Download Network of Vascular-Associated Dendritic Cells in Intima of Healthy

Network of Vascular-Associated Dendritic Cells in Intima of
Healthy Young Individuals
G. Millonig, H. Niederegger, W. Rabl, B.W. Hochleitner, D. Hoefer, N. Romani, G. Wick
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
Abstract—In earlier studies, our group has established a new “immunological” hypothesis for atherogenesis supported by
experimental and clinical studies showing that inflammatory immunological reactions against heat shock protein 60
initiate the development of atherosclerosis. In the present study, we describe the discovery of a so-far-unknown network
of dendritic cells in the innermost layer of arteries, the intima, but not veins of healthy humans and rabbits. The number
of these dendritic cells is comparable to that of Langerhans cells in the skin, and dendritic cells show a similar phenotype
(CD1a⫹ S-100⫹ lag⫹ CD31⫺ CD83⫺ CD86⫺ and no staining for von Willebrand factor or smooth muscle cell myosin).
These vascular-associated dendritic cells accumulate most densely in those arterial regions that are subjected to major
hemodynamic stress by turbulent flow conditions and are known to be predisposed for the later development of
atherosclerosis. These results open new perspectives for the activation of the immune system within the arterial wall.
(Arterioscler Thromb Vasc Biol. 2001;21:503-508.)
Key Words: atherosclerosis 䡲 intima 䡲 arteries 䡲 immunofluorescence 䡲 dendritic cells
A
therosclerosis is a multifactorial disease depending on
various exogenous factors that become effective on an
appropriate genetic background. For many years, atherosclerosis research has concentrated on lipid accumulation and the
proliferation of smooth muscle cells. However, more recently, the results of numerous studies have emphasized the
pivotal role of the immune system in atherogenesis.1–3 Clinical and experimental data from our own group have provided
evidence that atherosclerosis starts as an inflammatory immunological disease that is due to an (auto)immune reaction
based on humoral and cellular immunity against microbialhuman cross-reactive epitopes of heat shock protein 60.4 – 6
Immunohistochemical studies on frozen sections of arteries
revealed mononuclear cell infiltrations not only in early and
late atherosclerotic lesions but also in the arteries of healthy
infants and children at sites subjected to major hemodynamic
(turbulent) stress that are known to be predisposed for the
development of atherosclerotic lesions later in life. These
accumulations consist of activated T cells (most of them
carrying the ␣/␤ T cell receptor and a considerable number
expressing ␥/␦ T cell receptor as well), macrophages, dendritic cells (DCs), some scattered mast cells, and a very few
natural killer cells or B cells.7 In analogy to the mucosaassociated lymphoid tissue, we tentatively called these accumulations of mononuclear cells in healthy children “vascularassociated lymphoid tissue” and assumed a similar function
for these as a system of local defense of the vascular system.8
During the last decade, great progress has been made in the
field of DC research.9 Because it is now generally accepted
that the immune system plays an important role in atherogenesis, it was deemed of interest to perform a critical study on
the occurrence and distribution of DCs in the vascular system
with special emphasis on the sites of the newly discovered
vascular-associated lymphoid tissue.
One of the typical features of DCs is their presence at the
borderline of the body to its environment. At these locations,
they are present in an immature stage, characterized by high
endocytotic activity and low T-cell stimulatory potential
because of the lack of costimulatory molecules, such as
CD40, CD54, and CD86.10 Thus, DCs are present in the
skin,11 in the intestine (mainly in the Peyer’s patches),12 and
in the respiratory tract.13 But only recently have there been
suggestions that atherosclerotic lesions may also be populated
by DCs.14 –16 DCs in the atherosclerotic vessel wall express
human lymphocyte antigen-DR, CD1a, and S-100 protein14,16
and are positive for intercellular adhesion molecule-117 and
vascular cell adhesion molecule-1.18
Previous studies investigating vascular DCs concentrated
only on atherosclerotically altered arteries, but there were no
data available concerning the presence of DCs in the healthy
intima, as we had demonstrated to be the case in earlier
preliminary work.7,8
We have now focused our attention on the exact distribution and abundance of the vascular-associated DCs.
Received November 28, 2000; revision accepted January 23, 2001.
From the Institute for Biomedical Aging Research, Austrian Academy of Sciences (G.M., G.W.), and the Institute for General and Experimental
Pathology (G.M., H.N., G.W.), the Department of Forensic Medicine (W.R.), the Department of Transplant Surgery (B.W.H.), the Department of Cardiac
Surgery (D.H.), and the Department of Dermatology (N.R.), University of Innsbruck, Innsbruck, Austria.
Correspondence to Georg Wick, MD, Institute for General and Experimental Pathology, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria. E-mail
[email protected]
© 2001 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
503
504
Arterioscler Thromb Vasc Biol.
April 2001
Methods
sion), shock-frozen, and stored in liquid nitrogen until being cut into
4-␮m-thick frozen sections.
Arterial Specimens
Staining Procedures
Rabbits
Five female New Zealand White rabbits between 8 and 12 weeks of
age were obtained from Savo/Charles River Co, Kisslegg im Allgäu,
Germany. They were housed individually under conventional conditions in wire-bottomed cages at 22°C with a relative humidity of
60% in the Central Experimental Animal Facilities of the University
of Innsbruck, Medical School. They received water ad libitum and
were fed a standard diet (T775, Tagger & Co). All animal experiments were performed according to the institutional guidelines.
The animals were euthanized under ketamine (25 mg/kg) and
xylazine (5 to 10 mg/kg) anesthesia by heart puncture. The aortas
were carefully removed from the surrounding tissue from the
beginning of the aortic arch to the bifurcation into the 2 iliac arteries.
Then, they were immersed in ice-cold PBS, pH 7.2, and immediately
processed for further immunohistochemical or immunofluorescence
studies.
Humans
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
Human arterial samples from 14 infants and children aged 8 months
to 16 years were obtained from the Department of Forensic Medicine, University of Innsbruck, Medical School. Causes of death were
sudden infant death syndrome, polytraumata, intoxication, drowning,
pulmonary embolism, and aspiration pneumonia.
For investigations on vascular DCs in adults, we used human
arterial samples from the carotid arteries, aortas, and iliac arteries of
20 young adults between 17 and 34 years of age, whose death was
due to suicide, homicide, or accident (obtained from the Department
of Forensic Medicine, University of Innsbruck, Medical School) or
who were candidates for organ explantation (obtained from the
Department of Transplant Surgery, University of Innsbruck, Medical
School) and did not suffer from any clinical manifestation of
cardiovascular disease.
In 5 of the above-mentioned cases, veins were collected in
addition to the arteries (either the jugular vein or the iliac vein as a
pendant to the respective arterial sample).
The samples for frozen sections were collected, immersed in
transport buffer as described by Michel et al,19 and transported to the
laboratory on ice. Samples for the preparation of intimal sheets were
immersed in ice-cold PBS instead of transport buffer. The surrounding connective tissue was removed, and each artery was filled with
freezing medium (O.C.T. Tissue Tek, Miles Inc, Diagnostic Divi-
Sources and Dilutions of Antibodies
Antibody
Source
Dilution Catalogue No.
CD1a
DAKO
1⬊30
M0721
CD83
Serotec
1⬊50
MCA1582
S-100
Neomarkers
1⬊100
MS-296-PO
CD86
Ancell
1⬊50
307-040
N. Romani
1⬊10
DAKO
1⬊50
䡠䡠䡠
M0616
Lag (supernatant)
von Willebrand factor
CD31
DAKO
1⬊20
M0823
Smooth muscle myosin heavy chain
DAKO
1⬊25
M3558
CD68
DAKO
1⬊50
M718
Rabbit MHC II
ATCC
1⬊10
CRL 1760
Isotype IgG1 (hPL37)
P. Berger
1⬊50
䡠䡠䡠
Isotype IgG2a (hFSH)
P. Berger
1⬊30
Rabbit anti-mouse Ig
DAKO
1⬊30
䡠䡠䡠
Z0259
APAAP complex mouse
DAKO
1⬊60
D0651
Rabbit anti-mouse Ig-FITC
DAKO
1⬊30
F0261
Animal research kit
DAKO
䡠䡠䡠
K3954
Ig indicates immunoglobulin.
Immunohistochemistry on Frozen Sections
Slides with frozen sections were air-dried for 30 to 60 minutes at
room temperature. Blocking of nonspecific antibody binding was
achieved by incubation with 10% normal human serum (heat
inactivated at 56°C for 30 minutes) in blocking reagent (No.
1096176, Boehringer-Mannheim) for 15 minutes. Excess serum was
blotted off; the primary antibody was diluted in Tris-buffered saline
(TBS), pH 7.4, and applied directly without any washing procedure;
and the sections were incubated for 30 minutes. Optimal dilutions for
all antibodies were determined in pilot studies. Incubation took place
in a humidified chamber at room temperature. The sections were
then rinsed 3 times in TBS, the secondary antibody was applied, and
the sections were incubated for another 30 minutes. They were rinsed
as described above, and incubation with the alkaline phosphatase/
anti–alkaline phosphatase (APAAP) complex followed at room
temperature for 30 minutes. The incubation step with the secondary
antibody and the APAAP complex was repeated to increase the
staining intensity. After 3 further changes of TBS, visualization with
Fast Red Naphthol (Sigma) and counterstaining with Mayer’s
hemalaun was performed. Slides were finally mounted in Kayser’s
glycerol gelatin (Merck). The Table shows the source and dilution of
the antibodies used.
Double Staining on Frozen Sections
Double staining was achieved by combination of the APAAP
technique and the DAKO animal research kit. Immunohistochemistry was performed as described above for single staining. After
visualization with Fast Red, the second antibody was treated with the
biotinylation reagent according to the manufacturer’s instructions
and developed with horseradish peroxidase–labeled streptavidin
contained in the test kit.
En Face Immunofluorescence on Human or Rabbit
Intimal Sheets
Fresh arterial samples were washed in cold PBS to remove blood.
They were opened longitudinally, cut into 2 halves, and dissected
into small pieces of ⬇0.7⫻0.7 cm, followed by incubation in 0.5
mol/L NH4SCN (Merck) at 37°C for 60 minutes.20 After a wash in
PBS at room temperature, the intima was gently lifted off from the
rest of the vessel wall with delicate forceps. After they were rinsed
in PBS for 30 minutes, the samples were ready to undergo the
staining procedure.
Sheets were acetone-fixed for 10 minutes at room temperature
(with the exception of those that were to be stained for CD1a) and
rinsed in PBS for 20 minutes thereafter. Then incubation in the
optimally diluted primary antibody took place overnight at 4°C.
Washing was performed in PBS/1% BSA (Sigma) for 4 hours at
room temperature on a multiaxle rotator, and the sheet was incubated
in diluted FITC-labeled secondary antibody for the next 4 hours at
room temperature. Finally, the sheets were washed in PBS/1% BSA
on the rotator at room temperature overnight and mounted the
following morning. Incubation in NH4SCN, acetone fixation, and
incubation with antibodies were performed in micro–test tubes (No.
0030120.086, Eppendorf-Netherler-Hinz), whereas the washing took
place in 50-mL tubes (No. 252070, Falcon, Becton-Dickinson
Labware) to guarantee excess washing fluid. The specimens were
examined with a the laser scanning confocal fluorescence microscope (Zeiss), with magnifications ranging between 100- and
630-fold.
Negative Controls
For all experiments, negative controls were carried out either by
omitting the first antibody or by using an irrelevant isotype-matched
antibody.
Photographic Documentation
Stained sections were examined by light microscopy (Optiphon-2,
Nikon), and pictures were taken on the same microscope with use of
Millonig et al
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
Figure 1. Tangential section through intima of carotid artery of
8-year-old boy. CD1a⫹ DCs are stained with double-sandwich
technique with use of secondary antibody and APAAP complex.
Note that typical cytoplasmic processes extend in all directions.
Original magnification ⫻250.
automatic exposure equipment (UFX-DX, Nikon) and daylight films
(Fujichrome Velvia-50).
Intimal sheets were analyzed by confocal microscopy, and pictures were stored and printed on a laser printer.
Network of Dendritic Cells in Healthy Intima
505
Figure 2. Conventional frozen section of carotid artery of the
same 8-year-old boy. DCs are stained for lag antigen to demonstrate that they are Langerhans cell–like DCs. Original magnification ⫻200.
von Willebrand factor and smooth muscle cell myosin. This
pattern of marker expression is similar to that of immature
Langerhans cells in the epidermis.
En Face Immunofluorescence on Intimal Sheets
Results
Immunohistochemistry on Frozen Sections
CD1aⴙ Cells Are Present in All Human
Arterial Specimens
CD1a⫹ DCs were found in all human arterial samples. In
arterial specimens showing incipient atherosclerotic development, this finding was expected; therefore, these served as
positive controls for all further investigations.
CD1aⴙ Cells Are Present in Intima of Children
In children, DCs were found in all arterial specimens of the
intima in the subendothelial location, showing the typical
morphological features of DCs with long cytoplasmic processes. They are found in higher density in areas of bifurcation and more sparsely distributed in regions without a
deviating flow pattern. The best staining impressions were
obtained in tangential sections of the intima, displaying the
intimal area more broadly (Figure 1).
After demonstration of the presence of DCs in the intima of
healthy children by staining for CD1a, further analysis
concerning the characterization of the vascular-associated
DCs was performed to determine the subset of DCs. We
stained for CD86 (B7.2) as a marker for mature DCs, CD83,
S-100 (Figure 2), lag (a marker for Birbeck granules that are
specific for Langerhans cells), and CD31. von Willebrand
factor as an endothelial cell–specific molecule not expressed
on any subset of DCs was included as a control.
To exclude a type of cell showing smooth muscle cell21 or
macrophage phenotype with additionally acquired DC markers, we also stained for smooth muscle cell myosin and
CD68, respectively, in double-staining experiments or on
serial sections. These experiments showed that vascularassociated DCs have the following phenotype: CD1a⫹ S-100⫹
lag⫹ CD31⫺ CD68⫺ CD83⫺ CD86⫺, and they are negative for
Major Histocompatibility Complex Class IIⴙ Cells With
DC Morphology Are Found Abundantly in Intima of
Normal Rabbits
Rabbit intimal sheets were analyzed by immunofluorescence
for Ia expression. Ia⫹ cells were found spread over the intima
in all stained specimens. Considering the shapes of their cell
bodies and their long processes, they were primarily recognized as either macrophages or DCs (Figures 3 and 4), and
they formed a network in areas of dense accumulation,
particularly areas of Ia⫹ cell accumulation, such as those parts
of the rabbit aorta at which arteries branched off. Ostia of
branching arteries were not surrounded by stained cells, but
Figure 3. Ia⫹ cells in rabbit aortic intima. Intimal sheets, where
intima is separated from the rest of the vessel wall, are stained
by indirect immunofluorescence with use of a secondary antibody conjugated to FITC. Laser scanning confocal microscopy
was used. Original magnification ⫻200.
506
Arterioscler Thromb Vasc Biol.
April 2001
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
Figure 4. Single Ia⫹ cell in rabbit intima displaying typical DC
morphology. Cell is stained in intimal sheet prepared from rabbit
aorta by indirect immunofluorescence with use of a secondary
antibody conjugated to FITC. Laser scanning confocal microscopy was used. Original magnification ⫻630.
they were concentrated at those parts of the branch at which
the mechanical drag is most severe (Figure 5). Optical
sections in the laser scanning microscope showed that all Ia⫹
cells were lying under the endothelium and that none was
adherent to the endothelium. Endothelial cells did not stain
for Ia.
Closer scrutiny showed that Ia⫹ cells were orientated
longitudinally within the aorta except at those sites where
turbulent predominates over laminar blood flow.
CD1aⴙ Cells Are Found in Human Intima in Amounts
Equivalent to Those in Iaⴙ Cells in the Rabbit
Because no specific marker exists for rabbit DCs, identification of these cells was performed on human arterial intimal
sheets from individuals without macroscopic atherosclerotic
lesions. CD1a⫹ cells were found in most parts of the arterial
Figure 5. View from rabbit aorta into ostium of offbranching
intercostal artery. Ia⫹ cells are stained by indirect immunofluorescence. On this intimal sheet, differential distribution of Ia⫹
cells can be clearly demonstrated. Stained cells accumulate at
those sites of the branching artery where the mechanical drag is
most intense. Laser scanning confocal microscopy was used.
Original magnification ⫻100.
Figure 6. CD1a⫹ DCs in human intimal sheet prepared from
common iliac artery. DCs are found in the subendothelial position embedded in intimal connective tissue. Staining was performed by indirect immunofluorescence, and examination was
performed with use of laser scanning confocal microscopy.
Original magnification ⫻200.
specimens examined. As already shown in the rabbit by
expression of major histocompatibility complex (MHC) class
II, CD1a⫹ DCs are not distributed regularly, but they aggregate in hemodynamically stressed areas, where they form a
network-like structure similar to Langerhans cells in the skin,
extending their processes in all directions (Figure 6).
A very striking finding was that DCs do not exist in veins
that can therefore be considered to be an integrated negative
control for the staining procedures.
Discussion
In the present study, we show that DCs are present in the
normal intima of children. In contrast to all present investigations, we prove not only that DCs appear in the vessel wall
on inflammatory stimuli during atherogenesis but also that
they preexist under the endothelium, even in arteries of young
individuals still unaffected by any form of atherosclerosis.
The staining pattern for various markers used in DC characterization suggests a similarity between vascular-associated
DCs and Langerhans cells, inasmuch as both of them are
CD1a⫹ S-100⫹ lag⫹ CD31⫺ CD68⫺ CD83⫺ and CD86⫺ in the
immature state. In contrast to other investigations involving
atherectomy specimens with atherosclerotic lesions in which
stellate cells21 or subsets of smooth muscle cells22 are found
to display some of these markers, we (1) used exclusively
unaffected arteries of children and adolescents that showed
no myxomatous tissue, (2) showed by double staining and by
use of serial sections that these CD1a⫹ cells are negative for
myosin and CD68, and (3) showed evidence that these cells
are DCs by staining for lag, a unique marker for Langerhans
cells.23
The highest density of vascular DCs was observed at areas
in which turbulence predominates over laminar hemodynamics. This is true for human arteries as well as arteries from
rabbits, which are a generally accepted experimental animal
model for atherosclerosis research into the role of lipids as
well as immunological processes. The location of vascular
DCs corresponds exactly to the sites prone to the later
Millonig et al
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
development of atherosclerotic lesions. Turbulent flow results
in complex secondary flows with flow reversal and dynamic
stagnation points, resulting in prolonged endothelial resident
times for large atherogenic particles (eg, LDLs) or blood
cells. An increased contact time between leukocytes and
endothelial cells could lead to increased transendothelial
migration and, therefore, explains the higher density of all
different cell types, including DCs, at these sites.
Inasmuch as our experimental rabbits were normocholesterolemic, these results emphasize that predisposed areas can
be recognized from their cellular makeup even without any
lipid deposition.
The absence of vascular DCs in veins could be another
explanation (besides the difference in blood pressure) of why
we develop arteriosclerosis and not venosclerosis. Another
striking finding from the present study concerns the localization of DCs in the arterial intima. They are orientated
longitudinally with the blood stream in areas of laminar flow
conditions, but they change their distribution pattern in areas
of turbulent flow. So far, we can only speculate that extracellular matrix proteins in the intima are involved in this
process. Further studies on intimal extracellular matrix patterns will show whether they are distributed differentially in
areas subjected to different hemodynamic stress.
Surprisingly, en face staining for DCs revealed a network
formed by vascular DCs similar to that seen with Langerhans
cells in the skin, and by this technique, we were able to show
that the vessel wall seems to be protected from blood-borne
pathogens in the same manner as other surfaces of the body
in close contact with the environment. The fact that the
vascular wall is a huge surface and a possible site of antigen
processing and presentation and our present demonstration of
DCs in the arterial intima open new perspectives in this
respect. This prospect is certainly due to the general belief
that blood is a sterile fluid not requiring immunosurveillance,
because possible noxious substances have already passed the
gate control of the skin or the mucosa. However, a closer look
shows that even in the blood, invasions of bacteria can occur.
Bacteremia not only occurs during septic diseases but also is
an intermittent physiological condition in healthy organs,
triggered by minitraumata (eg, tooth brushing),24 turning
pathological only when it lasts longer than a few hours.
This amount of professional antigen-presenting cells in the
arterial intima opens new perspectives to immune reactions
taking place in the vessel wall itself.
To date, it has been speculated that initialization of the
immune response leading to development of atherosclerosis
might take place in the para-aortic lymph nodes, but our
studies implicate local initiation of the priming of naive
lymphocytes. Macrophages, which are always found in atherosclerotic lesions, were proposed as possible candidates
because they are highly positive for MHC class II molecules.25 Endothelial cells are not able to present antigens in
the normal unaffected intima because they do not express
MHC class II molecules constitutively. Endothelial cells are
MHC II positive essentially only in those areas in which
activated T cells have accumulated subendothelially but not
in the normal intima.26 This phenomenon can be explained by
the release of interferon-␥ from infiltrating T cells.27–29 For
this reason, endothelial cells can be excluded as initiators of
an immune reaction by presenting antigen to lymphocytes,
Network of Dendritic Cells in Healthy Intima
507
whereas they could play a role in the promotion of immune
reactions and in the activation of memory T cells. We now
disclose the possibility that resident intimal DCs may be
effective as local antigen-presenting cells and could perhaps
also initiate (auto)immune processes in the vessel wall. Their
subendothelial position is very advantageous to this scenario,
inasmuch as the DCs can come in close contact with
blood-borne antigens as well as with lymphocytes that adhere
to the endothelial layer and migrate through the vessel wall.
However, whether arteries need be of certain size to be
populated by DCs or whether DCs are present even in
arterioles remains to be investigated. The arterial surface is a
huge area when calculated for the whole body; therefore, our
data identify a new “immunological space” at which important immunological reactions could take place and influence
physiological as well as pathological processes.
In the present study, we describe the natural habitat of
vascular DCs; functional investigations will follow on the
basis of the present morphological data.
From the present study, it is not yet clear whether these
DCs play an atherosclerosis-promoting or -inhibiting role.
Both mechanisms are possible, ie, induction of tolerance or
stimulation of an immune response to the antigen triggering
atherogenesis. However, our morphological data suggest an
atherosclerosis-enhancing function by the accumulation of
DCs in areas of the vascular tree that are predisposed to later
atherosclerotic development.
Acknowledgments
This study was supported by the Austrian Science Fund, project No.
P-14741/MED (to G.W.), the Hans und Blanca Moser-Stiftung (to
G.M.), and a “Förderungsstipendium” of the University of Innsbruck
(to G.M.). We would like to thank Dr Hermann Dietrich and Ernst
Rainer for expert help with animal experiments and Dr Heidrun
Recheis for confocal microscopic analyses of the rabbit specimens.
References
1. Libby P, Hansson GK. Involvement of the immune system in human
atherogenesis: current knowledge and unanswered questions. Lab Invest.
1991;64:5–15.
2. Hansson GK. Cell-mediated immunity in atherosclerosis. Curr Opin
Lipidol. 1997;8:301–311.
3. Ross R, Atherosclerosis: an inflammatory disease. N Engl J Med 1999;
340:115–126.
4. Xu Q, Willeit J, Marosi M, Kleindienst R, Oberhollenzer F, Kiechl S,
Stulnig T, Luef G, Wick G. Association of serum antibodies to heat-shock
protein 65 with carotid atherosclerosis. Lancet. 1993;341:255–259.
5. Wick G, Schett G, Amberger A, Kleindienst R, Xu Q. Is atherosclerosis
an immunologically mediated disease? Immunol Today. 1995;16:27–33.
6. Wick G, Perschinka H, Xu Q. Autoimmunity and atherosclerosis. Am
Heart J. 1999;138:S444 –S449.
7. Wick G, Romen M, Amberger A, Metzler B, Mayr M, Falkensammer G,
Xu Q. Atherosclerosis, autoimmunity, and vascular-associated lymphoid
tissue. FASEB J. 1997;11:1199 –1207.
8. Waltner-Romen M, Falkensammer G, Rabl W, Wick G. A previously
unrecognized site of local accumulation of mononuclear cells: the
vascular-associated lymphoid tissue. J Histochem Cytochem. 1998;46:
1347–1350.
9. Steinman RM. The dendritic cell system and its role in immunogenicity.
Annu Rev Immunol. 1991;9:271–296.
10. Banchereau J, Steinman RM. Dendritic cells and the control of immunity.
Nature. 1998;392:245–252.
11. Romani N, Schuler G. The immunologic properties of epidermal Langerhans cells as a part of the dendritic cell system. Springer Semin Immunopathol. 1992;13:265–279.
12. Liu LM, MacPherson GG. Rat intestinal dendritic cells: immunostimulatory potency and phenotypic characterization. Immunology. 1995;85:
88 –93.
508
Arterioscler Thromb Vasc Biol.
April 2001
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
13. Holt PG. Pulmonary dendritic cell populations. Adv Exp Med Biol. 1993;
329:557–562.
14. Bobryshev YV, Lord RS. S-100 positive cells in human arterial intima
and in atherosclerotic lesions. Cardiovasc Res. 1995;29:689 – 696.
15. Bobryshev YV, Lord RS. Ultrastructural recognition of cells with dendritic cell morphology in human aortic intima: contacting interactions of
vascular dendritic cells in athero-resistant and athero-prone areas of the
normal aorta. Arch Histol Cytol. 1995;58:307–322.
16. Bobryshev YV, Lord RS, Rainer S, Jamal OS, Munro VF. Vascular
dendritic cells and atherosclerosis. Pathol Res Pract. 1996;192:462– 467.
17. Bobryshev YV, Lord RS. Mapping of vascular dendritic cells in atherosclerotic arteries suggests their involvement in local immuneinflammatory reactions. Cardiovasc Res. 1998;37:799 – 810.
18. Bobryshev YV, Lord RS, Rainer SP, Munro VF. VCAM-1 expression
and network of VCAM-1 positive vascular dendritic cells in advanced
atherosclerotic lesions of carotid arteries and aortas. Acta Histochem.
1996;98:185–194.
19. Michel B, Milner Y, David K. Preservation of tissue-fixed immunoglobulins
in skin biopsies of patients with lupus erythematosus and bullous diseases:
preliminary report. J Invest Dermatol. 1972;59:449–452.
20. Juhlin L, Shelley WB. New staining techniques for the Langerhans cell.
Acta Derm Venereol. 1977;57:289 –296.
21. Tjurmin AV, Ananyeva NM, Smith EP, Gao Y, Hong MK, Leon MB,
Haudenschild CC. Studies on the histogenesis of myxomatous tissue of
human coronary lesions. Arterioscler Thromb Vasc Biol. 1999;19:83–97.
22. Babaev VR, Bobryshev YV, Stenina OV, Tararak EM, Gabbiani G.
Heterogeneity of smooth muscle cells in atheromatous plaque of human
aorta. Am J Pathol. 1990;136:1031–1042.
23. Kashihara-Sawami M, Horiguchi Y, Ikai K, Takigawa M, Ueda M,
Hanaoka M, Imamura S. Letterer-Siwe disease: immunopathologic study
with a new monoclonal antibody. J Am Acad Dermatol. 1988;18:
646 – 654.
24. Donley TG, Donley KB. Systemic bacteremia following toothbrushing: a
protocol for the management of patients susceptible to infective endocarditis. Gen Dent. 1988;36:482– 484.
25. Hughes DA, Townsend PJ, Haslam PL. Enhancement of the antigen-presenting function of monocytes by cholesterol: possible relevance to
inflammatory mechanisms in extrinsic allergic alveolitis and atherosclerosis. Clin Exp Immunol. 1992;87:279 –286.
26. Xu QB, Oberhuber G, Gruschwitz M, Wick G. Immunology of atherosclerosis: cellular composition and major histocompatibility complex
class II antigen expression in aortic intima, fatty streaks, and atherosclerotic plaques in young and aged human specimens. Clin Immunol
Immunopathol. 1990;56:344 –359.
27. Masuyama J, Minato N, Kano S. Mechanisms of lymphocyte adhesion to
human vascular endothelial cells in culture: T lymphocyte adhesion to
endothelial cells through endothelial HLA-DR antigens induced by
gamma interferon. J Clin Invest. 1986;77:1596 –1605.
28. Epperson DE, Pober JS. Antigen-presenting function of human endothelial cells: direct activation of resting CD8 T cells. J Immunol. 1994;153:
5402–5412.
29. Wagner CR, Vetto RM, Burger DR. Expression of I-region-associated
antigen (Ia) and interleukin 1 by subcultured human endothelial cells. Cell
Immunol. 1985;93:91–104.
Downloaded from http://atvb.ahajournals.org/ by guest on May 14, 2017
Network of Vascular-Associated Dendritic Cells in Intima of Healthy Young Individuals
G. Millonig, H. Niederegger, W. Rabl, B. W. Hochleitner, D. Hoefer, N. Romani and G. Wick
Arterioscler Thromb Vasc Biol. 2001;21:503-508
doi: 10.1161/01.ATV.21.4.503
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272
Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/21/4/503
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the
Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for
which permission is being requested is located, click Request Permissions in the middle column of the Web
page under Services. Further information about this process is available in the Permissions and Rights
Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online
at:
http://atvb.ahajournals.org//subscriptions/