During the last decade, the mechanisms responsible for the weakening and destruction of the aortic wall,
which lead to the formation of AAAs, have been intensively studied and the degradation of the extracel-
lular matrix in the medial layer associated with in- creased proteolytic activity have been implicated in the
breakdown of the structural integrity of the aortic wall [14 – 17]. In AAAs, inflammatory cells secrete cytokines
that are the main source of enhanced proteolytic activ- ity responsible for weakening the aortic wall [18].
Despite the significance of immune and inflammatory cells accumulating in the AAA wall, the histopathologi-
cal features of immune inflammation as well as the immunocompetent cell interactions in AAAs have re-
ceived limited attention. We now report the involve- ment of VALT in immune responses in the aortic
aneurysmal wall.
2. Methods
2
.
1
. Tissue specimens Material was collected in accordance with the princi-
ples outlined in the Declaration of Helsinki [19]. The present study was approved by the institutional review
board of St Vincent’s Hospital, Sydney. Samples taken from the anterior wall were collected
from 31 typical atherosclerotic infrarenal AAAs rang- ing in diameter from 5 to 8 cm. All the patients, whose
ages ranged from 55 to 84 years, were operated upon on an elective basis and had no ruptured or rapidly
expanding aneurysms. For immunohistochemical analy- sis, unfixed samples were immediately embedded in
OCT compound, rapidly frozen in liquid nitrogen and stored at − 70°C until cryostat sectioning. For electron
microscopy, small tissue pieces were fixed in 2.5 glu- taraldehyde in 0.1 M phosphate buffered saline PBS,
pH 7.4.
2
.
2
. Immunohistochemical identification of cell types T-cells were identified with anti-CD3 and their
helperinducer and suppressorcytotoxic subtypes were identified with anti-CD4 and anti-CD8, respectively.
B-cells were identified with anti-CD20. Dendritic cells were
identified with
S-100 and
anti-CD1a. Macrophages were identified with anti-CD68 antibody.
Granulocytes were identified with anti-CD15. Endothe- lial cells were identified with anti-von Willebrand factor
antibody. Smooth muscle cells were identified with antibody to smooth muscle a-actin. The sources and
working concentrations of the antibodies used are given in Table 1.
2
.
3
. Single immunostaining procedure The tissue specimens were cut at 7 mm thickness and
the sections air dried for 45 min. After eliminating endogenous peroxidase activity by 0.3 H
2
O
2
for 5 min, the sections were preincubated with normal goat
serum and then tested with avidin-biotin complex ABC using the peroxidase-anti-peroxidase PAP
technique [20]. The sections were incubated for 30 min with the primary antibody and after washing in tris-
phosphate buffered saline TPBS, pH 7.6, for 10 min, the sections were incubated for 20 min with the appro-
priate biotin-labeled secondary antibodies horse anti- mouse-Vector BA-2000, or goat anti-rabbit-Vector
BA1000. The sections were then washed in TPBS for 5 min and treated with avidin-biotin complex Elite-ABC,
Vector PK61 00 for 30 min. After washing for 10 min
Table 1 Antibodies used in the study
Working dilation Source
Type Cell types identified
Clone Specificity
Designation M
NA134 CD1a
CD1a Thymocytes, Langerhans cells, interdigitating cells,
DAKO 1:50
vascular dendritic cells –
P Glial cells, ependyma, Schwann cells, Langerhans cells,
S100 S-100A, S-100B
DAKO 1:700
interdigitating cells, vascular dendritic cells P
CD3 DAKO
T-cells CD3
– 1:100
M MT310
CD4 CD4
Helperinducer subtype of T-cells DAKO
1:10 CD8
M DK25
CD8 Supressorcytotoxic subtype of T-cells
DAKO 1:50
CD20 B-cells
DAKO CD20
1:50 M
L26 CD68
Macrophages DAKO
1:50 EBM11
M PG-MI
1:50 DAKO
Endothelial cells Von Willebrand M
Factor F886
factor VIII-related
antigen M
1A4 Smooth muscle
SMA Smooth muscle cells
DAKO 1:400
a -actin
1:50 DAKO
Granulocytes, Reed Stemberg cells CD15
M MMA
CD15 M, monoclonal antibody; P, polyclonal antibody.
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
