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Autosomal dominant cerebrovascular amyloidosis Properties of peripheral blood lymphocytes.

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Autosomal Dominant
Cerebrovascular Amyloidosis:
Properties of Peripheral Blood Lymphocytes
Kari Stefansson, MD, Jack P. Antel, MD, Joel Oger, MD, James Burns, MD,
Avertano B. C. Noronha, MB, MD, Raymond P. ROOS,MD,
Barry G . W. Arnason, MD, and G. Gudmundsson, M D
Selected properties of peripheral blood lymphocytes (PBLs) from five ambulatory affected individuals of a kindred
with autosomal dominant cerebrovascular amyloidosis were studied. The percentage of PBLs bearing surface membrane immunoglobulin (SmIg+ cells) was increased in the patient group (30 2 3% versus 20 +- 2%; p < 0.05).The
percentage of PBLs forming early and total E-rosettes was comparable in patient and control groups. Mitogenic
response to concanavalin A (Con A) was suggestively reduced in the patient group, measured both by total 3Hthymidine incorporation and by comparison of stimulation indices. Mitogenic response to phytohemagglutinin and
pokeweed was comparable in the two groups. Capping of Con A by PBLs was significantly reduced in the patient
group compared with the controls (13 f 1% versus 26 f 2 % ; ~< 0.01).The findings of reduced Con A response and
increased SmIg+ cells support the hypothesis that immune dysfunction contributes to the development of
amyloidosis. The reduced capping suggests altered membrane properties in this autosomal dominant disorder.
Stefansson K, Antel JP, Oger J, et al: Autosomal dominant cerebrovascular amyloidosis: properties of
peripheral blood lymphocytes. Ann Neurol 7:436-440, 1980
Amyloidosis is a general term for a group of diseases
in which proteins recognizable by their ability to bind
Congo red are deposited within tissues for unknown
reasons [6]. Clinically, amyloidosis may be divided
into primary and secondary forms [ l o ] .In primary
amyloidosis no additional disease is present. Inherited forms of primary amyloidosis occur, although
the tissues in which the amyloid is deposited vary
among kindreds [24]. Secondary amyloidosis is a
sporadic disease that appears against a background of
chronic systemic illness.
The nature of the amyloid material in primary
amyloidosis differs from that in secondary amyloidosis. In primary forms of the disease the amyloid is often composed of light chains of immunoglobulin (Ig) or fragments thereof [ S ] , and abnormal
Ig components may be found in the circulation.
In secondary amyloidosis a unique protein known as
protein A of amyloid is deposited in the tissues, and
in the serum a high-molecular-weight protein which
shares antigenic determinants with protein A can be
detected [ 2 , 171. The origin of this circulating protein
is not known; it has been found in embryonic fibroblasts and in plasmacyte-like cells in spleen and tonsils
[ 12,2 51. Secondary-type amyloidosis can be induced
experimentally in animals by casein injections [4].
Immunological dysfunction has been found in
human amyloidosis, both primary and secondary, as
well as in the animal model of the disease. Scheinberg
and Cathcart [2 11 found that peripheral blood lymphocytes (PBLs) from patients with either primary or
secondary amyloidosis responded poorly to the Tcell mitogen concanavalin A (Con A) but normally to
the B-cell activator pokeweed mitogen (PWM). In
animals developing amyloidosis after casein injections, reduced T-cell numbers and function with enhanced B-cell function have been found [19, 201.
These changes have been ascribed to deranged immunoregulation.
The inherited forms of amyloidosis are particularly
useful entities for studying amyloidosis in humans,
since within families the same pathogenetic mechanism will likely be operative. An Icelandic kindred in
which cerebrovascular amyloidosis is inherited as an
autosomal dominant disease has been reported previously [7]. In this kindred, amyloid deposits are
confined to vessels of the brain, and the clinical presentation is one of intracerebral hemorrhage in early
From the Department of Neurology, The University of Chicago
Division of Biological Sciences and The Pritzker School o f
IL, INSERM, France, and the Department Of
Neurology, University of Iceland, Reykjavik, Iceland.
Received June 23, 1979, and in revised form Sept 28. Accepted
for publication Oct 18, 1979.
Address reprint requests
Dr Antel, Departmentof Neurology,
University of Chicago, 950 E 59th St, Chicago, IL 60637.
436 0364-5134/80/050436-05$01.25 0 1979 by Jack P. Antel
adulthood. The nature of t h e amyloid that accumulates is unknown. Cerebrovascular amyloidosis (congophilic angiopathy) also occurs sporadically and is
being recognized as the cause of a great proportion of
cases of intracerebral hemorrhage in normotensive
individuals [ 111. No explanation for the selective involvement of cerebral vessels, either in the autosomal dominant form of cerebrovascular amyloidosis or i n t h e sporadic cases, is at hand.
We have studied selected properties of PBLs obtained from the Icelandic kindred affected with
dominantly inherited cerebrovascular amyloidosis.
Cell subpopulations have b e e n enumerated using
surface marker techniques, and lymphocyte response
t o mitogens has been determined. To evaluate lymphocyte membrane properties, lymphocyte capping
of Con A was measured. Previously we found that
C o n A capping is reduced in Huntington’s chorea,
another autosomal dominant disease affecting the
brain [14].
Patients and Methods
Five normotensive patients with a mean age of 29 years
(range, 26 to 40 years), of the Icelandic pedigree previously
described [7], were studied. All had had two or more
episodes of cerebral hemorrhage. The mean duration since
the initial episode was three years (range, one to seven
years). In two of the patients, brain biopsies revealed
amyloid in vessel walls by Congo red staining. Total and
differential white blood cell counts, serum and cerebrospinal fluid Ig concentrations, and serum immunoelectrophoresis were dl within normal limits. At the time of
study the patients were ambulatory and were judged to be
in good general health, with no obvious diseases except for
sequelae from cerebral hemorrhages. As controls we used
healthy Icelanders age matched with the patients. In addition, 32 healthy American volunteers aged 20 to 40 (mean,
32 years) were studied in lymphocyte capping experiments.
Venous blood was drawn into tubes containing
ethylenediamine tetraacetic acid and kept at room temperature until the lymphocytes were isolated on FicollHypaque gradients 24 hours later. Cells were washed twice
in Hanks balanced salt solution without magnesium or calcium (modified HBSS).
T o detect lymphocytes with surface membrane immunoglobulin (SmIg+), 3 x lo6 lymphocytes in 100 p1 of
modified HBSS were mixed with an equal volume of a
1: 10 dilution of fluorescein isothiocyanate (FITC)conjugated goat antibody against human immunoglobulin
G (Huntingdon Research Center, Inc). The mixture was
shaken gently for 30 minutes at room temperature, washed
three times in modified HBSS, suspended in glycerol/
phosphate-buffered saline mounting fluid, and examined
under epifluorescent illumination. Three hundred lymphocytes were counted, and the percentage of cells that stained
was assessed. All counts were done on coded samples.
To quantify early E-rosettes, lo6 lymphocytes (0.25 ml)
were mixed with 0.25 ml of sheep red blood suspension
(0.5%) at room temperature and incubated in a water bath
at 37°C with constant shaking for 10 minutes. Cells were
then spun at 180 g in a refrigerated centrifuge for 5 minutes, the supernatant was removed, and the cell pellet was
gently resuspended. An aliquot was then examined under a
microscope. A rosette was defined as a white blood cell
having three or more attached red blood cells.
For total E-rosettes, the cell pellet obtained after the
centrifugation just detailed was kept overnight on ice as
previously described [IS]. The cells were then gently resuspended and an aliquot was examined.
For mitogen stimulation studies, cells were suspended at
1 x lo6 cells per milliliter in minimum essential medium
(Microbiological Associates) supplemented with 20% fetal
calf serum, glutamine ( 4 mmol/dl), and gentamicin (10
mgidl). Next, 0.2 ml of cells was incubated in microtiter
wells (Falcon) with either Con A, 3 pg/ml final concentration (Sigma Chemical Company), phytohemagglutinin
(PHA), 10 pg/ml (Sigma), or PWM, 10 pg/ml (Grand Island Biological Company). Quadruplicate cultures containing Con A or PHA were maintained at 37°C in a 5%
carbon dioxide humidified incubator for 72 hours. Cultures
containing PWM were maintained for seven days. The
cultures were then pulsed with 1 pCi of tritiated thymidine
(specific activity, 6.7 Ci/mol) (New England Nuclear) for
five hours and harvested with a Mash I1 apparatus (Microbiological Associates). The dry filters were placed in toluene containing Omnifluor (New England Nuclear), and
radioactivity was measured in a liquid scintillation counter.
Results are expressed both as counts per minute (cpm) with
standard error of the mean (SEM) and as stimulation indices (SI).
Our technique for Con A capping of lymphocytes has
been described [ 141. Briefly, 3 x lo6lymphocytes in 100 p1
of modified HBSS were reacted with an equal volume of
FITC-conjugated Con A (Miles Laboratories, Inc, Elkhart,
IN) to produce a final concentration of 100 pglml, and
gently agitated for five minutes at 37°C. Then 200 pl of 4%
paraformaldehyde was added to fix the cells. The cells were
washed twice, resuspended in mounting fluid, and examined under epifluorescent illumination. Five hundred cells
were counted and the percentage of cells with a cap was
determined. Caps were defined as distinct areas of fluorescence confined to less than one-half of the circumference of
a cell. A few macrophages, polymorphonuclear leukocytes,
and cell clumps were present but were not counted. Coded
cell samples from all patients and all controls were tested at
the same time.
Data were analyzed by Student’s t test.
T h e mean percentage of SmIg+ cells was 30 2 3%
for patients compared to 20 2 2% for controls @ <
0.05) (Table). Four of the five patients had m o r e than
25% SmIg+ cells, compared with n o n e of t h e six
controls (Fig 1). T h e r e was no significant difference
between patients and controls in t h e percentage of
lymphocytes that formed either early or total rosettes
with sheep erythrocytes (see the Table).
Data o n 3H-thymidine uptake in response t o Con
A, PHA, and PWM are given in t h e Table. T h e re-
Stefansson et al: Lymphocytes in Cerebrovascular Amyloidosis
Lymphocyte Capping, Lymphocyte Subsets, and Mitogenic Reactivity in Icelandic Patients
with Autosomal Dominant Cerebrovascular Amyloidosis and Icelandic Controlsa
Con A capping
SmIg+ cells
Mitogenic reactivity
* 1%
* 3%
32 t 5%
52 ? 4%
Con A
3,734 t 771
34,829 ? 5,848
13,178 3,549
* 4%
* 7%
p <: 0.2
9.6 -t 3.5
94.4 t 35.8
12.8 3.0
599 219
* 11.6
-t 52.8
* 1.3
* 87
p < 0.2
p < 0.2
aData are expressed as mean values ? SEM of results from five patients and six controls.
Con A = concanavalin A; SmIg+ = surface membrane immunoglobulin; P H A = phytohemagglutinin; PWM
stimulation index.
p < 0.01
p < 0.05
8,791 3,166
40,272 6,567
17,916 t 1,303
Con A
Unstimulated cells
80 1
pokeweed mitogen; SI
Fig 1 . Each circle indicates, for a single individual, the percentage of peripheral blood mononuclear cells with suyface
membrane immunoglobulin (SmIg+)
F i g 2. Each circle indicatej, for a single individual, the reJponse, expressed as stimalation index, of peripheral blood
mononuclear cells t o concanavalin A.
sponse to the dose of Con A employed was suggestively reduced ( p < 0.2). When expressed as SI,
the mean mitogenic response by patients’ cells to
Con A and PHA mitogens was suggestively reduced
compared with controls (p < 0.2). The SI for Con A
was less than 10 for four of the five patients and
above 10 for all controls (Fig 2).
The mean percentage of patients’ lymphocytes
with a fluorescent cap (13 % 1%) was significantly
lower (p < 0.01) than the percentage of capped cells
in the controls (26 ? 2%) (see the Table). For all six
controls, the percentage of cells capping was greater
than 20%; the percentage of cells capping for all five
patients was less than 20% (Fig 3). Healthy American
controls had a mean capping value of 22 k 1%.
438 Annals of Neurology Vol 7 No 5 May 1980
H 20-
L -
F i g 3 . Each circle indicates, for a single individual, the percentage of peripheral blood mononuclear cells that show concanavalin A capping.
No correlations between percentage capping,
mitogenic reactivity, and percentage SmIg+ or Erosetting cells were discerned.
In the present study we found evidence for lymphocyte dysfunction in patients with the dominantly inherited form of cerebrovascular amyloidosis. An imbalance of lymphocyte subsets and reduced T-cell
mitogenic reactivity were present in the patient
group. The increased percentage of SmIg+ cells may
reflect a rise in B-cell numbers o r increased adherence of antibody to non-B-cells via Fc receptors. Fc
binding of FITC-labeled goat antisera is reported
to be minimal [I]. T-cell numbers were comparable
to controls, whereas T-cell function, as measured by
mitogen reactivity to Con A, was suggestively reduced. Response to P H A and particularly to PWM
was better preserved. The lymphocyte transformation data appear to parallel those noted previously,
both in primary and secondary forms of human
amyloidosis [2 11 and in the experimentally induced
disease in mice [ 19,201. Neither our patients nor the
familial cases of amyloidosis studied by Scheinberg
and Cathcart [21] showed an M peak on serum immunoelectrophoresis, whereas in their nonfamilial
cases a peak was seen.
The finding of reduced lymphocyte capping of Con
A suggests that a membrane property of lymphocytes
may be aberrant. The reduced capping is unlikely to
be explained by an altered ratio of B- and T-cells
since both types reportedly cap equally [ 131. The
phenomenon of capping is dependent on a number of
cell properties, including fluidity of plasma membrane and integrity of microtubules and microfilaments [221. Capping is also energy dependent. The
defective lymphocyte capping in the patients with
amyloidosis may reflect a more generalized membrane abnormality. Capping abnormalities have been
found in genetic diseases in which generalized membrane abnormalities have been postulated [3, 181, including Huntington’s disease [ 141, and in several
forms of muscular dystrophy [ 161, although the latter
finding has been disputed [8, 231.
From this study we postulate that immune dysfunction, perhaps related to a membrane defect affecting cell-cell interaction, occurs in the autosomal
dominant form of cerebrovascular amyloidosis. A
similar pattern of altered immunoreactivity as well as
reduced Con A capping by lymphocytes is also
characteristic of aged individuals [9, 141. It is of
interest that congophilic angiopathy [261, benign
monoclonal gammopathies associated with amyloid
deposition [6], and sporadic generalized amyloidosis
[ 101 are disorders of the elderly.
Whether the in vitro tests of lymphocyte function
reported here can be used to identify asymptomatic
affected individuals and then to follow sequentially
the cellular changes underlying the disease will require further study. Increased knowledge of the immune abnormalities in autosomal dominant cerebrovascular amyloidosis may lead to an effective
means of preventing amyloid deposition.
Supported by a fellowship from the Iceland Science Foundation
(Kari Stefansson) and in part by Grant NS13526-02 from the National Institutes of Health, Bethesda, MD.
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autosomal, cerebrovascular, periphery, properties, dominantly, amyloidosis, lymphocytes, blood
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