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Papers of the Month - November 1997
I didn't see much in the published literature this month to bring to
your attention, so I am posting a preprint of the abstract for a lecture
I am giving at the NIH in February. It will eventually be published in
J Vasc Surgery. Why does the LifeLine Foundation/NIH put on the annual
vascular biology meeting during President's Birthday week, year after
year. For my family, it is my once a year ski week in Vermont, because
that is when the kids have their winter holiday. We and another family
rent a house at Okemo, and mercifully, I am scheduled to speak on Friday,
so I will only loose one day of skiing.
Addendum: the work that is described in the following abstract was
prospectively proposed to the NIH in a grant application several years
ago. The grant was *not* funded, and now they have the nerve to invite
me to Washington to present the work. According to the last info I could
find at the NIH website, they are only supporting *one* grant in the
United States to study diseases that kill more people annually than AIDS.
ONE PAGE ABSTRACT
INFLAMMATORY AND IMMUNE MECHANISMS IN ANEURYSM FORMATION
M. David Tilson, MD
Columbia Univ & St. Luke's/Roosevelt Hospital Center
1000 Tenth Avenue, NY, NY 10019
e-mail: mdt1@columbia.edu
Features of autoimmunity are commonly present in patients
with non-specific abdominal aortic aneurysms (AAA's). These
features include infiltration of the adventitia with chronic
inflammatory cells, aggregates of immunoglobulin in the aortic
wall (Russell bodies), destruction of matrix (including collagen-
associated microfibrillar proteins), activation of matrix
metalloproteinases, and antibodies in serum and aortic wall that
are immunoreactive against aorta-specific proteins.
Since susceptibility to another autoimmune disease of
maturity, rheumatoid arthritis (RA), is associated with specific
MHC Class II DR-B1 alleles, we have evaluated the hypothesis
that the genetic basis for AAA development may also be specified
by the DR-B1 genotype.
AAA's are rare in African-Americans, perhaps because they
require a double dose of the susceptibility gene to express the
phenotype, so we analyzed ten DR-B1 haplotypes from five patients
of color as a first step towards predicting a susceptibility
allele. Only three DR types were observed: 2, 12, and 13. This
result had a probability of 3 x 10-4 by chance alone. Inspection
of the amino acid sequences reveals that these three types have
phenylalanines (PHEs) at positions 31 and 47 of the second
hypervariable region. Reviewing the data on 21 additional North
American patients of mixed descent, 16 (75%) had an allele with
PHEs at positions 31 and 47; and 5 patients had a double dose of
putative susceptibility genes. Thus, based on a total of 52
mixed North American haplotypes, 81% of patients have at least
one allele with PHE residues in the second hypervariable region
at positions 31 and 47; and 35% have a double dose of the
putative susceptibility alleles. Word Count 1869
INFLAMMATORY AND IMMUNE MECHANISMS OF ANEURYSM FORMATION
M. David Tilson, MD
Columbia University and St. Luke's Roosevelt Hospital Center
1000 Tenth Ave, NY, NY 10019 email: mdt1@columbia.edu
Summary. Features of autoimmunity are commonly present in
patients with non-specific abdominal aortic aneurysms (AAA's).
Since susceptibility to another autoimmune disease of maturity,
rheumatoid arthritis (RA), is associated with specific MHC Class
II DR-B1 alleles, we have evaluated the hypothesis that the
genetic basis for AAA development may also be DR-B1 specific.
Our pilot study, based on 52 mixed North American haplotypes,
suggests that 81% of patients have alleles with phenylalanine
residues in the second hypervariable region at positions 31 and
47; and 35% have a double dose of the putative susceptibility
alleles.
Background. The first report of familial clustering of AAA was
by Clifton in 1977. In 1984 Tilson and Seashore reported fifty
families with two or more affected first-order relatives, and
Norrygard et al reported another large series of families. A
recent study predicts that the disease is caused by an autosomal
dominant gene.
Features of autoimmunity in AAA disease. Because of limitations
on length of text and number of citations, the author will focus
on findings from his own laboratory, acknowledging that several
others, especially William Pearce at Northwestern University,
have made important contributions.
1. Description of Russell bodies and purification of
immunoglobulin from AAA wall.
Although immunoglobulins (Ig's) are detectable in the wall
of atherosclerotic aortas, they are six to eight-fold more
abundant, class by class, in extracts of AAA specimens.
2. Quantitative description of the number and types of
inflammatory cells present in AAA wall.
Koch and coworkers were the first to identify the full range
of resident inflammatory cells immunohistochemically. They
reported higher semi-quantitative scores for CD-11c+
(macrophages) and CD-19+ (B-cells) in AAA versus occlusive
disease controls. We have quantitatively confirmed these
findings in our laboratory by fluorescence-activated cell sorting
(FACS).
3. Identification of a prominent elastolytic activity of AAA
wall as Matrix MetalloProteinases #9 (MMP-9), also known as
Type IV collagenase or Gelatinase-B.
MMP's are a family of enzymes, of which collagenase is a
member, that are active against the major components of
connective tissue (including collagen, elastin, fibronectin,
laminin, and proteoglycans). MMP's are important mediators of
tissue destruction in most diseases of autoimmunity. Plasmin, a
major activator of the MMP's, is also increased in AAA wall.
4. Identification of the cell of origin for MMP-9 as the
macrophage, and assignment of the cells of origin for
collagenase (MMP-1) as endothelial cells in the
neovascularizing adventitia and fibroblasts.
Macrophages are prominent in the infiltrate, and among their
numerous secretory products are several MMP's. The inflammation
and angiogenesis are reminiscent of rheumatoid pannus.
5. Increased concentration of cytokines (IL-1 beta and TNF-
alpha) in extracts of AAA wall.
We have isolated leukocytes from AAA specimens by
Ficoll/Paque separation; and we find that mononuclear cells
adhere to culture wells, remain viable for up to 2 weeks, and
have histological features typical of macrophages. We have
called these cells "AIM-cells" (for "AAA-infiltrating
monocyte/macrophages"). We have found that IL-1á is initially
produced by the cultured AIM cells; but as the cells become
quiescent, the level of constitutive secretion decreases.
However, the cells can be stimulated to make IL-1á again by
soluble factors extracted from AAA specimens.
6. Patients with AAA have IgG (in both aortic wall and serum)
that is immunoreactive with a fibrillar self-protein in the
aortic adventitia.
We have recently found immunohistochemically that the
immunoreactive protein is associated with collagen, not elastin.
This finding was unexpected, because most microfibrillar
glycoproteins distribute ubiquitously with elastin.
7. Purification of an autoantigenic aortic protein in man.
Our research group has recently reported the partial amino
acid sequence of an aortic adventitial collagen-associated
fibrillar protein, which appears to be one of the targets of an
autoimmune response. Our initial report suggested that the
MW was approximately 80 kDa; but we have found that the 80 kDa
form is dimeric. Thus, we have named it Aneurysm-Associated
Antigenic Protein-40 kDa (AAAP-40). It has similarities to
Microfibril-Associated Glycoprotein-36 kDa (MAGP-36), which was
reported by Kobayashi et al to have a tissue distribution limited
to the aorta in pig. If AAAP-40 is the human homolog of MAGP-
36, it would explain how an autoimmune reaction against this
protein might have consequences more or less limited to the aorta
and its branches.
The similarities of AAAP-40 and MAGP-36 to all three
fibrinogen chains are significant, and the homology to
fibrinogen-beta (FB) is particularly extensive. It is believed
that the fibrinogen chains diverged from a single common ancestor
in the course of evolution. Accordingly, AAAP-40 and MAGP-36
have an ancient evolutionary history, going back prior to the
divergence of the fibrinogens from their common ancestor,
probably in invertebrates. AAAP-40 also has a 12 residue
sequence that is identical to a sequence in vitronectin (VN).
8. Cloning of cDNA's for additional antigenic proteins in man,
one of which we have found to be aorta specific.
An expression library was made from mRNA of human aneurysmal
aortic adventitia and screened with anti-VN and anti-FB
antibodies. Two of the first five clones (#1 and #5) have novel
features and strongly resemble each other in domain structure.
While both have some features that occur in the families of
MAGP's and other matrix proteins (like collagen), their most
remarkable property is that each begins with a lengthy amino acid
sequence (~100 residues) that is homologous to members of the Ig
kappa family. The probability that the degree of similarity is
due to chance alone (as calculated by BlastP) is in the range of
1 x 10-50.
Antibody against Ig Kappa is strongly immunoreactive with
the aortic adventitial fibril, while immunoreactivity of the
antibody is less conspicuous in the matrix of ovary, prostate,
cervix, liver, testis, breast, and skin.
9. Serum antibody titer against recombinant-clone 1 (r-Cl-1),
as measured by ELISA, may be a simple and clinically useful
blood test for AAA susceptibility.
R-Cl-1 has been purified by elution from SDS/PAGE gel, and a
checkerboard titration was performed to determine optimal
concentrations of antigen and antibody for ELISA. We have now
studied 20 AAA patients and 10 controls. Ninety % (18/20) of the
patients were positive, and none (0/10) of the controls. The
differences in the means of the two groups was statistically
significant at the p = .0001 level.
10. Molecular mimicry may explain interesting reports of
associations of infectious agents with aneurysmal diseases
in man (cytomegalovirus) and primates (herpes virus).
AAA is associated with specific MHC Class II DR alleles.
Since AAA's are rare in African-Americans (perhaps because
they require a double dose of the susceptibility gene to express
the phenotype), we analyzed ten DR-B1 haplotypes from five
patients of color. Only three alleles were observed: 2, 12, and
13. This result had a probability of 3 x 10-4 by chance alone.
Inspection of the amino acid sequences reveals that these three
alleles have phenylalanines (PHEs) at positions 31 and 47 of the
second hypervariable region. Reviewing the data on 21 additional
North American patients of mixed descent, 16 (75%) had an allele
with PHEs at positions 31 and 47; and 5 patients had a double
dose of putative susceptibility alleles. Thus, 81% of a total of
26 patients fit the same pattern.
A former student has returned to Japan and studied 46 native
Japanese with AAA and 50 controls, matched approximately for age
and sex. DR-15 (which is a subset of DR-2, also sharing PHE's at
31 and 47) has been found in 59% of 46 AAA's, versus 28% of 50
control subjects (p < 0.005). Thus, in the much more
homogeneous population of Japan, one of the same genotypes
appears to be a susceptibility factor.
REFERENCES