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Blood First Edition Paper, prepublished online March 9, 2009; DOI 10.1182/blood-2008-10-184556
Scientific Category: VASCULAR BIOLOGY
CEACAM1+ myeloid cells control angiogenesis in inflammation
Running foot: CEACAM1 in inflammation and angiogenesis
Andrea K. Horst‡1
, Thomas Bickert‡*1, Nancy Brewig*, Peter Ludewig‡, Nico van
Rooijen+, Udo Schumacher++, Nicole Beauchemin#, Wulf D. Ito**, Bernhard Fleischer*,
Christoph Wagener‡, and Uwe Ritter*§
‡Institute of Clinical Chemistry, University Medical Center Hamburg-Eppendorf,
Martinistrasse 52, D-20246 Hamburg, Germany. *Department of Immunology, BernhardNocht-Institute for Tropical Medicine, Bernhard-Nocht-Straße 74, D-20359 Hamburg,
Germany. +Vrije Universiteit, VUMC, Department of Molecular Cell Biology, Faculty of
Medicine, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
++
Department
of Anatomy II: Experimental Morphology, University Medical Center Hamburg-Eppendorf,
Martinistrasse 52, D-20246 Hamburg, Germany. #McGill Cancer Centre, McGill University,
3655 Promenade Sir-William-Osler, Montreal, H3G1Y6, Canada. **University of Ulm,
Dept. Internal Medicine II, Robert-Koch-Straße 8, 89081 Ulm, Germany. §Department of
Immunology, Franz-Josef-Strauss-Allee-11, University of Regensburg, Germany.
1
contributed equally to the work
to whom correspondence should be addressed: A.K.H. and U.R.
communication with editorial office:
Uwe Ritter, Ph.D.
Andrea Kristina Horst, Ph.D.
Dept. of Immunology
Inst. of Clinical Chemistry, Diagnostic Cemter
University Regensburg
University Medical Center Hamburg Eppendorf,
Franz-Josef-Strauss-Allee 11
Martinistrasse 52
D-93053 Regensburg
D-20246 Hamburg
phone: 0049-941-944-5464
phone:0049-40-42803-1905
fax: 0049-941-944-5462
fax:0049-40-42803-4971
e-mail: [email protected]
e-mail:[email protected]
1
Copyright © 2009 American Society of Hematology
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The work presented in this manuscript was performed at the Department of Immunology,
Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany, and the Institute of
Clinical Chemistry, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Abbreviations: BM: bone marrow; BMT: bone marrow transplant; B6.WT: C57BL/6 wild
type mice; B6.Ceacam1-/- mice: Ceacam1-deficient mice on C57BL/6 background;
CEACAM1: Carcinoembryonic antigen-related cell adhesion molecule 1; ConA:
concanavalin A; H&E: hemaotxylin/eosin; L-Ag: Leishmania major antigen; Ly-6C: late
monocyte precursor differentiation antigen; LYVE-1: lymphatic vessel endothelial hyaluronic
acid receptor 1; mAb: monoclonal antibody; p.i.: post infection; PROX-1: prospero
homeobox-related protein 1; Th1 cell: T helper cell 1
2
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Abstract
Local inflammation during cutaneous leishmaniasis is accompanied by accumulation of
CD11b+ cells at the site of the infection. A functional role for these monocytic cells in local
angiogenesis in leishmaniasis has not been described so far. Here, we show that CD11b+ cells
express high levels of the myeloid differentiation antigen carcinoembryonic antigen-related
cell adhesion molecule 1 (CEACAM1). In experimental cutaneous leishmaniasis in C57BL/6
wild type (B6.WT) and B6.Ceacam1-/- mice, we found that only B6.Ceacam1-/- mice develop
oedemas and exhibit impairment of both hem- and lymphangiogenesis. Since CEACAM1
expression correlates with functional angiogenesis, we further analysed the role of the
CD11b+ population. In B6.Ceacam1-/- mice, we found systemic reduction of Ly6Chigh/CD11bhigh monocyte precursors. To investigate whether CEACAM1+ myeloid cells are
causally related to efficient angiogenesis, we used reverse bone marrow transplants (BMT) to
restore CEACAM1+ or CEACAM1- bone marrow in B6.Ceacam1-/- or B6.WT recipients,
respectively. We found that angiogenesis was restored by CEACAM1+ BMT only. In
addition, we observed reduced morphogenic potential of inflammatory cells in Matrigel
implants in CEACAM1- backgrounds or after systemic depletion of CD11bhigh macrophages.
Taken together, we show for the first time that CEACAM1+ myeloid cells are crucial for
angiogenesis in inflammation.
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Introduction
Inflammatory cells such as CD11b+ monocytes have been in the focus of controversial
discussions concerning their role in neo-vascularization 1-6. Bone marrow (BM) is a source for
precursor cells that exhibit considerable plasticity as they can replenish tissue-resident pool
leukocytes or merge or trans-differentiate into vascular structures
1-4
. Functional correlations
between the plasticity and angiogenic properties of specific myelomonocytic populations or
macrophage-precursors have been detailed in recent reports
5-9
. Carcinoembryonic antigen-
related cell adhesion molecule 1 (CEACAM1) is engaged in homotypic and heterotypic
adhesion processes during cellular growth and proliferation or innate and inflammatory
immune responses
10-12
. Recently, we demonstrated that CEACAM1 modulates angiogenesis
and vascular remodelling
13
. However, the involvement of CEACAM1 in angiogenesis has
been described from an endothelial-centric view so far. It has remained unclear if
CEACAM1-expressing progenitors from blood or BM may play a role in angiogenesis in
inflammation.
To address this question we used the experimental model of cutaneous leishmaniasis, known
to produce a severe local inflammation at the site of infection mainly caused by infiltrating
CD11bhigh cells
14-16
. After subcutaneous inoculation, the obligatory intracellular parasite
Leishmania (L.) major is engulfed by macrophages, where it enters its replication cycle. In
later phases of the infection, macrophages can eliminate the parasites following activation by
IFN-γ producing T helper (Th) 1 cells 17-19. Contrary to the well documented adaptive immune
response in experimental leishmaniasis, the impact of local leukocyte turnover on
angiogenesis in this model is unknown. Here, we addressed the question whether myeloid
cells modulate angiogenesis in the model of cutaneous leishmaniasis and demonstrate that
CEACAM1-expression on CD11bhigh cells is essential for angiogenesis in inflammation.
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Materials and Methods
Mice
6 to 8 week old B6.WT (CD45.1+ or CD45.2+ on C57BL/6 background) and B6.Ceacam1-/mice were housed under standard conditions in the animal facilities of the Bernhard-NochtInstitute for Tropical Medicine, and the University Medical Center Hamburg-Eppendorf.
Experiments were performed according to the guidelines for the care and the use of
experimental animals and were approved by the University’s ethical boards. B6.Ceacam1-/mice
were
generated
as
described
20,21
.
Infections
with
Leishmania
major
(MHOM/IL/81/FE/BNI) Virulent parasites were propagated in blood agar plates, and 3x106
promastigotes were subcutaneously injected into the right hind footpad of the mice as
described 22.
Matrigel implantation assays and cell preparations
Matrigel implantation assays containing Leishmania parasites (4x106 L. major per implant)
plus VEGF-C (R&D Systems, Wiesbaden-Nordenstadt, Germany, 500ng/implant) were
performed as described 13. Single cell suspensions were obtained after enzyme digestion in
Dispase (BD Biosciences, Heidelberg, Germany) and Collagenase D (Roche, Mannheim,
Germany).
Immunohistochemistry and quantification of oedematous areas and vessels
Matrigel implants or footpads were cryopreserved and embedded in OCT compound (Tissue
Tek, Diatec, Hallstadt/Bamberg, Germany).
Ten µm sections were fixed in ice cold acetone and stained with hematoxylin/eosin (H&E) or
the following antibodies: anti-LYVE-1- (Biomol, Lörrach, Germany), biotinylated antiMECA-32-, and anti-CD11b-antibodies (BD Biosciences). As secondary antibodies, we used
anti-rabbit TRITC- or anti-rat Cy5-labelled antibodies from Dianova (Hamburg, Germany),
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and Alexa-488-labelled streptavidin (BD Biosciences, Karlsruhe, Germany). Nuclei were
stained with DAPI (Sigma-Aldrich, Deisenhofen, Germany). Slides were blinded and
analysed on a Zeiss Axioskop 2 plus microscope. Pictures were processed with Openlab
software (Improvision) and ADOBE PhotoshopCS3. For quantification of the oedematous
tissue, the DAPI- areas within the infiltrate of the inflamed footpads were calculated using
ADOBE PhotoshopCS3. Similarly, lymphatic and blood vessel growth was evaluated by
quantifying LYVE-1+ and MECA-32+ areas. For each parameter, slides were photographed in
a meandering pattern. Per specimen (n=6 each), at least 20 pictures per footpad were taken for
computer-assisted processing.
Flow cytometry analyses
Either single cell preparations from footpads, digested with collagenase D (Roche), 106
leukocytes or 2x105 cells from Matrigel implants were analysed with the monoclonal
antibodies (mAbs) mentioned above; additionally, anti-Ly-6C mAb (BMA Biomedicals,
Augst, Switzerland), Alexa488-conjugated anti-CEACAM1 (CC1, a kind gift of K. Holmes,
Colorado), PerCP-Cy5.5-conjugated anti-CD11b (BD Biosciences), and PE-conjugated antiCD45.1 (BD Biosciences) were used. Flow cytometry was performed with a FACSCalibur
flow cytometer (BD Biosciences). Data were processed with CellQuestPro software (BD
Biosciences). For quantification of cells isolated from footpads, latex beads (Sigma-Aldrich)
served as internal standards.
Tube formation assay
Cells recovered from Matrigel plugs on day 7 were used in a tube-formation assay adapted
from Maruyama et al. 5. 100µl Matrigel (BD Biosciences) were diluted with 100µl endothelial
cell medium EBM-2 (Cambrex, Milan, Italy), added to 4-chamber slides (Lab-Tek II, Nunc,
Roskilde, Denmark) and allowed to gel for 30 minutes at 37°C. Different amounts of cells
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(5x105, 2.5x105, 1x105, 0.5x105) were seeded onto the gel in 500µl of EBM-2 containing 3%
FCS. Tube formation was monitored on day 2 by counting arborizing structures manually.
Bone marrow transfer
B6.WT and B6.Ceacam1-/- mice were irradiated with 11Gy. One day post irradiation, mice
received 107 BM cells from appropriate donors. BMT was evaluated in flow cytometry
analyses 60 days post transplantation to confirm reconstitution. In our experiments,
CEACAM1+ BM from B6.WT mice expressing CD45.1 was transferred into B6.Ceacam1-/recipients expressing CD45.2 (“rescue” BMT). For the “un-rescue” BMT, we used
B6.Ceacam1-/- mice as donors for B6.WT recipients (CD45.1).
Generation of polyclonal anti-CEACAM1 antiserum P1
Rabbit polyclonal antiserum was raised by subcutaneous injection of purified murine
CEACAM1 (four injections with 1 mg/ml pure CEACAM1 in incomplete Freund´s adjuvans).
Soluble CEACAM1 only containing the extracellular portion of murine CEACAM1
23
plus a
hexahistidine tag was expressed in Sf21 cells and affinity purifed. Purity was >95% according
to SDS-PAGE and Coomassie staining. The anti-CEACAM1-antiserum “P1” was harvested
after the fourth immunization by terminal bleeding. Potency and specificity of the antiserum
was characterized by Western Blotting and in immune histochemistry.
Proliferation assay and quantification of interferon-γ
Lymph node cells (3x105) from naive or infected mice were cultured in 200 µl either
unstimulated or incubated with ConA (2 µg/ml, Sigma-Aldrich) or L-Ag (derived from 9x105
L. major parasites) in 96-well culture dishes at 37°C and 5 % CO2 for 3 days. The amount of
IFN-γ in supernatants was quantified employing DuoSet ELISA Development system
according to the manufacturer´s instructions (R&D Systems). Proliferation was measured by
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[H3]-thymidine incorporation (0.02 µCi/well; Amersham/GE Healthcare, München, Germany)
for 18 hours following stimulation 24.
Quantification of anti-L. major-specific immunoglobulins by ELISA
ELISAs for the quantification of L.major-specific immunoglobulins in mouse serum were
performed as described 24.
Depletion of CD11bhigh macrophage precursors
B6.WT mice received liposomes either loaded with PBS or clodronate (Cl2MDP) on the day
before Matrigel implantation, and on days 2 and 5 after Matrigel injections. Flow cytometry
analyses to monitor CD11b+ population dynamics were performed on days 2, 5, and 7 in
peripheral blood. Cl2MDP (or clodronate) was a gift of Roche Diagnostics GmbH,
Mannheim, Germany. It was encapsulated in liposomes by N.v.R. as described earlier 25-27.
Statistical analyses
Statistical analyses were performed using the Student´s T-Test.
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Results
Cutaneous Leishmaniasis provokes oedema formation and impairment of local
angiogenesis in B6.Ceacam1-/- mice
In our model of cutaneous leishmaniasis, we inoculated L. major parasites into the right hind
footpads of B6.WT and B6.Ceacam1-/- mice. Following infection, footpad swelling was
recorded weekly through the entire course of the infection and calculated relative to the
diameter of the uninfected footpad. As shown in Figure 1A, footpad swelling reached a
maximum 3-4 weeks post infection (p.i.). In B6.Ceacam1-/- mice, footpad swelling was
significantly more pronounced compared to B6.WT controls and did not reach normal levels
until 125 days p.i. (Figure 1A). In contrast, no differences between the infected and
uninfected footpads were noticeable approx. 60 days p.i. in B6.WT mice (Figure 1A). In
addition to marked increases in footpad swelling, footpads from B6.Ceacam1-/- but not
B6.WT mice exhibited ulcerations, documented by photographs taken on day 41 p.i. (Figures
1B and 1C). Since increases in footpad swelling were most significant around day 21 p.i., we
chose this timepoint for all subsequent experiments, if not stated otherwise. To further
investigate the underlying causes for differences in the inflammatory phenotype, we
performed histological analyses. In H&E stains and detection of blood and lymphatic vessels
by immunohistology, we found tissue compaction and extensive vascularization in infected
footpads of B6.WT mice (Figure 1D, 1F). Newly formed lymphatic and blood vessels in
infected footpad of a B6.WT mouse are visible in the infiltrate (Figure 1F, green and red
arrowheads). In infected footpads of B6.Ceacam1-/- mice, cell-free spaces are visible in the
H&E stains (Figure 1E) and in immunofluorescence (Figure 1G). Poor vessel formation was
detectable in the infiltrate (Figure 1G). Blood and lymphatic vessels were mainly found in the
skin, (Figure 1G). The boundaries between the skin and the inflammatory infiltrate are
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indicated by white dotted lines (Figures 1F, 1G). Hence, the oedemas in the B6.Ceacam1-/mice result from the accumulation of liquid in tissues caused by impaired vascular drain of
interstitial fluids. The oedematous areas (quantified as areas devoid of DAPI-staining) in the
inflamed footpads of B6.WT and B6.Ceacam1-/- mice are given in Figure 1H. Whereas
approx. 50% of total areas in the infected footpads are devoid of DAPI staining in
B6.Ceacam1-/- mice, cell-free areas comprise up to approximately 35% in inflamed footpads
of B6.WT mice only (Figure 1H). The initial impression of inefficient local angiogenesis in
B6.Ceacam1-/- mice was corroborated by quantitative analyses of lymphatic and blood
vessels. Since lymphatic vessels may be dilated under inflammatory conditions and exhibit
variable diameters, we expressed lymphatic vessel densities in relation to the total area
analysed (Figure 1I). We found that LYVE-1+ areas or areas occupied by lymphatic vessels
were significantly higher in B6.WT compared to B6.Ceacam1-/- mice. Similarly, the area
claimed by MECA-32+ structures representing blood vessels was significantly reduced in the
infected footpads of B6.Ceacam1-/- mice compared to B6.WT animals (Figure 1J).
Since leishmaniasis is accompanied by a strong inflammatory response, we next characterized
the humoral and cellular immune response in order to elucidate whether differences in the
immune responses might influence angiogenesis in CEACAM1+ and CEACAM1backgrounds.
The adaptive immune response against L. major is not affected by loss of CEACAM1–
expression
To evaluate the humoral and cellular immune response against L. major, we measured
parasite-specific immunoglobulin production on days 20 and 40 p.i.. As summarized in
Figures 2A and 2B, comparable levels of anti-L. major-specific IgG1 and IgG2b antibodies
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were detected in B6.WT and B6.Ceacam1-/- mice. Hence, CEACAM1-deficiency does not
affect the B cell response against L. major parasites. To further analyse whether CEACAM1deficiency results in alterations of the cellular immune response, we determined proliferation
of whole cell preparations from the popliteal lymph nodes of B6.WT and B6.Ceacam1-/- mice
21 days p.i.. As shown in Figure 1C, proliferation of skin-draining lymph node cells is not
reduced in B6.Ceacam1-/- mice. Although cell proliferation is commonly accepted as a readout for T cell-priming and clonal expansion, cytokine secretion does not necessarily correlate
with proliferation. Thus, we quantified IFN-γ in supernatants from these cell cultures
indicative for an efficient Th1 response. We found comparable levels of IFN-γ in supernatants
from nodal cells derived from B6.WT and B6.Ceacam1-/- mice that had been re-stimulated
with L. major antigen (L-Ag) or concanavalin A (ConA; Figure 2D). Lymph node cells from
naïve B6.WT mice were used as controls. In conclusion, CEACAM1-deficiency does not alter
the proliferative response and IFN-γ production. Thus we conclude that the protective Th1type immune response against L. major is not altered in the absence of CEACAM1.
Therefore, the differences in the pathological phenotype between B6.WT and B6.Ceacam1-/mice are not a result of an inadequate adaptive immune response.
Ly-6Chigh/CD11bhigh cells that co-express CEACAM1 increase systemically after infection
with L. major
In addition to the adaptive immune response, we analysed the macrophage populations at the
site of the infection. During the early phases of leishmaniasis, we found a substantial increase
in total cell counts in the footpads of both mouse lines (Figure 3A), accompanied by enhanced
myelopoiesis and a massive influx of CD11b+ cells into the infected areas (Figure 3B) 27.
The inflammatory tissue largely consisted of CD11b+ cells, as detected in cross sections of the
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inflamed footpads, using fluorescently labelled anti-CD11b antibodies (Figures 3C and 3D,
shown in purple). In Figure 3C, CD11b+ cells are grouped around a large LYVE-1+ vessel in
the footpad of a B6.WT mouse. In footpads from B6.Ceacam1-/- mice (Figure 3D), CD11b+
cells form a lose infiltrate that contains a discontinuous structure of LYVE-1+ cells (Figure
3D). Since both B6.WT and B6.Ceacam1-/- mice exhibit substantial influx of CD11b+ cells
into the inflamed areas, but show differences in cellular distribution that coincide with
oedema formation in B6.Ceacam1-/- mice, we subjected the CD11b+ populations to more
detailed analyses. As shown in qualitative analyses in Figure 3E, Ly-6Chigh/CD11bhigh
monocytic precursors are detectable in both mouse lines, but importantly, in B6.WT mice,
this population is also highly positive for CEACAM1. Moreover, the Ly-6Chigh/CD11bhigh
population appeared to be diminished in the peripheral blood of B6.Ceacam1-/- mice (Figure
3E). Quantitative analyses of the monocytic precursor populations in bone marrow (Figure
3F) and peripheral blood (Figure 3G) of naïve and infected mice confirmed that B6.Ceacam1/-
mice exhibit a significant reduction in their Ly-6Chigh/CD11bhigh populations prior to and
during infection with L. major. Both mouse lines, however, equally respond to the infection
with expansion of the Ly-6Chigh/CD11bhigh population both in bone marrow and peripheral
blood (Figures 3F and 3G). This indicates that reduction in monocytic precursors is inherent
to a CEACAM1- phenotype.
Taken together, we observed that B6.Ceacam1-/- mice exhibit significant differences in their
innate immune response and their inflammatory phenotype as well as alterations in
angiogenesis after infection with L. major. However, we could not detect any differences in
the humoral or T-cell responses between B6.WT and B6.Ceacam1-/- mice. Therefore, we
decided to focus further functional analyses on the myeloid population positive for
CEACAM1 and CD11b.
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Reverse BMT using CEACAM1+ and CEACAM1- BM donors reveals an essential role for
Ly-6Chigh/CD11bhigh/CEACAM1+ monocytic progenitors in angiogenesis
To evaluate the impact of CEACAM1+ myeloid cells on angiogenesis, we performed reverse
BMTs using B6.WT donors (expressing CD45.1) for B6.Ceacam1-/- recipients (expressing
CD45.2; named “rescue” experiment hereafter) and B6.Ceacam1-/- donors for B6.WT mice as
recipients (named “un-rescue” experiment in the following). After reconstitution, the mice
were infected with L. major as described before, including B6.WT and B6.Ceacam1-/- mice as
controls. Ly-6Chigh/CD11bhigh progenitors were quantified in the BM and peripheral blood of
infected mice on d21 p.i., respectively (Figures 4A and 4B). Analysing the Ly6Chigh/CD11bhigh fraction in BM, we showed that this population is significantly reduced after
transfer of CEACAM1- BM into B6.WT recipients. Conversely, in the “rescue” experiment,
the numbers of Ly-6Chigh/CD11bhigh cells were restored to comparable levels as in B6.WT
mice (Figure 4A). Comparably, in peripheral blood, Ly-6Chigh/CD11bhigh monocyte precursors
were replenished to levels in B6.WT mice after transfer of CEACAM1+ BM into
B6.Ceacam1-/- mice (“rescue” BMT, Figure 4B). However, no significant differences between
Ly-6Chigh/CD11bhigh monocytic precursors were found between the “rescue” or “un-rescue”
experiments. To evaluate the effects of the BMTs on angiogenesis, we analysed histological
sections of the infected footpads after H&E and immune fluorescent labelling of blood and
lymphatic vessels, respectively (Figures 4C-4F). As shown in the H&E stain, the infiltrate
contained rather high cellular densities after the “rescue” BMT (Figure 4C), but exhibited
large cell-free areas in footpads of mice that had undergone the “un-rescue” BMT (Figure
4D). These findings are congruent with the staining patterns found in the B6.WT and
B6.Ceacam1-/- mice, shown in Figure 1D-1G. Here, the infected footpads from the “rescued”
B6.Ceacam1-/- mice showed a similar pattern as in the B6.WT mice, and in footpads of the
“un-rescued” B6.WT animals, we found oedema formation and reduction of angiogenesis
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comparable to our previous observations in B6.Ceacam1-/- mice (Figures 4E and 4F).
Vascular densities and oedematous areas were quantified as mentioned above and data are
summarized in Figures 4G-4I. In the B6.Ceacam1-/- mice that had undergone the “rescue”
transfer, LYVE-1+ areas in the footpad were significantly increased compared to the
B6.Ceacam1-/- and “un-rescued” mice (Figure 4G). Contrary, lymphatic vessels areas were
diminished in the B6.WT mice that had received CEACAM1- BM (“un-rescue” BMT). Here,
the lymphatic vascular areas were significantly smaller than in animals that had undergone the
“rescue” transfers (Figure 4G). Similarly, blood vessel formation was restored in the
“rescued” animals, and the areas claimed by MECA-32+ vessels were significantly increased
compared to footpads of B6.Ceacam1-/- mice (Figure 4H). Likewise, after the “un-rescue”
BMT, the extent of vascularization was reduced. In agreement with these observations, we
found that the oedematous areas in the footpads of mice after the “rescue” BMT were reduced
by half compared to the oedemas observed in footpads of B6.Ceacam1-/- animals (Figure 4I).
In line with these findings, B6.WT mice that had received CEACAM1- BM (“un-rescue”
BMT) prior to infection, exhibited cell-free areas to the same extent as B6.Ceacam1-/- animals
(Figure 4I). In conclusion, the BMT experiments demonstrate that the presence of CD11b+
cells with the potential to express CEACAM1 is crucial for angiogenesis in inflammation.
Lymphatic vessels co-express CEACAM1 and LYVE-1 in inflammation.
Since CEACAM1-expression on common myeloid cells infiltrating the site of infection with
L. major appears to be causally related to vessel formation under inflammatory conditions, we
investigated expression of CEACAM1 in the footpads of infected mice before and after
reverse BMTs. The three different panels (Figure 5) show single staining for CEACAM1
(yellow, left panels), LYVE-1 (red, middle panels) and the overlay plus MECA-32 (green)
and DAPI (blue; right panels). In infected B6.WT mice, a highly CEACAM1+ cellular
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infiltrate is seen in infected footpads (Figure 5A) as well as in lymphatic vessels (Figure 5B).
In the overlay (Figure 5C), co-expression of CEACAM1 on the lymphatic vessel as well as
partial co-expression on a MECA-32+ blood vessel was detected. In the infected B6.WT mice,
all of the newly formed lymphatic vessels were co-expressing CEACAM1 and LYVE-1. In
footpads of B6.Ceacam1-/- mice (Figure 5D-5F), only few LYVE-1+ structures are visible
(Figure 5E), but they are negative for CEACAM1 as well as the infiltrate formed in response
to the infection (anti-CEACAM1-staining, Figure 5D, and overlay, Figure 5F). Similarly to
our observations in B6.WT mice, expression of CEACAM1 is detected in footpads of mice
that had been subjected to the “rescue” BMT with CEACAM1+ BM (Figure 5G), and the
majority of the lymphatic vessels (Figure 5H) are also positive for CEACAM1 in the overlay
(Figure 5I). Contrary, after the “un-rescue” BMT using B6.Ceacam1-/- mice as donors and
B6.WT as recipient mice, CEACAM1-expression in the infiltrate (Figure 5J) as well as on
few lymphatic vessels was absent (Figures 5K, and overlay, Figure 5L). In summary, our
BMT experiments demonstrate that vessel formation within the infiltrate is only efficient if
CEACAM1 is expressed on myeloid cells.
CEACAM1-deficient macrophages fail to trigger angiogenesis in vitro
From our experiments conducted here, we cannot deduce whether CEACAM1-expression on
CD11b+ macrophage precursors contributes directly or indirectly to angiogenesis in
inflammation. Therefore, we sought for a suitable in vitro setting that allows mimicking
leishmaniasis and also studying macrophage behaviour. We performed Matrigel implantation
assays with growth factor enriched Matrigel also containing L. major parasites. In this model,
we intended to study formation of cellular assemblies and neo-vascularization in B6.WT and
B6.Ceacam1-/- mice. The implants were retrieved 7 days post implantation and analysed for
presence of vessel-like structures. In Figure 6A and 6B, immunohistochemical analyses of the
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implants are shown. In Figure 6A, an implant retrieved from a B6.WT mouse is shown. Here,
we found MECA-32+ structures (shown in green) and elongated, lumenizing LYVE-1+
structures (red). In specimens from B6.Ceacam1-/- mice, we only found single LYVE-1+ cells
and neither lymphatic nor blood vessel-like structures (Figure 6B). This was also confirmed in
H&E stainings and transmission electron microscopy analyses, with lumen-forming structures
in Matrigel implants only present in implants retrieved from B6.WT mice (Supplementary
Figure S1).
To investigate whether implant-derived inflammatory cells are able to form cellular
assemblies or adopt a tube-like morphology, we performed top-Matrigel tube formation
assays (Figure 6C-6G, Supplemental Figure S2). As shown in Figure 6C and enlargement in
Figure 6E, implant-derived cells from B6.WT mice form large cell clusters and thick tube-like
branches. In contrast, cells from B6.Ceacam1-/- mice only form smaller colonies and thin
tubular protrusions (Figure 6D and 6F). Arborizing structures are quantified in Figure 6G,
indicating that implant-derived cells from B6.Ceacam1-/- mice showed a significantly reduced
tube-formation potential compared to B6.WT control experiments. These findings were also
corroborated by counting the numbers of junctions with three or more branches in arborizing
entities, using different cell numbers seeded and by evaluation of different time points postseeding (Supplementary Materials and Methods, and Figures S2 and S3).
To confirm that CD11b+ macrophages may directly or indirectly assist vessel formation under
inflammatory conditions in our model system, we performed Matrigel implantation assays
under macrophage depletion. Clodronate-loaded liposomes have been reported to interfere
with lymphangiogenesis in vivo
28
. We used clodronate-loaded liposomes and PBS-loaded
liposomes as controls that were injected on the day before and on days 2, and 5 after Matrigel
implantation. As shown in Figures 6H through 6J, the CD11bhigh monocyte population was
16
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diminished significantly by treatment with clodronate-loaded liposomes, not by PBS-loaded
liposomes throughout the entire time course. Sections of Matrigel implants were analysed for
the presence of vessel-like structures on day 7 post implantation. In Matrigel implants from
mice that had received PBS-loaded liposomes, we found single LYVE-1+ cells but we also
observed cell clusters that resembled anastomosing vessel-like structures (Figure 6K). Only
few MECA-32+ structures were found (data not shown). In implants from mice that were
subjected to clodronate-mediated monocyte depletion, we only found single cells that
expressed LYVE-1, but we did not detect any extensive cell clusters or vessel-like structures
(Figure 6L). Total numbers of LYVE-1+ lumenizing structures in cross sections of Matrigel
implants are compared in Figure 6M. Here, we show that depletion of CEACAM1+/CD11b+
monocytic cells or absence of CEACAM1 equally affects the formation of lumenizing
LYVE1+ structures in Matrigel implants. From these data, we conclude that reduction of the
CD11bhigh monocyte population in peripheral blood concurs with reduction in cellular
clustering in the Matrigel implants. Since CD11bhigh cells co-express CEACAM1 on high
levels (cf. Figure 3E), a depletion of CD11b+ monocytes also includes depletion of
CEACAM1+ monocytes that are potentially involved in angiogenesis. Additionally, our
observations about cellular clustering and structure formation in implants from B6.WT mice
that were subjected to clodronate treatment are comparable to implants from B6.Ceacam1-/mice (Figures 6B and 6L). In conclusion, these in vitro data revealed that CEACAM1 and
CD11b double positive myeloid cells are pivotal for vessel formation.
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Discussion
In our report, we used the experimental model of cutaneous leishmaniasis to study
inflammatory lymph- and hemangiogenesis in CEACAM1+ and CEACAM1- mouse
backgrounds. Both mouse lines mounted an efficient adaptive immune response to L. major.
Still, the B6.Ceacam1-/- mice exhibited a different inflammatory phenotype in that they
responded to the local inflammation provoked by L. major with extended and increased
footpad swelling and oedema formation. These oedemas resulted from accumulation of liquid
in tissues caused by impaired vascular drain of interstitial fluids. Congruently, we found
inefficient hem- and lymphangiogenesis in the inflamed footpads of B6.Ceacam1-/- mice.
Since leishmaniasis triggers myelopoiesis and local accumulation of CD11b+ cells, we
analysed infiltrating cell populations in greater detail
27
. We found that in footpads of the
B6.Ceacam1-/- mice, local densities of inflammatory leukocytes were reduced, although the
total numbers of CD11b+ cells in the footpads of B6.WT or B6.Ceacam1-/- mice were
comparable. In B6.WT mice, these CD11b+ inflammatory cells within the infiltrate and their
Ly-6Chigh/CD11bhigh progenitors express high levels of CEACAM1. Importantly, these Ly6Chigh/CD11bhigh monocyte progenitors were diminished quantitatively in BM and peripheral
blood of B6.Ceacam1-/- mice prior to and after infection with L. major. The quantitative
differences in the CD11bhigh/Ly-6Chigh population were maintained throughout the course of
the infection, indicating that CEACAM1-deficiency might reduce myeloid progenitor
differentiation potential and maturation. Thus, CEACAM1-deficient mice respond to
leishmaniasis with inefficient replenishment of myeloid precursors. This result reinforces the
role of CEACAM1 as a differentiation antigen on myeloid cells but it also corroborates
previous reports that not only maturation and monocyte survival but also presentation of
CD11b on the cell surface are affected by CEACAM1 expression 29-31.
Therefore, it is most likely that besides its endothelial expression, CEACAM1 expression on
18
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inflammatory CD11b+ myeloid cells is of functional relevance during neo-vascularisation.
CEACAM1 expression becomes up-regulated on the leukocyte surface once cells have been
activated by pathogens or engagement of stimulatory receptors on their surface 11,32,33. For the
first time, we describe a causal relation between CEACAM1-expression on inflammatory
cells and local angiogenesis during an inflammatory response. Additionally, we found that
lymphatic vessels in the inflamed footpads co-expressed LYVE-1 and CEACAM1, whereas
the majority of lymphatic vessels in the skin of naïve mice did not. This indicates that
lymphatic vessels in inflammation express different surface markers than “quiescent” steadystate lymphatics. Also, CEACAM1 expression on blood endothelium in mice or humans is
not homogenous, and shows some variations 34 (A.K.H:, unpublished data).
A definite functional role for CD11b+ myeloid cells in neo-vascularization during hypoxia
and tumour progression has been verified regarding their potential to produce pro-angiogenic
cytokines or proteinases 28, 35-37. However, it has been in the focus of controversial discussions
how and if myeloid cells contribute physically to the formation of new blood and lymphatic
vessels.
We cannot deduce from our data that myeloid cells positive for CD11b and CEACAM1 either
fuse or integrate or trans-differentiate into lymphatic vessels and that they constitute definite
lymphatic or blood endothelial cell precursors. Whether CEACAM1 expression on
inflammatory lymphatic endothelial cells designates their origin, and whether or not it is
indicative or supportive of progenitor integration will need further investigation. Different
reports describe incorporation of Tie2+ or CD11b+ macrophages into newly formed vessels or
co-expression of macrophage and lymphatic markers during early stages of vessel formation
and in experiments ranging from 3–7 days 1,3,5,35,38,39. Still, these integration events were often
documented only for singular cells in tissue sections, which either emphasizes that they are
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rather rare events or only occur in a specific time window. However, we cannot exclude the
involvement of homophilic interactions between CEACAM1 molecules on leukocytes and
blood or lymphatic endothelium leading to extravasation or close endothelial interaction and
subsequent incorporation into the vasculature, since CEACAM1-derived peptides enhance
adhesion of neutrophilic granulocytes to immobilized human endothelial cells
40
.
Furthermore, it is not known if CEACAM1-mediated adhesion influences autocrine or
juxtracrine signalling processes during inflammation or whether high cellular densities are
required to create an angiogenic milieu. We have previously described that CEACAM1 is
implicated in hemangiogenesis and vascular remodelling and that CEACAM1 expression
enhances tissue recovery and capillary formation after femoral artery ligation
13,41
. These
reports and observations by others suggest that CEACAM1-dependent angiotrophic effects
are elicited when the endothelium is challenged e.g. in inflammation, hypoxia, or tumour
growth
13,41-43
. Also, CEACAM1 expression changes in lymphatic endothelial cells after
infection with Kaposi-associated herpes virus or alterations in growth factor homeostasis in
their environment
44,45
. In turn, CEACAM1 expression on endothelial cells appears to
influence lymphatic lineage marker expression and it has been described itself as a lymphatic
marker, which was identified in the context of comparative gene expression profiling of
lymphatic or blood endothelia 45, 46. However, CEACAM1 transcription levels did not respond
to virally induced expression of prospero homeobox-related protein 1 (PROX-1)47. This raises
the question whether CEACAM1 may be an upstream regulator of lymphatic lineage-specific
signalling pathways or could be involved in progenitor commitment. The observation that
CEACAM1 is expressed on blood endothelia in vivo, however, challenges this hypothesis.
To further explore a causative role for CEACAM1-expression on myeloid cells and the
formation of new blood vessels and lymphatic vessels, we performed BMT from CEACAM1+
donors into CEACAM1- recipients. Repeatedly, we analysed angiogenesis following infection
20
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with L. major. Strikingly, angiogenesis in the inflamed footpads was restored in the chimeras
and oedemas were absent. These data were also corroborated using reverse BMT of
CEACAM1- BM into CEACAM1+ recipients, which resulted in absence of local
angiogenesis. This provides evidence that CEACAM1+ common myeloid progenitors are
crucial for efficient inflammatory angiogenesis, and that CEACAM1 is expressed either in
newly formed lymphatics or in vessels embedded in inflammatory tissues. Under all
conditions examined, blood vessels in the mice express CEACAM1 heterogeneously both
under physiological and pathological conditions, congruent with human tissues 34,48,49.
To further extend our investigations on monocyte/macrophage contribution to angiogenesis in
vitro, we performed tube formation assays and Matrigel implantations with and without
clodronate liposome-induced macrophage depletion. We found that in B6.Ceacam1-/- mice,
neo-formation of vessel-like structures was impaired, and that CEACAM1-deficient
inflammatory cells exhibit reduced aggregation and tube-forming capacities in vitro.
Interestingly, Matrigel implant residing cells also showed impaired capabilities in the
formation of cell aggregates and anastomosing structures under clodronate treatment,
phenocopying the results we obtained in B6.Ceacam1-/- mice. Since clodronate liposomes
specifically deplete CD11bhigh macrophage precursors positive for CEACAM1, we also
depleted a CEACAM1+ population. Therefore, we suggest that expression of both CEACAM1
and CD11b is required on inflammatory cells to catalyze angiogenic processes (cf. Figure 3B)
27
.
In conclusion, we support the data that CEACAM1 is expressed on lymphatic endothelia
under specific conditions
44,45,50
.
Here, we show for the first time that CEACAM1+/CD11b+
cells control angiogenesis during inflammation, and that both blood and lymphatic vessel
formation are affected by loss of CEACAM1+/CD11bhigh cells. Further studies will need to be
21
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conducted to understand the detailed mechanisms how vascular formation is controlled by
CEACAM1+ common myeloid precursor cells.
Acknowledgements. The authors thank Christa Frenz, Krimhild Scheike (University Medical
Center Hamburg-Eppendorf) and Alexandra Veit, Ulricke Richardt and Dr. Christel Schmetz
(Bernhard-Nocht-Institute, Hamburg) for expert technical assistance and support with
transmission electron microscopy. We also thank Dr. Sven Mostböck (University of
Regensburg) for critical review of the manuscript. This work was supported by the Priority
Program of the German Research Foundation (DFG) SPP1190: The tumour-vessel interface
to A.K.H. and C.W., in part by DFG grant IT-13/3 to A.K.H. and W.D.I., and support of Jung
Foundation of Science and Research, Hamburg, to U.R..
Author Contributions
A.K.H. and U.R.: designed and performed research, wrote manuscript
T.B. and Na.Br.: performed research
N.v.R.: provided liposomes for macrophage depletion
P.L.: performed computer-assisted vessel quantification, analyzed data
U.S., B.F., W. D. Ito, and C.W.: analyzed and interpreted data
Ni.Be.: provided B6.Ceacam1-/- mice, analyzed data
Authors declare no conflict of financial interests.
22
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Figure Legends
Figure 1. Course of cutaneous leishmaniasis in B6.Ceacam1-/- and B6.WT mice and
characterization of local lymphatic and blood vessel formation. (A) Footpad swelling
following infection with L. major. Weekly recordings of footpad swelling are shown for
B6.Ceacam1-/- (white circles) and B6.WT mice (black squares). Footpad swelling is expressed
as percent increase of the infected over the non-infected footpad, respectively. Data shown
here summarize the mean ± SEM from 6 individuals each; the experiment was repeated three
times. *P< 0.05, **P< 0.01. Photographs in (B) and (C) of footpads from a B6.WT (B) and a
25
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B6.Ceacam1-/- mouse (C) document ulcerations (indicated with black arrowheads) in the
B6.Ceacam1-/- animal on day 41 p.i.. (D)-(G) Representative histological analyses of cross
sections of the infected footpads of B6.WT (D and F) and B6.Ceacam1-/- mice day 21 p.i. (E
and G). Cryostat sections were analysed by H&E staining (D and E) and immune fluorescent
labelling (F and G) of lymphatic vessels (anti-LYVE-1 antibody; shown in red), blood vessels
(anti-Meca-32 antibody, shown in green) and nuclei (DAPI; shown in blue). The dotted white
line indicates the border line between skin and inflammatory infiltrate. Magnification x200.
(H)-(J) Quantification of oedematous areas and lymph- and blood vessel formation in cross
sections of infected footpads of B6.WT (black bars) and B6.Ceacam1-/- mice (white bars).
Data shown here represent means ± SEM from at least 6 specimens. (H) Cell-free areas were
determined by quantifying the DAPI-free areas, expressed relative to the total inflammatory
area analysed; ***P< 0.001. (I) Quantification of lymphatic vessels by calculating percent
LYVE-1+ areas in cross sections of infected footpads; ***P<0.001. (J) Quantification of
MECA-32+ areas relative to the total areas analysed and expressed as percent MECA-32+
areas, **P< 0.01.
Figure 2. Characterization of the adaptive immune response towards L. major in
B6.Ceacam1-/-
and
B6.WT
mice.
(A)-(B)
Quantification
of
L.
major-specific
immunoglobulins in serum , IgG1 (A) and IgG2b (B) on days 20 and 40 p.i. by ELISA. The
amounts of the L. major-specific antibodies are expressed as relative ELISA units. Each
symbol represents data from one B6.WT (black squares) or B6.Ceacam1-/- mouse (white
circles; n=6 each). (C) Proliferation of lymph node cells from naïve B6.WT mice and
B6.Ceacam1-/- and B6.WT mice on day 21 p.i. after treatment with cell culture medium
(white bars), ConA (black bars) and L-Ag (shaded bars) expressed in counts per minute (cpm)
after H3-thymidine incorporation. Data shown here summarize means ± SEM from 6
26
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individuals per group. (D) Quantification of IFN-γ -production in supernatants from lymph
node cells from naïve B6.WT mice and B6.WT and B6.Ceacam1-/- mice following infection
with L. major on day 21 p.i. in response to medium (white bars), ConA (black bars) or L-Ag
(shaded bars). Data are summarized as means ± SEM from 6 mice per group (n.d.: not
detectable).
Figure 3. Characterization of the dynamics of the CD11b+ population in B6.Ceacam1-/and B6.WT mice during leishmaniasis. (A) and (B) Quantification of cellular influx into
infected footpads in B6.Ceacam1-/- and B6.WT mice by flow cytometry. Increase in total cell
counts (A) and CD11bhigh cells (B) in the infected footpads following infection with L. major
on days 0, 7 and 21 p.i. in B6.WT (black squares) and B6.Ceacam1-/- mice (white circles). (C)
and (D) Representative images following immune fluorescence staining of CD11b+ cells
(anti-CD11b-antibody; shown in purple), lymphatic vessels (anti-LYVE-1 antibody, shown in
red) in cross sections of the infected footpads in a B6.WT mouse (C) and a B6.Ceacam1-/mouse (D). Nuclei are stained with DAPI (blue). Magnification X400. (E) Representative
histograms showing high CEACAM1-expression on the Ly-6Chigh/CD11bhigh population from
peripheral blood of a B6.WT mouse (upper histograms, upper right square in the dot plot), but
not on Ly-6Chigh/CD11bhigh population in B6.Ceacam1-/- mouse (lower histograms). Note that
the Ly-6Chigh/CD11bhigh population is diminished in the peripheral blood of a naïve
B6.Ceacam1-/-mouse (upper right square in the lower dot plot histogram). (F) and (G)
Quantification of Ly-6Chigh/CD11bhigh monocyte precursors in the bone marrow (F) and
peripheral blood (G) in naïve and infected B6.WT (black bars) and B6.Ceacam1-/- mice
(white bars). Note that naïve B6.Ceacam1-/- mice harbour a significantly smaller Ly6Chigh/CD11bhigh progenitor population in the bone marrow prior to infection compared to
B6.WT animals, **P<0.01. In peripheral blood, B6.Ceacam1-/- mice maintain a significantly
27
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reduced Ly-6Chigh/CD11bhigh population before and after infection with L. major, P<0.05.
Data are presented as mean ± SEM from at least 9 mice each.
Figure 4. Analysis of the CEACAM1+/Ly-6Chigh/CD11bhigh progenitor population and
local angiogenesis after reverse transfer of CEACAM1+ and CEACAM1- BM into
recipient mice. (A) and (B) summary of quantitative flow cytometry analyses of Ly6Chigh/CD11bhigh populations in bone marrow (A) and peripheral blood (B) on day 21 p.i. with
L. major following CEACAM1+ and CEACAM1- BMT into B6.Ceacam1-/- and B6.WT mice,
respectively. Quantifications of Ly-6Chigh/CD11bhigh monocytic precursors after infection with
L. major and transfer of CEACAM1+ BM into CEACAM1- recipients (“rescue”, dark grey
bars) and transfer of CEACAM1- BM into B6.WT recipients (“un-rescue”, shaded bars) are
shown. Data from B6.WT and B6.Ceacam1-/- mice are depicted in black and white bars,
respectively. Data are represented as means from at least 6 individuals each, ± SEM. *P<0.05;
**P<0.01. (C)-(F) Histological analyses of cross sections of infected footpads on day 21 p.i.
in H&E stainings (C and D) and immune fluorescence (E and F) after BMT of CEACAM1+
BM into B6.Ceacam1-/- mice (“rescue”, C and E) and CEACAM1- BM into B6.WT mice
(“un-rescue”, D and F). In (E) and (F), lymphatic vessels are shown in red (anti-LYVE-1labelling) and blood vessels are coloured in green (anti-MECA-32 labelling). Nuclei are
shown in blue (DAPI). The white dotted line indicates the boundary between the skin and the
inflammatory infiltrate. Magnification X200. (G)-(I) Quantification of LYVE-1+ (G) and
MECA-32+ areas (H) as well as cell-free spaces (I) in inflamed areas of infected footpads in
control mice and after BMT as indicated. *P<0.05; **P<0.01; ***P<0.001.
Figure 5. Labelling of CEACAM1, LYVE-1, MECA-32 and nuclei in cross sections of
infected footpads. (A)-(C) Labelling of CEACAM1 (A, anti-CEACAM1 polyclonal
antiserum P1; yellow) reveals that the cellular infiltrate in the footpad is CEACAM1+. LYVE28
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1 is expressed on lymphatic vessels in the infiltrate (B, shown in red). In the overlay (C), coexpression of CEACAM1 and LYVE-1 on lymphatic vessels is shown in B6.WT mice. Blood
endothelium (anti-MECA-32 labelling, green) also expresses CEACAM1 (C). In footpads
from B6.Ceacam1-/- mice, no CEACAM1-labelling is present (D), and LYVE-1+ structures
(E) are CEACAM1- (F). (G)-(I) After BMT of CEACAM1+ BM into B6. Ceacam1-/- mice
(“rescue”), CEACAM1-labelling is restored in the infected footpads (G) and LYVE-1+
structures (H) co-express CEACAM1 (I). In the “un-rescue” experiment following
CEACAM1- BMT into B6.WT mice, CEACAM1 expressing cells are absent in the infiltrate
(J), and lymphatic vessels (K), (L). Representative photographs from 6 individuals each are
shown, magnification X630.
Figure 6. Analysis of morphogenic properties of CEACAM1+ and CEACAM1inflammatory cells in vitro and in vivo. (A) and (B) Matrigel implants were retrieved on day
7 and cross sections were analysed for MECA-32+ (shown in green) and LYVE-1+ structures
(shown in red). In implants from B6.WT mice (A), lumenizing MECA-32+ and LYVE-1+
structures are visible (arrows), whereas implants from B6.Ceacam1-/- mice only contain single
LYVE-1+ cells and no MECA-32+ structures (B; n=6, magnification X630). (C)-(F) TopMatrigel tube formation assays using implant-derived cells from B6.WT (C and E) and
B6.Ceacam1-/- mice (D and F) were documented 2 days post seeding 5x105 cells per well,
magnification X50 (C and D) and X200 (E and F). (G) Quantification of arborizing structures
from one representative experiments out of four. Cells from B6.WT mice are shown in black
squares, those form B6. Ceacam1-/--derived cells are shown in white circles. ***P<0.001.
(H)-(L) Analysis of CD11bhigh monocytes and their morphogenic properties following
treatment with PBS- or clodronate-loaded liposomes: Dot plot histograms of CD11bhigh cells
in gate R3 after treatment with control liposomes (H) or clodronate-loaded liposomes (I). (J)
Quantification of CD11bhigh populations in peripheral blood of B6.WT mice after treatment
29
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with PBS-loaded liposomes (black squares) or clodronate-loaded liposomes (white triangles).
Data shown here present the mean ± SEM of 6 individuals each, with samples analysed on
days 2, 5 and 7 during Matrigel implantation; ***P<0.001. (K) and (L) show cross sections of
Matrigel implants retrieved from B6.WT mice undergoing treatment with PBS-loaded
liposomes (K) and clodronate-loaded liposomes (L). Cross sections were stained for LYVE-1
(shown in red) and DAPI (blue), n=6 specimens each, magnification X1000. (M)
Quantitatification of LYVE-1+ structures in implants from B6.WT (black squares) or
B6.Ceacam1-/- mice (white circles), and B6.WT mice treated with PBS-loaded liposomes
(black triangles) or clodronate-loaded liposomes (white diamonds); **P<0.01, *P<0.05. Each
symbol represents data from one implant. Experiments were performed with at least 5
individuals.
30
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Prepublished online March 9, 2009;
doi:10.1182/blood-2008-10-184556
CEACAM1+ myeloid cells control angiogenesis in inflammation
Andrea K. Horst, Thomas Bickert, Nancy Brewig, Peter Ludewig, Nico van Rooijen, Udo Schumacher,
Nicole Beauchemin, Wulf D. Ito, Bernhard Fleischer, Christoph Wagener and Uwe Ritter
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