(Stroke. 1997;28:1351-1356.)
© 1997 American Heart Association, Inc.
Articles |
From the Departments of Psychopharmacology (A.E., W.E.M.) and Psychiatry (H.F.), Central Institute of Mental Health, and the Department of Neurology (M.O., M.H.), Klinikum Mannheim of the University of Heidelberg, Mannheim, Germany.
Correspondence to Prof Dr Walter E. Müller, Department of Pharmacology, University of Frankfurt, Biocenter, Geb N260, D-60439 Frankfurt, Germany. E-mail psymail{at}as200.zimannheim.de
| Abstract |
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Methods [Ca2+]i was recorded in 26 patients with SVE, 26 age-matched nondemented control subjects, and 26 age-matched patients with AD. Basal [Ca2+]i and [Ca2+]i after lymphocyte activation with the mitogen phytohemagglutinin (PHA) were measured with the fura 2 method. In addition, modulation of the Ca2+ signaling by the peptide ß-amyloid and the potassium channel blocker tetraethylammonium was studied.
Results Basal [Ca2+]i was not different between patients and control subjects. After stimulation with PHA, however, a significant reduction of the Ca2+ response could be observed in lymphocytes of SVE patients compared with control subjects and with AD patients, providing evidence that the Ca2+ homeostasis of lymphocytes is impaired in SVE. The effect of the peptide ß-amyloid, the major constituent of senile plaques in AD brain, on Ca2+ signaling was similar in SVE patients and nondemented control subjects but typically reduced in cells of AD patients. Potassium channels were not involved in the impaired Ca2+ response of SVE lymphocytes after cell activation.
Conclusions [Ca2+]i is not only one of the most important second messengers in signal transduction of many cells but also an early event in the signal cascade of cell proliferation as a reaction to antigen recognition. This mechanism seems to be impaired in SVE. These findings may result in new insights regarding the pathogenesis of this disease and the possible involvement of inflammatory or immunologic disturbances.
Key Words: calcium cerebrovascular disorders dementia lymphocytes encephalopathy
| Introduction |
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Free [Ca2+]i is elevated by several second messenger generating systems. Because of its broad and crucial role in signal transduction pathways, [Ca2+]i has attracted specific attention. In lymphocytes, [Ca2+]i represents an early event in the intracellular signal cascade of cell proliferation and cytokine activation.12 Therefore, measurement of [Ca2+]i appears to be a valuable tool to detect early disturbances in the immunologic function of these cells.
The goal of the present study was to assess specific changes of [Ca2+]i regulation in a large group of patients with SVE13 and to distinguish between SVE, AD, and nondemented control subjects.
| Subjects and Methods |
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Blood cells from 26 patients (14 men and 12 women) with "probable" or "possible" AD according to the NINCDS-ADRDA criteria17 were used as a second demented group to elucidate differences in pathogenesis. Patients were recruited from an ongoing longitudinal study.18 These patients were also followed for at least a 2-year period, and confirmation of the diagnosis was established before entry into the present study. With regard to the aforementioned criteria, particular care was taken to separate vascular from degenerative dementia disorders and to exclude patients with mixed diseases. The mean age was 68±8.6 years (range, 51 to 86 years; median, 68 years).
Blood cells from 26 nondemented individuals of similar age (mean age, 69±6.7 years; median, 70 years; range, 56 to 84 years; 14 men and 12 women) were used as controls. These subjects also underwent standardized neurological and neuropsychological examinations, as previously mentioned, and brain imaging disclosed cerebral infarction, brain atrophy, or significant white matter lesions.
Approximately half of the AD patients, half of the aged control subjects, and most of the SVE patients were taking cardiovascular and antidiabetic drugs. Nine SVE patients were taking calcium antagonists. None of the drugs used are known to interfere with [Ca2+]i homeostasis in lymphocytes. This is also the case for calcium antagonists, since lymphocytes do not express L-type Ca2+ channels.19
Cell Separation
Peripheral blood lymphocytes were separated from
heparinized blood by centrifugation with Ficoll-Hypaque
at 400g for 40 minutes, as previously
described,20 according to the method of
Boyum.21
[Ca2+]i Measurements
Fura 2-AM (3 µmol/L) loading and measurements of
[Ca2+]i were performed as described
previously.22 Freshly prepared lymphocytes were stimulated
with PHA (Sigma) at a final concentration of 15 µg/mL.
[Ca2+]i was calculated according to the
method of Grynkiewicz et al.23 To investigate the
involvement of potassium channels in PHA-induced Ca2+
response, cells were exposed for 2 minutes to TEA (50 mmol/L)
before stimulation. To study the effects of preaggregated ßA25-35
(1 µmol/L; Sigma) on PHA-induced increases in
[Ca2+]i, freshly prepared lymphocytes were
preincubated with ßA25-35 for 60 seconds.20 22
Ca2+ responses to PHA in the presence or absence of
ßA25-35 are expressed as
Ca2+, which is the maximal
increase in [Ca2+]i over baseline.
Ca2+ßA25-35 is the ßA-induced increase in
[Ca2+]i over maximal stimulation by PHA.
Statistical Analysis
Significance was tested with ANOVA (SAS Institute), Student's
t test, or the paired t test.
| Results |
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Ca2+) after PHA (15
µg/mL) stimulation in lymphocytes from aged control subjects and SVE
patients. The maximum Ca2+ signal was reached after 1
minute in both groups (Fig 1
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In contrast, the PHA-induced Ca2+ increase was not
different between AD patients and control subjects (Fig 2
inset,
Table
), confirming previous findings.26 27 28 29
Moreover, measurement of basal [Ca2+]i in
lymphocytes revealed no difference among the three groups (Table
).
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Approximately half of the AD patients and half of the aged control
subjects were taking drugs, mainly for cardiovascular
diseases, but none were taking calcium antagonists. For
each group, the basal [Ca2+]i and the
PHA-induced Ca2+ increase in lymphocytes of individuals on
drugs versus the drug-free individuals were not significantly different
(basal [Ca2+]i: control subjects, 107.7±19.4
versus 95.8±19.1 nmol/L; patients, 107.7±11.3 versus 99.0±18.6
nmol/L;
Ca2+: control subjects, 84.5±22.3 versus
102.8±15.7 nmol/L; patients, 106.7±27.1 versus 99.0±26.3 nmol/L,
respectively). The majority of the SVE patients were taking drugs for
cardiovascular diseases, and 9 of 26 patients were
taking calcium antagonists. There was no difference in
basal [Ca2+]i levels or in PHA-induced
Ca2+ increase between patients taking calcium
antagonists and calcium antagonistfree
individuals (basal [Ca2+]i: 97.2±12.0 versus
101.9±14.3 nmol/L;
Ca2+: 82.1±25.6 versus 83.7±25.1
nmol/L, respectively), which is in agreement with the report that
peripheral human lymphocytes do not express functional
voltage-gated L-type Ca2+ channels.19
Moreover, there was no significant difference in
Ca2+ to
PHA in lymphocytes of SVE patients with normotonus, mostly after
medication, compared with SVE patients with either hypotonus or
hypertonus. Furthermore, plasma cholesterol levels did not
differ among the three groups (Table
).
The time to reach the maximum Ca2+ peak after stimulation
with PHA was unaltered between SVE patients and age-matched control
subjects (control subjects: 68.1±26.6 seconds; SVE patients:
76.7±29.8 seconds) (Fig 3
). In contrast, the time to
reach the maximum Ca2+ peak was significantly delayed
(P<.01) in lymphocytes of AD patients compared with control
subjects (AD: 96.4±42.4 seconds; control subjects: 68.1±26.6 seconds)
(Fig 3
inset) by unaltered Ca2+ response between both
groups (Fig 2
inset), confirming our previous
findings.28
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As previously described,20 the Ca2+-amplifying
effect of ß-amyloid is not restricted to neuronal cells but could
likewise be demonstrated on the PHA-induced Ca2+ increase
in lymphocytes. Preincubation of freshly prepared lymphocytes obtained
from aged nondemented control subjects with the ß-amyloid fragment
25-35 (ßA25-35) (1 µmol/L) resulted in a significant
amplification (
Ca2+ ßA25-35: 13.4±9.4 nmol/L, the
difference between the Ca2+ response to PHA alone and the
Ca2+ response to PHA in the presence of ßA25-35) (Fig 4
). A similar amplification of the PHA-induced
Ca2+ increase was observed in lymphocytes from SVE patients
after exposure to ßA25-35 (
Ca2+ ßA25-35: 8.3±10.5
nmol/L) (Fig 4
). In contrast, ßA25-35 amplification was absent in
lymphocytes from AD patients (0.6±5.9 nmol/L; P<.001
compared with the
Ca2+ ßA25-35 values of the aged
control subjects, P<.01 compared with the
Ca2+ ßA25-35 values of the SVE patients) (Fig 4
inset,
Table
), confirming our previous findings about the marker function of
this effect in AD.22 30 31 The effect of ßA25-35 on
PHA-induced Ca2+ increase in lymphocytes was not different
between SVE patients taking calcium antagonists and calcium
antagonistfree individuals (
Ca2+
ßA25-35: 10.3±8.8 versus 7.2±11.4 nmol/L, respectively).
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Furthermore, we investigated the effect of the potassium channel
blocker TEA on the PHA-induced Ca2+ increase in
lymphocytes. Preincubation of lymphocytes with TEA depolarizes the
membrane potential, which reduces the Ca2+ current into the
cell after PHA stimulation. Preincubation of cells with TEA (50
mmol/L) for 2 minutes caused a similar inhibition of the
Ca2+ increase in lymphocytes of nondemented control
subjects and SVE patients (51.9±9.9% inhibition versus 48.3±12.4%
inhibition, respectively) (Fig 5
). Lymphocytes of AD
patients, however, showed a tendency toward diminished effects of TEA
(42.2±14.6% inhibition) compared with nondemented control subjects
(Fig 5
inset), supporting the assumption of a pure AD-specific
potassium channel dysfunction.32 33 34
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| Discussion |
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On the other hand, SVE patients could be distinguished from AD patients in two experiments that appear to determine AD-specific alterations. In agreement with our previous findings22 28 30 and those of others,31 the lymphocyte sensitivity for the Ca2+-amplifying effect of ß-amyloid is strongly reduced in AD patients compared with nondemented control subjects and compared with SVE patients. The specific reduction of the effect of ß-amyloid on [Ca2+]i and even the complete absence of the Ca2+-amplifying effect are striking and represent an important peripheral marker of AD.22 28 30 37
One possible explanation for the impaired ß-amyloid sensitivity of lymphocytes in AD might be a potassium channel dysfunction, as previously described by Etcheberrigaray et al32 33 from skin fibroblasts of AD patients. The authors demonstrated that the effect of ß-amyloid on potassium channel function and the subsequent modulation of [Ca2+]i were reduced in skin fibroblasts of AD patients as a result of the absence of TEA-sensitive potassium channels in AD fibroblasts.33 A similar reduction of the effects of TEA has been in lymphocytes from AD.19 29 By blocking potassium channels, TEA is also able to indirectly influence the Ca2+ influx into lymphocytes. Preincubation of lymphocytes with TEA depolarizes the membrane potential, which reduces the Ca2+ influx into the cell after PHA stimulation.38 Consistent with our data19 and those of others,29 lymphocytes from AD patients showed a tendency to diminished effects of TEA compared with control subjects. This mechanism was unaltered in lymphocytes from SVE patients compared with control subjects. Therefore, K+ channels appear not to be involved in the impaired Ca2+ response of lymphocytes from SVE patients after PHA stimulation. Whether this potassium channel alteration can be linked to the reduced ß-amyloid sensitivity of AD lymphocytes is currently under investigation.
In lymphocytes, an increase in [Ca2+]i represents an early event in the intracellular signal cascade of cell proliferation after PHA stimulation.12 Very importantly, Tarkowski et al6 demonstrated that during stroke the proliferative responses of T lymphocytes to the mitogens PHA and concanavalin A were reduced compared with healthy control subjects. Therefore, we can assume that findings of an impaired Ca2+ activation in SVE lymphocytes are in accordance with these data and provide new insights into the pathogenesis of SVE with respect to a probably altered immunologic function. The nervous system and the immune system are known to communicate with soluble factors such as cytokines and interact at various levels. The cytokines are upregulated in patients with ischemic stroke and suggest an early inflammatory response in human cerebrovascular disease.7 8 Thus, modulators of cell-mediated immunity may alter T lymphocyte and macrophage invasion or function, as seen after vasospasm of superficial cerebral arteries and cerebral injury.10 11 The cellular invasion characterized by the alteration of the CD4+/CD8+ ratio in cerebral tissues is seen in many forms of inflammation. Even in AD an increase in CD8+ cytotoxic/suppressor T cells has been found,39 probably initiating an activation of brain microglia40 ; in addition, overexpression of two cytokines may be involved in HIV neuropathogenesis with similarities to the neuronal cell loss in AD.41
In conclusion, our results show that [Ca2+]i regulation was specifically impaired after cell activation during SVE. This effect was detected in circulating peripheral lymphocytes obtained from these patients. There was some overlap between the SVE and the AD groups. Several of the SVE patients showed low responses to the Ca2+-amplifying effect of ß-amyloid and after TEA treatment, as occurred in lymphocytes from AD patients. When one considers the difficulties of differentiating between vascular disease and the coexistence of vascular and degenerative dementia,4 overlap of some results between patient groups should be expected. Nonetheless, our work offers a new approach to partially discriminate between two different groups of patients with either cerebrovascular disease or AD.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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Received February 5, 1997; revision received April 21, 1997; accepted April 21, 1997.
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