitation with M-450 beads
(Dynal) coated with goat anti-mouse IgG at a five:one bead to cell
ratio. Cells bound to beads were recovered using a magnet, as described
above for Stro-1+ cells, and lysed for 15 minutes at
4°C in the dark in PBS, 0.1% (vol/vol) Triton X-100, 0.5%
(wt/vol) deoxycholate to solubilize membrane molecules.46
After three washes with PBS/BSA 0.4%, membrane molecules complexed
with beads were incubated for 30 minutes at 4°C in the dark, under
gentle agitation, with intact marrow stromal cells or fresh BM
CD34+ cells, CD3+ PBL, or K562 cells (three to
five beads/cell). Cells with bound beads were separated magnetically as
described above. Both cells coated with beads and uncoated cells were
recovered and counted using a Malassez chamber. The former represented
cells capable of binding HCA membrane molecules solubilized from
stromal cells, ie, cells bearing either HCA or CD6 membrane antigens
(homophilic and heterophilic interactions, respectively).
After numeration, each cell fraction (bound to beads or unbound) was
labeled with an anti-CD45 antibody or with anti-CD34-PE and
anti-CD38-FITC antibodies, before being passaged through the flow
cytometer to check for the presence of remaining intact CD45-negative cells, caused by insufficient solubilization of stromal cell
membranes, and to analyze the phenotype of the bound and unbound
CD34+ marrow cells.
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RESULTS |
HCA expression in early embryonic blood-forming tissues.
In the course of human early ontogeny, the onset of intraembryonic
hematopoiesis is marked by the appearance, by the beginning of the
fifth week, of CD34+ pluripotential progenitors on the
ventral wall of the aorta.47 Aorta-associated
CD34+ cells are arranged in clusters of several hundreds of
primitive hematopoietic precursors, as inferred from phenotype and in
vitro behavior.47 These cell clusters likely arise
from a primitive hematogenous territory derived from the paraaortic
splanchnopleura, a region of the embryo that contributes stem cells for
definitive hematopoiesis in birds and mice.48,49 Therefore,
aorta-associated ventral tissues in the 4- to 6-week human embryo are a
site in which to study initial stem cell emergence and
expansion.50 Hybridization of an HCA probe on cross
sections of early human embryos in the brachial region showed the
presence, immediately subjacent to the aortic endothelium, of a
discrete population of HCA-expressing mesenchymal cells. In all seven
cases analyzed, corresponding to 30- to 42-day embryos, the HCA message
was restricted to the ventral, and, to a lesser extent, lateral
mesenchyme which surround the CD34+ cell clusters, but was
seldom present dorsally in regions of the aorta not associated with
hematopoiesis (Fig 1A). In
all instances, strong labeling of the notochord and neuronal precursors
(not shown) confirmed previous observations of HCA expression by these nonhematopoietic tissues.15,23
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| Fig 1.
HCA distribution in human embryonic and fetal
blood-forming tissues. (A) Cross-section in the trunk region of a
5-week human embryo hybridized with a DIG-labeled antisense HCA probe
(dark blue) and subsequently stained with the HPCA-1 antibody against
the CD34 surface molecule (red). Note HCA-expressing mesenchymal cells
(arrows) subjacent to a cluster of CD34+ hematopoietic
progenitors associated with the ventral aortic endothelium, which also
expresses CD34. A few HCA+ cells are also present on the
lateral aspect of the aorta (arrowhead) (original magnification × 100). (B) Seventeen-week fetal liver. Hematopoietic cells are
intermingled with polygonal hepatocytes displaying strong HCA surface
staining (original magnification × 100). (C) Eight and one-half-week
fetal thymus. The thymic epithelium appears uniformly stained (original
magnification × 25). (D) Close-up view of an 8.5-week fetal thymus
that shows stronger HCA density on epithelial cells in the outermost
region of the rudiment (original magnification × 250). (E)
Seventeen-week fetal thymus. Virtually all epithelial cells beneath the
capsule are HCA+. Stained cells are also present within
the inner cortex (original magnification × 100).
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At the sixth week of development, intraaortic clusters gradually
disappear and the liver becomes the main hematopoietic organ. The
proliferation and differentiation of blood cell progenitors occur at
the contact of hepatocytes and of endothelial cells lining blood
sinuses. Histochemical stainings were performed with the anti-HCA
antibody on liver samples ranging from 5 weeks of gestation to 3 weeks
after birth. In all 5-week liver samples analyzed, most hepatocytes
were already HCA positive. HCA expression was then observed at even
higher levels in all liver samples up to late fetal stages, at which
fully differentiated hepatocytes were easily identifiable by their
characteristic polygonal shape (Fig 1B). Strong expression of HCA was
maintained in postnatal hepatocytes at 2 and 3 weeks after birth, a
period when the hematopoietic activity of the liver has declined (not shown).
HCA expression by the thymic epithelium.
Pharyngeal endoderm contributes the epithelial rudiment of the thymus,
the lymphoid development of which ensues on iterative colonization by
migrating hematopoietic cells.48 Stem-cell seeding to the
embryonic avian and mouse thymus has been timed accurately in
experimental chimeras and in vitro organ cultures,51-53
whereas immunodetection of hematopoietic cell antigens on tissue
sections showed the onset of human embryonic thymus colonization at
about 8 weeks of development.54 At 8.5 weeks, the earliest
stage tested in the present study, the undifferentiated thymic
epithelium is still homogenous in appearance. A strong HCA signal was
then detected on most epithelial cells (Fig 1C), and the most external
cell layers appeared more strongly labeled when examined at higher magnification (Fig 1D). Specialization into cortical and medullary compartments, which occurs at 13 to 15 weeks,55 was
accompanied by a restriction in the expression pattern of HCA to the
cortical epithelium. Thus, at the fourth month of gestation, HCA
expression was mainly restricted to epithelial cells at the periphery
of thymic lobules, in the external cortical region. Subcapsular
epithelial cells in a 3- to 4-cell-thick layer appeared strongly
stained, although some epithelial cells scattered over the cortex were also positive (Fig 1E). Hassall's bodies in the medullary region were
also HCA+ as were a few epithelial cells (not shown). This
pattern remained unchanged at late fetal and early postnatal stages
(not shown).
HCA expression by fetal and postnatal BM stromal cells.
BM is the last primary blood-forming tissue that forms in human
development, at the expense of the cartilaginous rudiments of long
bones. After active and rapid chondrolysis in the diaphyseal area,
invading osteoblasts, vascular sprouts, and mesodermal precursor cells
establish from 8.5 to 10.5 weeks the cellular environment in which
hematopoiesis emerges during the 11th week56 from precursor cells believed to migrate from the liver.57 Because of the
simple structure of the BM at these early stages, the microenvironment of incipient hematopoiesis could be described as the "primary logette," a mesodermal islet protruding into the sinusal lumen. That
structure is a loose network of mesoderm-derived supporting cells
organized around an arteriole and lined with CD34+
endothelial cells.56 However, bone calcification has
already started at these early stages and we were unable to define
fixation/embedding conditions compatible with the preservation of both
marrow structure and HCA epitope. Therefore, in situ hybridization
experiments were performed on bone anlagen younger than 16 weeks of
gestation, the stromal cell organization in logettes being obscured at
later stages as a result of extensive blood cell proliferation.
Hematopoietic cells develop within logettes in contact with three main
stromal cell types, namely, endothelial cells, abluminal pericytes, and intratrabecular myoid cells that express smooth muscle-specific -actin (-SM actin) and extend long processes throughout the logette matrix.56 In situ hybridization on sagittal
long-bone sections at 11 and 12 weeks of development showed high levels of HCA mRNA in elongated structures, parallel to the central artery, within the marrow cords (Fig 2A). Stained
cells were most frequently aligned, but isolated positive cells were
also observed. Because bone sections endure less well than other
tissues the high temperatures required for hybridization, the resulting
tissular structures were poorly preserved. To circumvent this problem,
sections from the same BM samples were labeled with an anti-CD34
antibody and counterstained with hematoxylin (Fig 2B). Comparison of
both series of slides suggested that the HCA-expressing elements
identified by in situ hybridization corresponded to nonendothelial
intratrabecular flattened cells, interspersed among developing blood
progenitors. In addition to intrasinusal stromal cells, osteoblasts
were also found to express the HCA message (not shown).
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| Fig 2.
In situ hybridization analysis of HCA expression in fetal
BM. (A) Eleven and one-half-week fetal BM logette hybridized with a
DIG-labeled HCA probe. HCA+ cells (dark blue staining)
can be seen running along both sides of the central arteriole. (B)
Adjacent section labeled with the same CD34 antibody as in (A) and
counterstained with Gill's hematoxylin. Endothelial cells lining the
central arteriole and the primary logette express CD34. The
HCA+ cells in the upper section roughly correspond to
rows of flattened cells located among developing blood progenitors
(arrows).
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HCA expression was next examined on BM stromal layers established in
vitro and endowed with the ability to support hematopoiesis. Primary cultures were first established from ABM. On enzyme
dissociation and flow cytometry analysis, these cultures were observed
to contain CD45+ hematopoietic cells as well as
CD45 stromal cells, most of which showed a high
level of autofluorescence as judged from diagonal distribution on
dot plots (Fig 3A). The majority of these
stromal cellsif not all of themwere stained by antibodies to HCA
and to human Thy-1, whereas no expression of rat HCA (MoAb 11-59),
CD34, or CD6 could be detected at their surfaces (Fig 3A).
Thus, two surface molecules expressed by the most primitive
hematopoietic stem cells, HCA and Thy-1, are also present on BM
nonhematopoietic stromal cells established in long-term culture.
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| Fig 3.
HCA expression by human BM stromal cells in primary
cultures derived from (A) total ABM cells: the cells were stained with
MoAbs against rat HCA (11-59), HCA, Thy-1, CD34, and CD6, and with
anti-CD45 to discriminate hematopoietic cells; and (B)
Stro-1+ stromal progenitors sorted from ABM. For each
antibody the fluorescence histogram is presented along with that of an
irrelevant antibody of the same isotype (heavy line).
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HCA expression was also studied on stromal cells grown in vitro either
from Stro-1+ progenitors isolated by immunoaffinity from
ABM or from stromal colonies selected in methylcellulose. Both methods
allow the development of adherent, hematopoiesis-supporting, vascular
smooth muscle-like cells expressing -SM actin and other cytoskeletal
proteins specific of myoid cells.39,40 All stromal cells
grown in these conditions were HCA+. The intensity of
fluorescence was strong with a mean of 25.5, versus 1 for the isotype
control (Fig 3B). On in situ immunostaining of confluent layers, all
cells appeared fluorescent, with more densely stained long streaks
probably corresponding to membrane ruffles (not shown). Such structures
are compatible with the expression of an adhesion molecule.
These data clearly indicate that vascular smooth muscle-like stromal
cells developed in culture are HCA+, with a percentage of
positive cells and antigen density higher than those observed after
labeling with the 6-19 and 1B10 MoAbs (Fig 3B), previously shown by Li
et al58 to strongly stain stromal cells. On
the contrary, and as previously reported,58 expression of
antigens recognized by Stro-1 in established stromal layers was
restricted to a minority of cells showing weak mean fluorescence intensity (MFI = 10.3; Fig 3B). CD45+ and CD68+
hematopoietic cells were not detected in these cultures, confirming the
purity of the stromal cell populations present in Stro-1+
cell-derived layers and CDCLs.
Finally, HCA expression was studied in immortal marrow stromal cell
lines from human and nonhuman primates transformed by the early SV40
oncogenes L87/4 and L88/5,33 KM102,35
L2Ori,59 and PU-3434 or by
the human papilloma virus E6 and E7 oncogenes HS23 and
HS27A.32 All SV40-transformed and all E6-E7-transformed cell lines were HCA+ (Table 1),
but the MFI was fourfold to fivefold more intense for
L2Ori and KM102 (22 ± 5 and 17 ± 3, respectively) than for HS27A, HS23, L87/4, L88/5, and PU-34.
Lack of HCA expression by spleen and lymph node stromal cells.
Whereas the HCA molecule was expressed by stromal cells in all primary
hematopoietic organs and tissues analyzed, as summarized in
Table 2, it was absent from secondary ones.
No HCA+ cells were present on antibody-treated frozen
sections of 4- and 5-month spleen and mesenteric lymph nodes which at
that stage are already fully colonized by hematolymphoid cells (not
shown).
Surface expression of CD6 by a primitive subset of hematopoietic
CD34+ cells.
HCA is identical to ALCAM, a glycoprotein which mediates the binding of
thymocytes to thymic epithelium.15,31 A known ligand for
ALCAM at the surface of T cells is CD6.31 Because of the herein described wide distribution of HCA/ALCAM on stromal cells in all
primary blood-forming tissues, besides the thymus, we examined by flow
cytometry the expression of CD6 at the surface of hematopoietic precursor cells. Three-color immunofluorescence was performed as
previously described27 on mononuclear cells from ABM or
from G-CSF-mobilized blood. CD34 expression and rho123 uptake were examined together with the presence of Thy-1, HCA, CD6, and the 4
integrin chain (CD49d), a molecule known to form dimers with the 1
integrin that mediates adhesion of hematopoietic progenitors and stem
cells.3,6 Confirming our previous
observations,15,27 all rhomed/low
CD34+ cells express HCA in the marrow and mobilized
peripheral blood (Fig 4), whereas only a
subset of those are positive for Thy-1 expression. Interestingly, in
both marrow and blood compartments, CD34+
rhomed/low cells could be split into two subsets according
to CD6 staining, one of which expressed this antigen at a low but
significant level (Fig 4). Virtually all cells of both origins were
stained with the CD49d-specific antibody.
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| Fig 4.
Expression of adhesion-related antigens on
CD34+ cells. ABM mononuclear cells and
G-CSF-MPB were processed and stained with rho123 as
described in Materials and Methods. Staining profiles of
CD34+ gated cells with rho123 versus control isotype,
HCA, Thy-1, CD6, and CD49d are shown. The percentage of cells in each
quadrant of the plot is indicated.
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Marrow stromal cell HCA membrane molecules interact with BM stromal
and hematopoietic cells.
The F84.1 antibody recognizes the N-terminal region of the HCA molecule
without any inhibitory effect on the formation of HCA-transfected CHO
cell agregates.15 We took advantage of this property to
select HCA+ and/or CD6+ cells using
F84.1-HCA membrane molecule complexes. The procedure used comprised two
steps: (1) collection of HCA molecules from marrow stromal cells using
magnetic beads and a membrane solubilization protocol, and (2)
selection of HCA+ and/or CD6+ intact
cells by affinity for the HCA membrane molecules purified in step 1. HCA+ CD6 marrow stromal cells used in
step 1 were either from a stromal layer generated from
Stro-1+ cells, or from the L2Ori line. In step 2, we
used (1) HCA+ CD6 stromal cells, (2)
HCA CD6+ PBL, (3) HCA+
and/or CD6+ BM CD34+ cells, and (4)
HCACD6 K562 cells as a negative
control.31 No contaminating stromal cells from step 1 were
present among complexes because no CD45 intact
cells were detected after step 2 using flow cytometry.
As shown in Table 3, bead-coated intact
cells were eventually recovered only when the F84.1 antibody was used
in the first step. On L2Ori membrane extracts, we
recovered 20% and 30% L2Ori cells and PBL,
respectively. On Stro-1+ cell-derived stroma extracts, 20%
and 10% BM stromal and CD34+ cells, respectively, were
retained. Finally, as expected, no HCACD6 K562 cells bound to HCA
immune complexes. HCA+ and/or CD6+
cells can be therefore sorted by affinity for HCA molecules, indicating that HCA:HCA or HCA:CD6 interactions occurred between membrane complexes and intact cells.
Remarkably, CD34+ BM cells selected by this method were
almost exclusively CD38 (87% ± 2.3%; Fig
5). This indicates that selection by HCA
complexes was not random, but directed at a specific
CD38 cell population. Likewise, lymphocytes sorted
on the basis of their HCA affinity appeared as a homogenous population
with low forward and side scatters, further showing the specificity of the HCA interactions uncovered by our cell-binding assay.
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| Fig 5.
Flow cytometry analysis of CD34+ cells
selected on HCA-F84.1 complexes. Bound (A) and unbound (B) cells were
double-stained with anti-CD34-PE (FL2) and anti-CD38-FITC (FL1)
antibodies. The upper right region was delineated as excluding any
nonspecifically labeled cell after incubation with IgG1 irrelevant
controls.
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DISCUSSION |
We have in the present study investigated the distribution of the HCA
adhesion molecule in the stromal compartments of human embryonic and
fetal blood-forming tissues. A previous report by some of
us15 has shown that HCA is expressed by human
CD34+ hematopoietic stem cells and myeloid progenitors. We
also reported that HCA, like its avian homolog
SC1/BEN,17,28 is able to mediate homophilic cell-cell
adhesion.15 Based on these observations, we examined the
possibility that HCA might participate in adhesive interactions between
hematopoietic and stromal cells.
Hematopoietic cells develop first outside the embryo, in the wall of
the yolk sac, at the expense of mesoderm-derived hemangioblastic cell
clusters that also give rise to the first blood vessels. Hematopoiesis
starts in the human yolk sac around 18 days of
development,60,61 and we have not been able yet to assay
such early tissues for HCA expression; the earliest yolk sac we have
examined was 5 weeks old, a stage from which the frequency of
endogenous hematopoietic clonogenic progenitors drops
dramatically.62,63 HCA expression was then detected in
endodermal cells (not shown). The significance of this observation is
still unclear, although a key role of the yolk sac endoderm in blood
island induction has been described in the chicken and mouse
embryos.64-66 The finding that murine yolk sac
endoderm-derived cell lines are able to sustain hematopoiesis provides
additional evidence in favor of this notion,67 as does the
expression of CD34 by yolk sac endoderm cells in murine
embryos.68 Further examination of HCA expression in the
human hematopoietic yolk sac is now pending on the unpredictable
availability of 3- and 4-week samples.
Inside the 5-week embryo, HCA-expressing stromal cells were found both
in the liver and in the mesenchymal region surrounding the dorsal
aorta. There is now experimental evidence in animal models that the
latter territory supports the emergence and expansion of stem cells for
definitive hematopoiesis.69,70 At this stage of embryonic
development, intraluminal clusters of primitive CD34+ blood
precursors are indeed associated with the ventral walls of the human
aorta47 and vitelline artery (Tavian et al, submitted for
publication). Similarly, hematopoietic clusters described at an equivalent stage of development in avian and mouse embryos are
always in association with the endothelial floor of the aorta, but not
with its dorsal aspect.71,72 Interestingly,
HCA+ cells in the human mesenchymal territory comprising
the aorta were mainly restricted to the ventrolateral region lining the aortic endothelium. Thus, the distribution of HCA+
paraaortic mesenchymal cells is spatially correlated with the presence
of intravascular clusters of blood cell progenitors. The microanatomy
of hematopoiesis in the ventral wall of the embryonic aorta is not
precisely characterized yet. The discontinuous aspect of the
aortic endothelium at the level of attached stem cell clusters suggests
that dedifferentiated endothelial cells or underlying primitive
mesenchymal cells contribute the emerging stem cells (Tavian et al,
submitted for publication). Direct contact between subaortic
HCA+ cells and the immediate forerunners of hematopoietic
stem cells might mediate that earliest commitment to hematopoiesis.
These results, like those obtained by in situ hybridization on fetal BM
sections, should be interpreted cautiously, given that we could not
detect any HCA+ cells in this region on antibody staining.
A simple explanation for this may be that the HCA protein is not
expressed by periaortic cells underlying hematopoietic foci.
Nevertheless, although the anti-HCA antibody allowed the detection by
flow cytometry of positive hematopoietic cells,15 it failed
to do so on the corresponding tissue sections. This might reflect the
existence of distinct HCA isoforms exhibiting differential sensitivity
to the tissue-processing conditions used in this study.
HCA can be added to the following list of antigens and cell adhesion
molecules that have been detected on the surface of 8-week thymic
epithelial cells at the time of colonization by hematopoietic progenitors: LFA-3 (CD58), VLA-1, -2, -3, -4, -6, ICAM-1 (CD54), and
the TE-3 antigen.73 HCA was at this early stage uniformly expressed by undifferentiated thymic epithelial cells, but this pattern
evolved with the functional specialization of thymus compartments. In
the second trimester of gestation, HCA was detected on all subcapsular
cells in the cortical epithelium, which is the site of entry of
lymphoid progenitors, whereas scattered, often stellate, positive
epithelial cells were observed within both the cortex and the medulla.
This indicates that HCA-mediated adhesion to epithelial cells might
concern primarily undifferentiated T-cell progenitors but also
thymocytes at different maturation stages. This pattern is much the
same as that reported by Patel et al74 in the adult thymus,
where epithelial ALCAM mediates interactions with maturing thymocytes
through the CD6 molecule. Taken together, these observations suggest
that thymic HCA is involved in stem cell entry and thymocyte maturation
at embryonic, fetal, and postnatal stages.
We also detected the HCA molecule in stromal cells in both fetal and
ABM. In situ hybridization experiments on fetal bone sections with a
DIG-labeled probe showed expression of the hca gene by a subset of flattened intrasinusal stromal cells. Although the
cell morphology was poorly preserved, both the elongated aspect and the
distribution within marrow sinuses of the HCA labeling was similar to
that of myoid stromal cells described previously.56 These
results were confirmed on in vitro cultures of ABM stroma. Primary
stromal layers seemed considerably enriched in HCA+ cells,
contrasting with staining profiles of fresh total BM samples, where
few, if any, HCA+ CD45 cells could be
detected (not shown). HCA-expressing native stromal cells may represent
a very small subset of BM from aspirates, and the antigen density on
that population may be too low to be readily detected by flow
cytometry. BM culture in vitro may stimulate the preferential growth of
a rare subset of HCA+ cells, or upregulate HCA expression.
This is a likely explanation, because we could not detect HCA on
endothelial cells in embryonic and fetal tissue sections, whereas high
HCA levels have been reported in cultured endothelial
cells.29
Purified stromal populations were also found to exhibit high surface
levels of the HCA protein. We have reported before that under long-term
culture conditions, most stromal cells from the BM adherent layer are
vascular smooth muscle-like cells expressing -SM actin as well as
other markers of vascular smooth muscle differentiation.75
This myofibroblastic population can be derived either from
Stro-1+ cells40 or from stromal colonies
(CDCLs).39 Most likely these cells represent the in vitro
counterparts of the -SM actin+ cells described in the
hematopoietic environment of the adult and fetal BM.56,75
We have also shown that these cells support the expansion and
multilineage differentiation of CD34+
CD38 blood cell progenitors.39,76 Thus,
the results presented here establish that HCA is expressed in the BM by
a subset of vascular smooth muscle-like cells endowed with the ability
to support hematopoiesis. Almost all cells from primate immortalized
stromal lines also showed surface HCA, indicating that HCA expression
is not repressed after genomic insertion of SV40 (T and t) or human
papilloma virus (E6 and E7) oncogenes. Although transformation usually
dramatically affects the different components of the
fibronexus,77 including integrins, it is not
necessarily the case for HCA even if a low MFI can be detected for some
cell lines (Table 1). Finally, HCA expression by PU-34 stromal cells
indicates the presence of this molecule in Macaca mulatta as
well as humans.
We examined whether high HCA expression by stromal cells might be
involved in interactions with other HCA+ stromal cells or
HCA+ and/or CD6+ hematopoietic
primitive cells or lymphocytes. Using HCA molecules solubilized from
stromal cells in a cell-binding assay, we were able to select 20% of
intact BM stromal cells, 30% of PBL, and 10% of primitive
CD34+ hematopoietic cells. Relatively low percentages of
recovered cells may be because of technical reasons (steric hindrance
by 4.5-µm beads or low amounts of antibody-bound HCA molecules with the required conformation for ligand interaction) or because of the
fact that a fraction only of the target cells would be involved. Indeed, CD38 cells represented up to 87% of those
selected by HCA affinity from CD34+ BM cells, whereas only
17% were found within the HCA+ subset in the starting
population, as determined by double-staining with F84.1 and anti-CD38
antibodies (not shown). The preferential selection of
CD38 cells, together with the homogenous scatter
characteristics of the CD6+ lymphocyte population sorted on
F84.1-HCA complexes indicates that our immuno-affinity procedure was
effective to uncover HCA:HCA and HCA:CD6 molecular interactions.
Our previous15 and present studies show that HCA and CD6
are coexpressed on human rhomed/low CD34+
cells, a population enriched in hematopoietic stem
cells.26,27 Human and murine ALCAM expression is
transiently upregulated in CD6+ T cells on
activation.22,74 Coexpression of both members of a
ligand/receptor pair in the same cell has previously been described. For instance, the ICAM-1/LFA-1 pair is present on the surface of
CD34+ progenitors in cord blood and ABM,78 and
thymocytes are positive for both CD2 and its counterreceptor
LFA-3.79 The significance of such overlapping expressions
is unknown. The so far unreported expression of CD6 by hematopoietic
stem cells might be related to the phenomenon referred to as
"multilineage priming." According to this hypothesis,
hematopoietic stem and progenitor cells would simultaneously activate
several lineage-associated genetic programs before commitment into a
particular one, as suggested for murine FDCP-mix and CD34+
lin progenitors.80 If the same applied
to human cells, one would expect to find low levels of expression of
lymphoid-, myeloid-, or erythroid-associated markers in blood stem
cells. In this context, it is worth noticing that molecules classically
considered as markers of commitment into the lymphoid (CD2, CD7, CD10,
and CD19) and myeloid (CD33) lineages are expressed by uncommitted
CD34+ progenitors capable of multilineage differentiation
when cultured on supporting thymic stroma.81 Further
investigations are required to understand whether the low levels of CD6
we have detected on human hematopoietic stem cells are relevant to
their adhesion to stromal cells. It will be also important to assay the
respective hematopoietic potentials of CD6+ and
CD6 CD34+ cells over the long term, in
vitro and in vivo.
In conclusion, this set of data emphasizes a role for HCA among other
adhesion molecules expressed by the hematopoietic microenvironment. To
our knowledge, HCA is the first adhesion molecule described so far in
the hematopoietic system with such a widespread expressionin the
mesenchyme surrounding aortic hematopoietic nests, on hepatocytes at
the stage of hematopoietic development, on thymic epithelial cells, and
on fetal and adult myoid marrow cells. In striking contrast, no HCA
expression was detected in the framework of secondary lymphoid tissues.
It should be interesting to investigate the possible induction of this
molecule in pathological conditions when the adult spleen supports
primary hematopoiesis, such as myeloproliferative disorders and some
chronic anemias. VCAM-1 (CD102) and ICAM-1 (CD54) have also been
detected on the surface of thymic and marrow-supporting
cells.2,5,7,82 However, the expression of HCA is always
high on marrow vascular smooth muscle-like stromal cells, at variance
with that of VCAM-1 which is usually low82 unless
upregulated by proinflammatory cytokines,2,3 and that of
ICAM-1 which is very low if detectable.2,3,82 The intensity
of HCA expression by human stroma is similar to that of SH4 and Thy-1,
whose role in cell adhesion is not clearly defined, and to that of
CD44, VLA-1, and VLA-5 integrins, which are mainly involved in cell to
matrix adhesion and in the organization of the extracellular
matrix.82 In this context, the role played by HCA in
homotypic and heterotypic adhesion between stromas and hematopoietic
cells and lymphocytes is significant.
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ACKNOWLEDGMENT |
We are indebted to C. Carrière and to Drs F. Narcy, E. Aubeny,
and P. Blot for providing human embryonic and fetal tissues. We are
also grateful to Drs Beverley Torok-Storb, David Williams, Joel
Greenberger, Armand Keating, and Peter Dörmer for providing the
cell lines and to J. M. Certoux for procuring cultured lymphocytes. We
thank Dr Stallcup for the gift of the F84.1 antibody. We acknowledge the assistance of F. Viala, F. Beaujean, and H. San Clemente with the
preparation of figures, and we thank M. Scaglia for typing the manuscript.
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Top
Abstract
Introduction