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*
Laboratory of Tumor Immunology, Scientific Institute San Raffaele, Milan, Italy;
Laboratory of Pathology, Scientific Institute San Raffaele, Milan, Italy; and
Laboratory of Immunopathology, National Institute for Cancer Research and Advanced Biotechnology Center (IST-CBA), Genoa, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 integrin than
the CD14+CD34- counterpart, being dull
positive for other integrins. Using stably transfected
PECAM-1+, VCAM-1+, or ICAM-1+
cells, we found that PECAM-1 and, to a lesser extent, VCAM-1, could
support transmigration of CD14+CD34+ cells,
whereas the L-ICAM-1 interaction was involved in cell adhesion.
PECAM-1-driven transmigration was conceivably dependent on a
haptotactic gradient, as it was reduced by 80% across NIH/3T3 cells
transfected with the PECAM-1-
cyto deletion mutant. This mutant lacks
the cytoplasmic tail and displays a reduced tendency to localize at the
intercellular junctions, thus failing to form a molecular junctional
gradient. Once differentiated, dendritic cells derived from
CD14+CD34+ precursors retained their
transendothelial migratory capability, using both PECAM-1 and ICAM-1
for transmigration. We suggest that a subset of
CD14+CD34+ circulating leukocytes can localize
to peripheral tissues and differentiate into functional dendritic
cells, thus representing a functional reservoir of potential APC.
PECAM-1, constitutively expressed on vascular endothelium, is likely to
play a relevant role in the egress of this population from the
bloodstream. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transendothelial migration of hemopoietic progenitor cells occurs
during mobilization from bone marrow in response to cytokines and
during homing of circulating progenitors (10). CD34+
leukocyte precursors express several adhesion molecules, such as
selectin and integrins, which are known to mediate the different steps
of extravasation (3, 11, 12). The interaction between the CD34 molecule
itself and ligands of the selectin family is thought to be important
for the initial binding of hemopoietic progenitors to endothelial cells
(13). Likewise, members of the integrin family, such as the
4ß1 and the
Lß2 integrins, proved to be important for
progenitor cell trafficking both in vitro and in vivo (14, 15).
Recently, PECAM-1/CD31 has been reported to enhance the adhesivity of
4ß1 integrin expressed by hemopoietic
progenitor cells (16).
Mobilization of CD34+ cells from bone marrow is a rare event in the adult life, and it is conceivably due to changes in the expression, or affinity to their ligand, of different adhesion molecules during differentiation (17, 18, 19). Indeed, the adhesion molecules expressed on those progenitor cells that are committed to leave the bone marrow, should acquire a higher affinity for counterreceptors expressed by endothelial cells than by stromal cells and extracellular matrix (20, 21, 22). Likewise, the ability to localize to peripheral tissues depends, in mature leukocytes, on a sequence of molecular events that are tightly connected to each other (3, 4, 5, 6). These events can also be regulated by local conditions, such as cytokine production during inflammation, Ag sensitization in the immune response, and secretion of chemokines (23, 24).
We show that a subset of CD14+ PBMC, coexpressing the CD34 precursor marker, is able to migrate across endothelial cells and differentiate into immunostimulatory DC. Migration is apparently mediated by the PECAM-1 molecule through a haptotactic gradient. Once differentiated, DC can use both PECAM-1 and vascular cell adhesion molecule-1 (VCAM-1) for transmigration.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purified, or the FITC- or phycoerythrin (PE)-conjugated,
anti-CD14, anti-CD80 (B7.1), anti-CD86 (B7.2), and the
anti-surface Igs (sIg) mAbs were purchased from Becton Dickinson
(Sunnyvale, CA); the anti-ICAM-1 (CD54; 84H10), the anti-CD83
(HB15a), and the PE-conjugated anti-CD34 (HPCA-1) mAb were
purchased from Immunotech (Luminy, Marseille, France); the
anti-CD106/VCAM-1 (BBA6) mAb were purchased from British
Biotechnology (Oxford, U.K.). The anti-HLA-DR mAb (D1.12) was
kindly provided by R. Accolla (ABC, Genoa, Italy), the
anti-4 integrin (CD49d) mAb (HP2/1) was
provided by F. Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain),
the anti-CD36 mAb (NL07) was a kind gift of M. Alessio (Dibit,
Milan, Italy), and the anti-human mannose receptor (pan1) was a
gift of A. Mantovani (Istituto Farmacologico M. Negri, Milan, Italy).
The anti-CD11a/L integrin mAb 70H12 and the
anti-CD31/PECAM-1 mAb M89D3 were obtained in our laboratory (25, 26), and the anti-CD1a (OKT6) mAb was obtained from the American
Type Culture Collection (Rockville, MD). These mAbs were purified from
ascites fluids by affinity chromatography. All the purified Abs were
used at concentrations of 5 µg/ml in functional studies and at 1
µg/ml in immunofluorescence. When indicated, to avoid any unspecific
effect due to Fc
R binding, mAbs were pepsin digested.
F(ab')2 fragments were prepared according to the method of
Parham (27). PE-conjugated goat anti-isotype mouse Igs (GAM) was
from Zymed Laboratories Inc. (South San Francisco, CA).
Cell isolation and sorting
Peripheral blood CD14+ cells were isolated from healthy donors (buffy coats, kindly provided by the Blood Transfusion Department of our institute), after density gradient centrifugation, according to the method of Sallustio and Lanzavecchia (28). CD34+ and CD34- cell subsets were fractionated using immunomagnetic beads (Dynal, Milan, Italy) according to the manufacturer’s procedure. Purified T cells were obtained after two rounds of plastic adherence followed by immunodepletion of CD14+ and HLA-DR+ cells. When indicated, cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin (Biochrom, Berlin, Germany), 10% heat-inactivated FCS (PAA Labour, Linz, Austria), and 20 ng/ml recombinant granulocyte-macrophage CSF (GM-CSF) as described (29). Media were endotoxin free as shown by the Limulus lysate colorimetric assay (BioWhittaker, Verviers, Belgium).
Electron microscopy
Cells were fixed for transmission electron microscopy (EM) in 2.5% glutaraldehyde for 20 min at 4°C and washed three times in phosphate buffer. Postfixation was performed in 1% osmium tetroxide. Samples were then dehydrated in ethyl alcohol and propylene oxide and embedded in Epon-Araldite resin (Fluka (Sigma-Aldrich), Milan, Italy). Thin sections (80 nm) were then stained with uranyl acetate and lead citrate and analyzed under a Zeiss CEM 902 electron microscope.
PECAM-1, ICAM-1, and VCAM-1 transfectants
PECAM-1- and ICAM-1-stable transfectants were obtained in
NIH/3T3 murine fibroblasts as described (9). CD31/PECAM-1 was subcloned
into pc/DNAI/Neo expression vector (Invitrogen, San Diego, CA) at the
XhoI (5') NsiI (3') sites from the original pGEM7
vector (CD31/PECAM-1/pGEM7 kindly provided by Peter Newmann, Blood
Center of Southeastern Wisconsin, Milwaukee, WI). CD54/ICAM-1 was
subcloned into pcDNAI/Neo at the XhoI site from the original
pRc/CMV vector obtained from T. Springer (Harvard University, Boston,
MA). Transfection was performed by calcium phosphate-DNA
coprecipitation, and stable transfectants were selected by addition of
the neomycin analogue G418 to a final concentration of 0.8 mg/ml.
NIH/3T3 cells stably expressing a truncated form of CD31/PECAM-1
lacking the cytoplasmic tail (CD31/PECAM-1-cyto) were obtained as
described (9). The VCAM-1 transfectants in Chinese hamster ovary (CHO)
cells were kindly provided by A. Dobrina (Department of Physiology and
Pathology, University of Trieste, Trieste, Italy). Mock NIH/3T3 and CHO
cells, transfected with the vector alone, were used as controls.
Immunofluorescence
For immunofluorescence, 105 cells/sample were fixed with 3% paraformaldehyde in PBS (30 min of incubation) and stained with the various mAbs followed by the anti-isotype-specific PE-conjugated GAM. Double staining was performed using the FITC- or PE-conjugated anti-CD14 and the PE-conjugated anti-CD34 mAbs or the FITC-anti-sIg antiserum. After washing, samples were run on a FACStarPlus equipped with an argon ion laser (Becton Dickinson, Mountain View, CA), gated to exclude cell debris and nonviable cells. At least 5000 events/sample were analyzed. Results are expressed as mean red fluorescence intensity vs mean green fluorescence intensity (arbitrary units, a.u.) or mean log fluorescence intensity (a.u.).
Analysis of Ig gene expression
Ig heavy chain gene rearrangement was analyzed by PCR in CD34+ and CD34- cell subsets and in cell populations derived from the above mentioned subsets after culture with GM-CSF. Briefly, cell were digested overnight at 37°C in 100 µl of lysis buffer containing proteinase K (Sigma Chemical Co., St. Louis, MO; 200 µg/ml); after 10 min of heating at 95°C, 3 µl of each sample were used as template in 50 µl of a PCR mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 50 pmol of each primer, 200 µM concentrations of each dNTP, 1.25 U of Taq polymerase, and 1.5 mM MgCl2. A seminested reaction was applied for amplification of the Ig heavy chain gene, using the framework 3 V-region primer in conjunction with nested primers directed to the J regions (LJH and VLJH). Twenty microliters of the reaction products were analyzed by electrophoresis on a 3% agarose gel.
Adhesion and transmigration assay
HUVEC were isolated and cultured as described (30). Cells were
used within four passages. Adhesion or transmigration of radiolabeled
(51Cr, DuPont NEN, Boston, MA), unsorted
CD14+ or CD14+CD34+, and
CD14+CD34- sorted cells, through HUVEC,
ICAM-1, VCAM-1, PECAM1, or PECAM-1-cyto transfectants, was
performed with Transwell cell culture chambers (polycarbonate filters,
3-µm pore size, Costar, Cambridge, MA) as described (9). In some
samples, cells were preincubated for 15 min at 4°C with saturating
amounts (5 µg/ml) of the anti-CD31 (M89D3), anti-CD11a
(70H12), or anti-CD49d (HP/1) F(ab')2 and washed twice,
before the onset of the transmigration assay. At different time points
(4 or 12 h), migrated cells were recovered from the lower
compartment and lysed with 100 mM Tris buffer containing 0.1% Triton
X-100. The radioactivity of the samples was measured in a gamma counter
(Packard, Sterling, VA). Results are expressed as percent migrating
cells, calculated as described (9), i.e., (cpm of lysates from migrated
cells/cpm of lysates from cells of the total input) x 100. In some
experiments, both migrated and nonmigrated cells were recovered and
cultured for 3, 7, or 14 days in the presence of GM-CSF and tested in
the transmigration assay.
Mixed lymphocyte reaction and Ag-specific stimulation
To test the immunostimulatory potential of migrated and nonmigrated cells cultured with GM-CSF, after 7 days of culture cell derived from either CD14+CD34+ or CD14+CD34-, cell precursors were used as stimulators for allogeneic or tetanus toxoid (TT, kindly provided by S. Burastero, HSR-Dibit, Milan, Italy)-specific T lymphocytes at different T:DC ratios. TT-specific T cell lines were obtained by stimulation of syngeneic PBMC with TT (10 µg/ml), Percoll gradient separation of T cell blasts after 7 days, culture in T cell growth factor (Lymphocult, Biotest Diagnostics Inc., Dreieich, Germany), and weekly restimulation with TT. About 105 allogeneic or 2 x 104 TT-specific syngeneic T cells, washed, and maintained without Lymphocult for 48 h, were added to irradiated (4000 rad) DC (from 2 x 104 to 103) in 96-well round bottom microwell plates in RPM1 1640 culture medium supplemented with 10% heat-inactivated FCS and antibiotics as above. For TT presentation, DC were loaded with TT for 12 h before addition of specific T cells. After 3 days, for the allogeneic reaction (MLR), or 48 h for TT-specific response, cells were pulsed with 1 µCi of [3H]TdR (DuPont NEN) per well for the last 18 h of culture, harvested, and counted in a beta counter (Packard). Tests were conducted in triplicate, and results are expressed as mean cpm ± SD [3H]TdR uptake by stimulatory DC or responder allogenic T cells alone was <100 cpm; [3H]TdR uptake by TT-specific T cell lines alone was <1000 cpm (not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD14+ cells collected from peripheral blood of
healthy donors usually represent mature Mo; however, we found that a
small proportion of these cells, ranging from 5 to 10%, coexpress the
CD34 molecule (Fig. 1
A shows a
representative experiment of 10), which is a marker of hemopoietic
progenitors (31). CD34 expression in this cell population was also
confirmed by Western blot (not shown). Since CD34 is usually lost
during leukocyte maturation, the CD14+CD34+
cell population might represent an intermediate stage of
differentiation toward mature Mo. This subset of double positive cells
can transmigrate across endothelial cell monolayers: indeed, when
purified CD14+ cells were used in a transmigration assay,
almost all the cells (95%) recovered from the lower chamber after
12 h were CD34+ (Fig. 1B). This
transendothelial migration apparently occurs in the absence of
chemokines, because we did not find detectable levels of monocyte
chemoattractant protein 1 (MCP1), MCP2, or IL-8 in the supernatants of
HUVEC after 3 days of culture (not shown). The migration ability of
sorted CD14+CD34+ cells was compared with that
of CD14+CD34- cells. As shown in Figure 1C, only the former subset transmigrated without the
addition of exogenous chemokines, whereas the latter could migrate only
after addition of 50 nM MCP1 (not shown), in keeping with reported data
(32). Along this line, Table I shows that
CD14+CD34- cells displayed a higher tendency
to adhere to HUVEC, compared with CD14+CD34+
cells, and this adhesion is blocked by anti-CD11a mAbs.
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Both peripheral blood and bone marrow CD34+
precursors express ß1 or ß2 integrins and
PECAM-1 (15, 16). We found (Fig. 2 shows
a representative experiment) that the
CD14+CD34+ cell subset expresses higher levels
of PECAM-1 (mean fluorescence intensity (MFI) ± SD from 10 independent
experiments, 110 ± 4 a.u.) than the
CD14+CD34- counterpart (MFI 28 ± 3
a.u.). Likewise, the CD34+ cell population was brightly
positive for the 4 integrin (MFI 80 ± 5
a.u.), whereas the CD34- cells were weakly stained with
the anti-4 integrin mAb (MFI 25 ± 3
a.u.). Conversely, the L integrin was highly expressed
on CD14+CD34- cells (MFI 66 ± 7
a.u.), but not on the CD14+CD34+ cell subset
(MFI 22 ± 5 a.u.).
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4 integrin by CD14+CD34+ cells
was responsible for transendothelial migration, transmigration assays
were performed using purified CD14+CD34+ cells
and NIH/3T3 cell monolayers stably expressing PECAM-1, VCAM-1, or
ICAM-1. Both PECAM-1 (Fig. 3A) and, to a lesser
extent, VCAM-1 (Fig. 3B) transfectants proved to
support CD14+CD34+ cell migration, at variance
with ICAM-1+ cell monolayers (Fig. 3C).
Migrated cells were detectable in the lower Transwell compartment after
4 h (not shown), reaching the maximum after 12 h (Fig. 3), in
particular when transmigration was evaluated across PECAM-1-transfected
cell monolayers (Fig. 3A). Pretreatment of migrating
cells with the F(ab')2 of mAbs directed against either
PECAM-1 (Fig. 3A) or 4 integrin (Fig. 3B) prevented cell migration. Likewise, preincubation
of PECAM1+ transfectants with the F(ab')2
of M89D3 mAb abrogated cell migration (not shown), suggesting that a
CD31/PECAM-1-mediated homophilic adhesion was operating. This was also
supported by the observation that the vß3 integrin, another
reported ligand for PECAM-1 (33), was virtually absent from both
CD14+CD34+ cells and PECAM-1+
transfectants (not shown). Background migration across
mock-transfected NIH/3T3 or CHO cells was always <10%
(9).
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cyto deletion mutant. This mutant lacks the
cytoplasmic tail and displays a reduced tendency to localize at the
intercellular junctions, thus being homogeneously distributed along the
plasma membrane (9). Transmigration was reduced by 80% using
monolayers of NIH/3T3 cells transfected with the PECAM-1-cyto mutant
(Fig. 3D
), indicating that a PECAM-1 haptotactic gradient can drive
transmigration of CD14+CD34+ cells. Migrated CD14+CD34+ cells can differentiate into functional DC
Migrated CD14+CD34+ cells (Fig. 4, a and b)
expressed the CD80 Ag (Fig. 4c), which is a DC
hallmark (34), beside CD86 (not shown), and very low levels of CD36
(Fig. 4d), a marker of mature Mo or macrophages (35).
This would suggest that CD14+CD34+ cells might
represent a subset of precursors capable of differentiating into DC,
rather than Mo. Indeed, after 7 days of culture in the presence of
GM-CSF, migrated cells lost the CD14 (Figs. 4 and 4f), the CD34 (Fig. 4g), and the CD36 (Fig. 4i)
molecules and up-regulated HLA-DR (Fig. 4l),
remaining positive for both CD80 (Fig. 4h) and CD86
(not shown) costimulatory (34) molecules; furthermore, they were not
stained with dyes for the nonspecific esterases (not
shown). On the contrary, nonmigrating
CD14+CD34-CD36+ cells (Fig. 4, m–q), cultured in GM-CSF, gave rise to
CD14+CD34-CD80-CD36+
cells (Fig. 4, r–v), positive for the
nonspecific esterase staining (not shown), which conceivably represent
macrophages. However, activated B cells have been reported to share
some phenotypic features of monocyte/macrophages (36); indeed, we found
that about 2% of purified peripheral blood CD14+
leukocytes coexpressed sIg (Fig. 5A), at variance with
CD14+CD34+ migrated cells, which were sIg
negative (not shown). Thus, we analyzed for Ig heavy chain gene
rearrangement both CD14+CD34+ and
CD14+CD34- subsets, before and after culture
with GM-CSF. The electrophoresis of PCR products revealed the presence
of a smear, suggestive of polyclonal rearrangement, in the
CD14+CD34- population (Fig. 5B,
lane 2), while samples run in the remaining lanes,
from the same subset after culture in GM-CSF (lane
4) or from CD14+CD34+ cells, before
(lane 3) and after culture (lane
5), were essentially devoid of amplified DNA fragments.
That cells derived from migrating CD14+CD34+
leukocytes were actually DC was confirmed by electron microscopy.
Figure 6A shows that
CD14+CD34+ cells have few cytoplasmic
projections and high nucleo-cytoplasmic ratio, are roundish, and are
larger than CD14+CD34- leukocytes. Nuclei are
smooth-countered with finely dispersed chromatin and usually one small
nucleolus is usually present; the cytoplasmic organelles are mainly
represented by mitochondria, rare rough reticulum cisternae, and small
Golgi complexes (Fig. 6A).
CD14+CD34- cells show, in turn, slender
cytoplasmic projections, irregular nuclei with marginated
chromatin, and abundant cytoplasm containing some electron-dense
lysosome-like granules (Fig. 6B). Cells derived
from CD14+CD34+ precursors display many
dendritic-like cytoplasmic protrusions (filopodia) and a cytoplasm
poor of granules and lysosomes (Fig. 6C), thus being
bona fide DC, whereas cells derived from
CD14+CD34- leukocytes have ultrastructural
characteristics consistent with monocyte/macrophage lineage such as
short and thick membrane projections and a cytoplasm filled with dense
lysosome-like granules (Fig. 6D). We failed to
demonstrate Birbeck’s granules (usually found in Langerhans cells) in
our DC; however, unlike cells from CD14+CD34-
leukocytes (not shown), DC acquired CD1a (Fig. 7d) in 
50% of the
donors examined, and CD83, which is a DC marker (38), in all of the 10
donors analyzed (Fig. 7e) and expressed low levels of
mannose receptor (Fig. 7f), as reported in mature
DC (39).
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, DC
obtained from CD14+CD34+ migrating precursors
could stimulate both allogenic (Fig. 8A) and syngenic
TT-specific T cell proliferation (Fig. 8B). On the
contrary, CD14+ cells derived from nonmigrated peripheral
blood CD14+CD34- Mo were poor stimulators in
MLR (Fig. 8A), and they were less efficient than DC
in the stimulation of TT-specific T cell lines (Fig. 8B).
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One of the predictable characteristics of DC with potential Ag
presenting capacity is the ability to become veiled cells and
recirculate (2, 40). Thus, we addressed the question of whether DC
derived from CD14+CD34+ cells displayed
any migratory activity. To this aim, transmigration assays were
performed using both HUVEC, and the various transfectants
expressing either ICAM-1, VCAM-1, or PECAM-1. We found that DC
obtained from CD14+CD34+ precursors displayed a
higher migratory capacity through HUVEC monolayers than cells derived
from the CD14+CD34- counterpart (Fig. 9). This was confirmed by the finding
that DC obtained from CD14+CD34+ precursors
could efficiently migrate across PECAM-1 and VCAM-1 transfectants as
well, at variance with cells from CD14+CD34-
Mo (Fig. 9). Moreover, GM-CSF-cultured
CD14+CD34- cells could adhere to
ICAM-1-transfected cell monolayers more efficiently (35 ± 4%)
than the CD14+CD34+-derived DC (10 ±
3%), whereas adhesion of CD34- or
CD34+-derived populations to PECAM-1 or VCAM-1
transfectants was superimposable (not shown). These data support the
hypothesis that DC derived from CD14+CD34+
precursors show peculiar migratory properties.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This population possibly represents an intermediate stage of
differentiation from bone marrow precursors to mature Mo or DC.
Although DC maturation has been described as a single pathway, the
heterogeneity of DC within peripheral blood points to the existence of
two separate pathways in DC development, one committed directly from an
early precursor and the other from a CD14+ monocyte-like
stage (41). Our data would indicate that such a bipotent precursor is
present in peripheral blood, in keeping with a recent interpretation of
DC origin and maturation (41, 42). Unlike mature CD14+
leukocytes, the CD14+CD34+ subset shows high
expression of PECAM-1 and 4 integrin, being dull
positive for all the other integrins. Both ß1 and
ß2 integrins are expressed in lower levels by circulating
CD34+ cells, compared with noncirculating bone marrow
CD34+ progenitors (43). It is conceivable that the
preferential expression of certain molecules accounts for the different
adhesive/migratory behavior of the two cell populations. Indeed,
CD14+CD34+ precursors use both
PECAM1-dependent homophilic interaction and an
4-VCAM-1 adhesion system for transmigration, at variance
with CD14+CD34- cells which adhere, rather
than migrate, through the L-ICAM-1 receptor-ligand
pair.
These findings are in agreement with data from other authors showing that human CD34+ progenitor cells use both PECAM-1 and VCAM-1 adhesion systems (16). However, although they describe an indirect involvement of PECAM-1 in cell adhesion, as PECAM-1 engagement leads to up-regulation of integrin affinity for their ligands, we found that PECAM-1 can drive directly the migration of CD34+ precursors through a homophilic haptotactic gradient. It is tempting to speculate that in addition to modulation of integrin avidity state, PECAM-1 can regulate the adhesive and migratory properties of CD34+ precursors by two additional mechanisms: 1) up- or down-regulation of PECAM-1 surface expression during leukocyte maturation; 2) modulation of PECAM-1 surface distribution on endothelial cells, thus favoring either migration (haptotactic gradient) or adhesion (homogeneous distribution).
We have reported that PECAM-1 localization at the intercellular
junctions in endothelial cells depends on the connection with
cytoskeletal proteins and is tightly regulated by PECAM-1
phosphorylation (30). The balance between phosphorylated and
dephosphorylated endothelial PECAM-1, can be perturbed by TNF-,
through the activation of cellular kinases, both in vitro and in
vivo (30), thus implying that tissue microenvironment can influence
both PECAM1 distribution and function. Nevertheless, the finding
that PECAM1-driven transmigration of
CD14+CD34+ cells is apparently
chemokine-independent would favor the hypothesis that this
population is committed to localize to peripheral tissues, whatever the
microenvironment, to complete their maturation and exert their
function. This is further supported by the observation that both
migrated and sorted CD14+CD34+ cells can
differentiate into immunostimulatory DC during culture with GM-CSF,
which is known to enhance DC maturation (36, 44, 45).
DC precursors originating from the bone marrow migrate to peripheral
tissues and primary lymphoid organs, where they become professional APC
(46). After processing, Ag-carried DC migrate to the lymph nodes for
the induction of specific T lymphocytes (47). Accordingly, we found
that DC derived from migrating CD14+CD34+
precursors retain the ability to migrate using both PECAM-1 and
4-VCAM-1 adhesion systems in the absence of detectable
chemotactic stimuli. DC with an abundance of adhesion and accessory
molecules, able to stimulate quiescent T cells in MLR and to home to
the T cell areas of lymphoid tissues, have been described (48) in
keeping with our results. Moreover, a regulated expression and membrane
distribution of PECAM-1, in the bone marrow or in peripheral lymphoid
organs, might contribute to the progression of DC maturation and
recirculation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Elisabetta Ferrero, Laboratory of Tumor Immunology, Scientific Institute San Raffaele, Via Olgettina 60, I-20132 Milan, Italy.
3 Abbreviations used in this paper: Mo, monocytes; DC, dendritic cells; GAM, goat anti-mouse immunoglobulins; GM-CSF, granulocyte-macrophage colony-stimulating factor; PE, phycoerythrin; TT, tetanus toxoid; PECAM-1, platelet endothelial cell adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; sIg, surface immunoglobulin; EM, transmission electron microscopy; CHO, Chinese hamster ovary; MCP1, MCP2, monocyte chemoattractant proteins 1 and 2; MFI, mean fluorescence intensity.
Received for publication August 11, 1997. Accepted for publication November 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 and 5ß1 are differentially expressed during myelopoiesis and mediate the adherence of human CD34+ cells to fibronectin in an activation dependent way. Blood 81:344.vß3 integrin. requirement of intracellular and extracellular calcium. Eur. J. Immunol. 27:1893.[Medline]
-induced vascular leakage involves PECAM-1 phosphorylation. Cancer Res. 56:3211.vß3 integrin involved in adhesion of leukocytes to endothelium. J. Cell Biol. 130:451.. J. Exp. Med. 184:695.. J. Immunol. 154:5851.[Abstract]
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 C. H. Parsons, B. Szomju, and D. H. Kedes Susceptibility of human fetal mesencyhmal stem cells to Kaposi sarcoma-associated herpesvirus Blood, November 1, 2004; 104(9): 2736 - 2738. [Abstract] [Full Text] [PDF]
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A. Moldenhauer, M. Nociari, G. Lam, A. Salama, S. Rafii, and M. A.S. Moore Tumor Necrosis Factor Alpha-Stimulated Endothelium: An Inducer of Dendritic Cell Development from Hematopoietic Progenitors and Myeloid Leukemic Cells Stem Cells, March 1, 2004; 22(2): 144 - 157. [Abstract] [Full Text] [PDF]
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A. M. Woltman and C. van Kooten Functional modulation of dendritic cells to suppress adaptive immune responses J. Leukoc. Biol., April 1, 2003; 73(4): 428 - 441. [Abstract] [Full Text] [PDF]
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E. Ferrero, D. Belloni, P. Contini, C. Foglieni, M. E. Ferrero, M. Fabbri, A. Poggi, and M. R. Zocchi Transendothelial migration leads to protection from starvation-induced apoptosis in CD34+CD14+ circulating precursors: evidence for PECAM-1 involvement through Akt/PKB activation Blood, January 1, 2003; 101(1): 186 - 193. [Abstract] [Full Text] [PDF]
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 A. Poggi, M. R. Zocchi, R. Carosio, E. Ferrero, D. F. Angelini, S. Galgani, M. D. Caramia, G. Bernardi, G. Borsellino, and L. Battistini Transendothelial Migratory Pathways of V{delta}1+TCR{gamma}{delta}+ and V{delta}2+TCR{gamma}{delta}+ T Lymphocytes from Healthy Donors and Multiple Sclerosis Patients: Involvement of Phosphatidylinositol 3 Kinase and Calcium Calmodulin-Dependent Kinase II J. Immunol., June 15, 2002; 168(12): 6071 - 6077. [Abstract] [Full Text] [PDF]
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S. Hirohata, T. Yanagida, T. Nagai, T. Sawada, H. Nakamura, S.'i. Yoshino, T. Tomita, and T. Ochi Induction of fibroblast-like cells from CD34+ progenitor cells of the bone marrow in rheumatoid arthritis J. Leukoc. Biol., September 1, 2001; 70(3): 413 - 421. [Abstract] [Full Text] [PDF]
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S. J. Bell, R. Rigby, N. English, S. D. Mann, S. C. Knight, M. A. Kamm, and A. J. Stagg Migration and Maturation of Human Colonic Dendritic Cells J. Immunol., April 15, 2001; 166(8): 4958 - 4967. [Abstract] [Full Text] [PDF]
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E. Riedl, J. Stockl, O. Majdic, C. Scheinecker, K. Rappersberger, W. Knapp, and H. Strobl Functional Involvement of E-Cadherin in TGF-{beta}1-Induced Cell Cluster Formation of In Vitro Developing Human Langerhans-Type Dendritic Cells J. Immunol., August 1, 2000; 165(3): 1381 - 1386. [Abstract] [Full Text] [PDF]
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S. Gardella, C. Andrei, S. Costigliolo, L. Olcese, M. R. Zocchi, and A. Rubartelli Secretion of bioactive interleukin-1beta by dendritic cells is modulated by interaction with antigen specific T cells Blood, June 15, 2000; 95(12): 3809 - 3815. [Abstract] [Full Text] [PDF]
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E. Ferrero, K. Vettoretto, A. Bondanza, A. Villa, M. Resnati, A. Poggi, and M. R. Zocchi uPA/uPAR System Is Active in Immature Dendritic Cells Derived from CD14+CD34+ Precursors and Is Down-Regulated upon Maturation J. Immunol., January 15, 2000; 164(2): 712 - 718. [Abstract] [Full Text] [PDF]
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K. Crawford, D. Gabuzda, V. Pantazopoulos, J. Xu, C. Clement, E. Reinherz, and C. A. Alper Circulating CD2+ Monocytes Are Dendritic Cells J. Immunol., December 1, 1999; 163(11): 5920 - 5928. [Abstract] [Full Text] [PDF]
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 M. D. Denton, C. S. Geehan, S. I. Alexander, M. H. Sayegh, and D. M. Briscoe Endothelial Cells Modify the Costimulatory Capacity of Transmigrating Leukocytes and Promote CD28-mediated CD4+ T Cell Alloactivation J. Exp. Med., August 16, 1999; 190(4): 555 - 566. [Abstract] [Full Text] [PDF]
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A. Poggi, M. R. Zocchi, P. Costa, E. Ferrero, G. Borsellino, R. Placido, S. Galgani, M. Salvetti, C. Gasperini, G. Ristori, et al. IL-12-Mediated NKRP1A Up-Regulation and Consequent Enhancement of Endothelial Transmigration of V{delta}2+ TCR{gamma}{delta}+ T Lymphocytes from Healthy Donors and Multiple Sclerosis Patients J. Immunol., April 1, 1999; 162(7): 4349 - 4354. [Abstract] [Full Text] [PDF]
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, lane
4, and DC, lane 5, respectively) were tested for Ig
heavy chain gene rearrangement by PCR. Lane 1, DNA m.w.
marker V (Boehringer, Mannheim, Germany). One representative experiment
of six.
