in TPBS, visualisation of antigens was produced by treatment for 5 min with 3,3-diaminobenzidine DAB.
The PAP system with DAB chromogen yielded a brown reaction product at the site of the target antigens.
Alternatively, cell types were identified using the alka- line phosphatase anti-alkaline phosphatase APAAP
technique. Alkaline phosphatase-conjugated antibody Dako with Fast Red Dako as an enzyme substrate
were used. The APAAP system with Fast Red chro- mogen resulted in a rose precipitate at the site of the
identified antigens. All the incubations were completed at room temperature. For negative controls, the first
antibodies were omitted or the sections were treated with an immunoglobulin fraction of non-immune goat
serum Vector S-1000 as a substitute for the primary antibody. None of the negative control sections showed
positive immune staining. Counterstaining was per- formed with Mayer’s haematoxylin and the sections
were examined in an Olympus microscope at 10 × 10 and 10 × 40 magnifications.
2
.
4
. Double immunostaining procedure The
co-localisation of
dendritic cells
with lymphocytes was analysed by double immunostaining
combining the PAP and APAAP techniques. After visualisation of dendritic cells by the PAP technique,
the tissue sections were washed for 60 min with 0.1 M glycinehydrochloric buffer pH 2.2 at 4°C. The sec-
tions were then incubated with one of the second primary antibodies anti-CD3 or anti-CD20 for 60 min
at room temperature. Lymphocytes were visualised us- ing the APAAP technique, as described above for single
immunostaining. Negative controls were carded out as described above and none of the negative control sec-
tions showed positive immune staining. Counterstaining was performed with Mayer’s haematoxylin.
2
.
5
. Electron microscopic analysis After fixation in 2.5 glutaraldehyde in PBS pH
7.4, specimens were postfixed in 1 osmium tetroxide, dehydrated in graded ethanol and propylene oxide and
were then embedded in Araldite resin. Ultrathin sec- tions were cut on a LKB-III ultratome. Ultrathin sec-
tions were stained with uranyl acetate and lead citrate and examined with the aid of a Hitachi H7000 electron
microscope at an accelerating voltage of 100 kV.
3. Results
In all the specimens, atherosclerotic changes of the intima were present. Advanced atherosclerotic plaques
containing a necrotic core were observed in 24 of the 31 cases. Atherosclerofic lesions contained CD68 positive
foam cells and CD68 negative foam cells, the later being immunoposifive for the antibody against smooth
muscle a-actin. Amongst these foam cells, a large num- ber of T-cells CD3
+
and a few dendritic cells express- ing S-100 and CD1a were intermingled.
Scattered between smooth muscle cells in the under- lying media were macrophages CD68
+
and T-cells CD3
+
. Some medial smooth muscle cells showed low intensity staining for the antibody against smooth mus-
cle a-actin and the pycnotic appearance of their nuclei suggested cellular destruction. The media contained
many microvessels derived from the adventitial vasa vasorum, around which differing numbers of T-cells,
dendritic cells and macrophages were observed. B-cells CD20
+
were seldom observed in the media. In all the specimens, large numbers of immune and
inflammatory cells were observed in the adventitia. There were diffusely distributed and intermingled T-
cells CD3
+
, B-cells CD20
+
, dendritic cells CD1a
+
; S-100
+
, macrophages
CD68
+
and granulocytes
CD15
+
. T-cells CD3
+
were presented by their CD4
+
and CD8
+
subpopulations and in all the speci- mens, CD4
+
cells were a prevalent cell subtype. The numbers of CD4
+
cells were estimated as exceeding the numbers of CD8
+
cells by four – 16 times. Massive inflammatory cell accumulations were also observed in
the adventitia Fig. 1A – D. Within some of these infl- ammatory infiltrates, cells were distributed relatively
evenly throughout Fig. 1A while within others, an uneven distribution of cells was prominent Fig. 1C.
Different patterns of cell distribution within the infiltrates corresponded to the peculiarities of the distri-
bution of T-cells CD3
+
Fig. 1B, D; Fig. 2A, B. Infiltrates with a relatively even cell distribution con-
sisted mostly of T-cells CD3
+
with only a few den- dritic cells S-100
+
; CD1a
+
residing between them Fig. 2A, B.
Within inflammatory infiltrates where the cell distri- bution was uneven, one or several aggregates of CD3
negative cells were present and were usually located in the central areas of the infiltrates Fig. 1D; Fig. 3A, B.
These aggregates of CD3 negative cells were sur- rounded by CD3 positive cells Fig. 1D; Fig. 3A, B.
Cells which formed the aggregates of CD3 negative cells were found to stain positively with anti-CD20
antibody Fig. 3C, D. This type of cell infiltrate was, therefore, identified as a typical lymphoid follicle as it
contained a germinative centres consisting of B-cells Fig. 3A-D. In 17 of the 31 specimens, the lymphoid
follicles with B-cells forming germinative centres were observed. In eight specimens, several follicles united to
form lymph node-like structures Fig. 1C, D. We were not able to establish a correlation between the degree of
adventitilal inflammation and clinical data.
In T-cell rich zones of the lymphoid follicles, den- dritic cells expressing CD1a and S-100 were identified
Fig. 1. Typical appearance of massive inflammatory infiltrates A – C and the distribution of CD3 positive cells rose B, D seen at a low
magnification × 100 in the adventitia of the AAA wall. B, D: Sections were stained with anti-CD3 antibody using the APAAP
technique. A and B show a relatively even distribution of lymphocytes within the infiltrates while C and D represent consec-
utive sections containing an infiltrate with an uneven distribution of lymphocytes. In D, note aggregates of CD3 negative cells, which are
surrounded by CD3 positive cells rose within the infiltrate. In A, an atherosclerotic necrotic core is marked by an asterisk. M, media;
A, adventitia. A – C, counterstaining with Mayer’s haematoxylin.
Fig. 4A, B. Double immunostaining demonstrated that these dendritic cells contacted T-cells Fig. 4B.
Electron microscopic examination confirmed the forma- tion of contacts between dendritic cells and T-cells
within lymphoid follicles Fig. 4C. These contacts were mostly through dendritic cell processes extending from
the cell body. In the cytoplasm of dendritic cells which clustered with T-cells, hypertrophied cisterns of the
tubulovesicular system were prominent Fig. 4C.
In some inflammatory infiltrates, B-cells represented a predominant cell type, estimated as being as much as
60 – 80 of the total cell population of the infiltrate Fig. 5A, B. In the central areas of these infiltrates,
B-cells were usually closely packed while on the periph- ery, B-cells were more diffusely disseminated Fig. 5A,
B. Sparse dendritic cells expressing S-100 and CD1a were distributed amongst lymphocytes in the peripheral
zones of the infiltrates. Double immunostaining and electron microscopic examination of these infiltrates
demonstrated that dendritic cells contacted B-cells Fig. 5C, D.
Fig. 3. Distribution of T-cells A and B-cells C in a lymphoid follicle formed in the AAA adventitia. T-cells brown were identified
with anti-CD3 and B-cells brown were identified with anti-CD20 using the PAP technique. A and C are consecutive sections. B is
a detail of A. D is a detail of C. In C, D, a germinative centre formed by B-cells is shown by arrows. In C, M, media; A, adventi-
tia. A – C, Counterstaining with Mayer’s haematoxylin. Magnifica- tions: × 100, × 400, × 100, × 400.
Fig. 2. Immunohistochemical identification of dendritic cells and visualisation of their contacts with T-cells within an adventitial
inflammatory infiltrate. Dendritic cells brown were stained with S-100 antibody using the PAP technique and T-cells rose were
identified with anti-CD3 antibody using the APAAP technique. B is a detail of A. Note that the infiltrate consists of T-cells only. Note
also that dendritic cells contact T-cells on the periphery of the infiltrate as well as in its centre. In A, M, media; A, adventitia.
Counterstaining with Mayer’s haematoxylin. Magnifications: × 100, ×
400.
Fig. 4. Immunohistochemical A, B and electronmicroscopic C visualisation of dendritic cells and their contacts with T-cells at the
peripheral zone of lymphoid follicles. In A, dendritic cells brown were identified using anti-CD1a antibody and the PAP technique.
Counterstaining with Mayer’s haematoxylin. Magnification: × 400. B: Dendritic cells brown were stained with S-100 antibody using
the PAP technique and T-cells rose were identified with anti-CD3 antibody using the APAAP technique. Counterstaining with Mayer’s
haematoxylin.
Magnification: ×
400. C,
clustering of
T- lymphocytes asterisks around a dendritic cell star. Note that the
dendritic cell posseses a well-developed tubulovesicular system while it lacks lysosomes and phagolysosomes. Cisterns of the tubulovesicu-
lar system are shown by arrows. Electron micrograph. Magnification: ×
8100.
processes extending from the cell body, lacked lyso- somes and phagolysosomes and were characterised by
the presence of a well developed tubulovesicular system which is a unique feature of cells from the dendritic cell
family [21,22]. This unique tubulovesicular network may facilitate transport of antigens within the cell
cytoplasm [21,23].
T-cells require antigen to be processed and presented to them by antigen-presenting cells [21,24,25]. Dendritic
cells are involved in various diseases where they repre- sent a minor cell population [21,24,25]. Even though
their numbers are small, dendritic cells provide very efficient antigen-presentation and are the most potent
antigen-presenting cells known [21,24,25]. The associa- tion of T-cells with dendritic cells has been shown to
occur in atherosclerosis [26]. Dendritic cells are proba- bly also an important cell element in VALT activation
in AAAs. The present study is the first to demonstrate the formation of direct contacts between dendritic cells
and lymphocytes within inflammatory infiltrates in the
Fig. 5. B-cell rich inflammatory infiltrates in the adventitia of the AAA wall. A: A group of infiltrates containing large numbers of
B-cells CD20
+
. B-cells brown were identified using the PAP technique. M, media; A, adventitia. Counterstaining with Mayer’s
haematoxylin. Magnification: × 100. B is a detail of A. Magnifica- tion: × 400. C, dendritic cell located within an infiltrate where it
contacts B-cells. Double immunostaining in which dendritic cells were visualised with S-100 antibody by the PAP technique while B-cells
were visualised with anti-CD20 by the APAAP technique. Counter- staining with Mayer’s haematoxylin. Magnification: × 400. D: Elec-
tron micrograph demonstrating a contact of a dendritic cell star with a B-cells plasma cells asterisks. Magnification: × 6000.
4. Discussion
