Neurobiology of Aging, Vol. 19, No. 3, pp. 191–199, 1998
Copyright © 1998 Elsevier Science Inc.
Printed in the USA. All rights reserved
0197-4580/98 $19.00 ϩ .00

PII:S0197-4580(98)00050-5

Increased Natural Killer Cell Cytotoxicity in
Alzheimer’s Disease May Involve Protein Kinase
C Dysregulation
S. B. SOLERTE,1* M. FIORAVANTI,* A. PASCALE,† E. FERRARI,* S. GOVONI,‡ F. BATTAINI§
*Department of Internal Medicine, Geriatrics and Gerontology Clinic, University of Pavia, Pavia, Italy
†Institute of Pharmacological Sciences, University of Milano, Italy
‡Institute of Pharmacology, University of Pavia and IRCCS S. Giovanni di Dio, Alzheimer Unit, Sacred Heart
Hospital-FBF, Brescia, Italy
§Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Italy
Received May 20, 1997; Revised January 6, 1998; Accepted February 9, 1998
SOLERTE, S.B., M. FIORAVANTI, A. PASCALE, E. FERRARI, S. GOVONI, AND F. BATTAINI. Increased natural killer cell
cytotoxicity in Alzheimer’s disease may involve protein kinase C dysregulation. NEUROBIOL AGING 19(3) 191–199, 1998.—
Increased cytokine-mediated cytotoxic natural killer (NK) cell activity has recently been demonstrated in patients with senile dementia
of the Alzheimer’s type (SDAT). In the present study, we evaluated whether protein-kinase C (PKC), a main regulatory enzyme
involved in the mechanism of exocytosis by NK cells, has a role in the cytotoxic response of NK cells (during IL-2 and IFN-␤
exposure) from SDAT patients. Our data demonstrate the presence of an increased cytotoxic response by NK cells to IL-2 (mean
increase ϩ102%) and IFN-␤ (mean increase ϩ132%) in SDAT patients in comparison with healthy elderly subjects (ϩ75% and ϩ88%
for IL-2 and IFN-␤, respectively). A smaller suppression of NK cytotoxicity after cortisol was also observed in SDAT (mean decrease
Ϫ24%) than in the control group (Ϫ44%). The NK cell activity of SDAT patients was inversely correlated with the cognitive status
as evaluated by the analysis of MMSE (Mini Mental State Examination) score. A comparison of young and elderly healthy subjects
revealed no variations in NK cell activity. A physiological decrease in cytosolic PKC activity was demonstrated in healthy old subjects
after IL-2 and IFN-␤ incubation, but not in SDAT patients, while no variations in kinase activity were observed after cortisol
incubation. The decreased activity with cytokines was associated with reduced levels of PKC ␣ and ␤II isoforms. An alteration in
cytokine-mediated NK cell activity associated with PKC dysregulation is therefore suggested to occur in patients with SDAT. These
changes may indicate the existence of an immunological component to the pathogenesis and progression of the disease. © 1998 Elsevier
Science Inc.
Alzheimer’s disease
Dementia
Cortisol
Magnetic cell sorter

Aging

Protein kinase C

A growing body of evidence indicates that neuroimmune mechanisms, involving both immunological and neurobiological factors,
may be associated with the pathophysiology and progression of
Alzheimer’s disease (AD) (27,39,54). Among the cellular components of the immune system, natural killer (NK) cells are considered to be important effectors in the host defenses which express
non major histocompatibility complex (MHC)-restricted cytotoxic
activity against tumoral and viral targets (25,36,46,48,62,63). NK
cytotoxic activity is mainly dependent on biochemical events
leading to the exocytosis of preformed proteolytic granules (13,
53,67), and therefore can be modulated by cytokines and hormonal
factors (5,18,20,24,26,63,64).
A protein kinase C (PKC)-dependent signaling pathway has
been implicated in the Ca2ϩ-dependent mobilization of cytotoxic

Natural killer

Cytotoxicity

Cytokines

granules from NK cells (19,32,59,61). In fact, the PKC activator
phorbol myristate acetate (PMA) induces a prompt cytotoxic
response that is sensitive to the highly specific PKC inhibitor, PKC
pseudosubstrate antagonist peptide (61). Recent research has also
demonstrated that NK cell cytotoxicity toward tumor cell line
K562 is specifically regulated by a PKC-dependent pathway,
independently of other signal transduction systems (9).
There are two separate lines of evidence to support the
existence of altered immune response and PKC activity in the
brain and peripheral tissues of demented patients, suggesting that
there may be an interaction between these systems. Thus, changes
in NK number and function have been reported in normal aging
(1,8,30,31,35,43,57) and in patients with senile dementia of the
Alzheimer’s type (SDAT) (3,55,56). At the same time, PKC

1
Address correspondence to: Bruno Solerte, Department of Internal Medicine, University of Pavia, Ospedale S. Margherita, Piazza Borromeo, 2, 27100
Pavia, Italy.

191

192

SOLERTE ET AL.

alterations have been found in neuronal and non-neuronal peripheral tissues (14,17,21,44,51), while decreased PKC activity has
been demonstrated in cultured fibroblasts (22) and in platelets (10)
of patients with AD.
In light of these considerations, the aim of the present study
was to investigate whether the altered NK cell cytotoxic function
observed in SDAT was associated with disorders of the PKC
signal transduction pathway. To this end, NK cells derived from
SDAT patients and healthy elders were isolated by means of
negative magnetic separation and assayed for cytotoxicity against
K562 targets after incubation with IL-2, IFN-␤, and cortisol.
Calcium-dependent PKC activity in soluble and particulate fractions of NK cells, and NK cytolytic function were also evaluated.
METHODS

Subjects
The study was approved by the Ethical Committee of the
Department of Internal Medicine of the School of Medicine of the
University of Pavia. Written informed consent was obtained from
all subjects and patients or, where appropriate, from their caregivers.
Eleven patients with senile dementia of the Alzheimer type
(SDAT) were carefully selected from the Alzheimer’s Unit of our
department and were recruited for the study. The diagnosis of
SDAT was initially performed in agreement with the criteria
described in the Diagnostic and Statistical Manual of Mental
Disorders, 3rd edition-revised (DSM III-R) (2), and then each
patient was confirmed for probable or possible AD using the
diagnostic standards of the National Institute of Neurological and
Communicative Disorders and Stroke-Alzheimer’s Disease and
Related Disorders Association (NINCDS-ADRA) criteria (40).
The evaluation of the Hachinski ischemic score (23) was used to
exclude multi-infarct dementia, and all SDAT patients achieved a
score of 4 or less (mean Ϯ SD, 2.2 Ϯ 0.9). Diagnosis of SDAT was
further supported by physical and neurological examination, by
laboratory evaluation (including a complete biochemical assessment of nutritional status) and by the determination of brain
imaging (MRI or CT scan). Cognitive status and the severity of
dementia were also quantified by using the Mini Mental State
Examination (MMSE) test (16). The score of patients with SDAT
ranged from 6 to 15 (mean Ϯ SD ϭ 10.3 Ϯ 2.4). All patients with
SDAT were free of any medication (for at least 2 weeks) and no
diseases (e.g., respiratory and urinary tract infections) known to
affect immune function and PKC were observed 2 months before
the inclusion in the study.
Patients with SDAT were compared to 12 age and sex-matched
healthy elderly subjects. These subjects were recruited in agreement with the criteria of the SENIEUR protocol (34) in order to
exclude clinical and immunological alterations. The clinical and
biochemical characteristics of healthy elderly subjects and patients
with SDAT are reported in Table 1. Ten normal-weight healthy
young subjects (6 women and 4 men, age ϭ 32.2 Ϯ 4 years) were
also chosen as a further control group in order to compare the data
on NK cytotoxic activity obtained in SDAT and in healthy elders.
No differences concerning sex distribution, age, body weight
(expressed as body mass index ϭ BMI) and biochemical parameters were demonstrated between healthy elderly subjects and
patients with SDAT (Table 1).
Procedures
After an overnight fast, human peripheral blood mononuclear
cells (PBMC) were obtained from heparinized venous blood

TABLE 1
CLINICAL AND BIOCHEMICAL CHARACTERISTICS OF HEALTHY
SUBJECTS AND PATIENTS WITH SDAT (STANDARD DEVIATION
BETWEEN BRACKETS)
Healthy subjects

n
men:women
age (years)
BMI (kg/m2)
Ht (%)
Hb (g/dL)
serum albumin (g/L)
serum pre-albumin (g/L)
transferrin (g/L)
total lymphocytes (cells/mm3)

Patients with SDAT

12
7:5
78 (7)
22.5 (1.5)
44 (5)
13.9 (1.4)
43 (3)
0.33 (0.02)
3.1 (0.2)
1973 (51)

11
6:5
77 (6)
21.2 (1.7)
42 (3)
13.7 (1.3)
41.7 (2.6)
0.31 (0.02)
3.0 (0.3)
1925 (51)

samples (Vacutainer, Hemogard, lithium heparin, Becton Dickinson, Meyland, France).
PBMC were isolated by Ficoll-Hypaque density gradient
(Lympholyte-H, Cedarlane Laboratories Ltd., Ornby, Ontario,
Canada) (11). Plastic-adherent cells were removed by incubation at
37°C in Petri-culture dishes for 1 h.
The remaining non-adherent cell population was passed
through nylon wool columns preincubated for 1 h with RPMI 1640
supplemented with 10% heat-inactivated autologous serum
(RPMI/AS) at 37°C (5% CO2 in air). T/NK cells were obtained by
rinsing the columns with tissue culture medium, which leaves B
cells and remaining monocytes attached to the nylon wool (28).
The enriched fraction of PBMC containing T/NK cells was used
for the separation in the magnetic field.
The NK cells were separated under sterile conditions. The
magnetic cell separation system (MACS) and the NK cell isolation
kit (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) were
used for immunomagnetic separation of NK. Washed PBMC were
resuspended in 80 ␮L buffer (per 107 total cells), containing PBS
supplemented with 0.5% BSA, and incubated for 15 min. at 6°C
with 20 ␮L of reagent composed of modified CD3, CD4, CD19, and
CD33 antibodies of mouse IgG1 isotype in order to label non-NK
cells. Afterwards, PBMC were washed once in PBS and incubated
for 15 min. at 6°C with 20 ␮L of colloidal superparamagnetic
MACS microbeads recognizing non-NK cells. Labeled and unlabeled cells were separated in a high-gradient magnetic field,
generated in a steel wool matrix inserted into the field of a
permanent magnet (42,47). The columns were sterilized in an
autoclave at 120°C shortly before use.
The negative unlabeled cells, representing the enriched nonmagnetic NK cell fraction, were eluted from the separation column
outside the magnetic field in a laminar flow hood to ensure
appropriate asepsis. The efficiency of separation was evaluated by
flow cytometry, using a FACScan (Becton Dickinson, Mountain
View, CA, USA). The sample obtained from the negative fraction
was stained with FITC-conjugated NK cell antibodies (CD56,
CD16ϩ) and counted for total NK cell number. Anti-Leu 11b
(anti-CD16) and anti-Leu 19 (anti-CD56) were purchased from
Becton Dickinson. The MACS procedure allowed us to separate
the negative NK cell population in approximately 2 h with yields
of over 95% and purity of 97 Ϯ 1% for CD16, CD56ϩ NK cells.
Viability of the NK subpopulation was determined by Trypan blue
uptake prior to the cytotoxicity assay against K562 tumor cells and
PKC activity assay.
The human myelogenous leukemia cell line K562 was used as

NATURAL KILLER CELLS AND PROTEIN KINASE C IN AD
the target in the cytotoxicity assays (50,59) and was grown in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal
calf serum (FCS), 1% glutamine, and 100 ␮g/mL gentamycin. The
K562 cell line was maintained in suspension culture flasks at 37°C
in a 5% CO2 incubator (Heraeus BB 6220, Hanau, Germany).
All K562 target cells used were Ͼ 90% viable as measured by
Trypan blue dye exclusion (Trypan blue solution 0.4% SIGMA
Chimica, Milano, Italy).
Immunological procedures were performed in a completely
sterile manner in a class II biological safety cabinet (Microflow
51426, MDH Ltd., Andover, UK). Complete medium containing
RPMI 1640 medium (HyClone Laboratories Inc, Logan, UT,
USA) enriched with 10% heat-inactivated FCS (HyClone Laboratories Inc, Logan, UT, USA), 1% glutamine (HyClone Laboratories Inc, Logan, UT, USA) and 100 ␮g/mL gentamycin (Irvine
Scientific, Santa Ana, CA, USA) was used for cytotoxicity assays.
After magnetic separation, NK cells were washed three times
(with 0.9% saline and complete RPMI medium) and finally
resuspended to a measured (Sysmex Toa F800 Microcell Counter,
Dasit, Bareggio, Italy) density of 7.75 ϫ 106 cells/mL of complete
medium. NK cells were incubated for 20 h (18,26) at 37°C in a
humidified atmosphere of 95% air and 5% CO2, with cortisol,
IL-2, and IFN-␤ and without the use of modulators (to measure the
spontaneous basal cytotoxicity). Cortisol (Hydrocortisone,
SIGMA Chimica, Milano, Italy), IL-2 (recombinant human IL-2,
Proleukin, Chiron Corporation, Emeryville, USA), and human
IFN-␤ (Betantrone, Italfarmaco S.p.A., Milano, Italy) were diluted
in a complete fresh medium (in a 0.1 mL final volume) and used
at a final concentration of 10Ϫ6 M, 100 IU/mL, and 650 IU/mL,
respectively. After incubation, the cells were washed twice with
0.9% saline and then once with complete medium containing
Medium 199 modified and 5% fraction V bovine albumin (SIGMA
Chemical Co., St. Louis, MO, USA) counted, assessed for viability
by Trypan blue dye exclusion, and then assayed for cytotoxicity
against K562 target cells.
After washing three times with 0.9% saline and complete
medium (Medium 199ϩ5% Albumin fraction V), 3 ϫ 104 target
cells in 0.1 mL complete medium were mixed in triplicate with
various concentrations of NK effector cells in the wells of a
round-bottomed 96-hole standard microtiter plate (TPP®, Celbio,
Pero-Milano, Italy), at a final total volume of 0.2 mL. These
mixtures gave final effector:target ratios (E:T) of 25:1, 12.5:1,
6.25:1, 3.125:1. After a second incubation for 4 h at 37°C in a 5%
CO2 atmosphere, the microtiter plate was centrifuged and a fixed
aliquot (0.1 mL) of supernatant was extracted from each well and
transferred to the corresponding wells of a flat-bottomed microtiter
plate. The cytotoxicity assay of NK cells was based on the kinetic
measurement by a computer-assisted (Milenia Kinetic Analyzer
DPC, Los Angeles, CA, USA) microtiter plate reader of the
amount of lactate dehydrogenase (LDH) released into the supernatant of target cells, according to the calculation of Korzeniewski
and Callewaert (29). Subsequently, 0.1 mL of lactic acid dehydrogenase substrate mixture was added to each well at intervals of 3 s.
Data on NK cytotoxic activity were expressed as lytic units
(LU)/107 cells (49) and as percentage of increase or decrease of
specific lysis.
After a 20-h incubation at 37°C (with 5% CO2) with cortisol,
IL-2, and IFN-␤ and without the use of modulators, four different
aliquots of NK cells were frozen on dry ice and stored at Ϫ80°C
until the kinase assay (no more than 1 week). Cells were homogenized using a teflon glass homogenizer in 500 ␮L 0.32 M sucrose
buffered with 20 mM Tris-HCl pH 7.4 containing 2 mM EDTA,
0.5 mM EGTA, 50 mM mercaptoethanol, 0.3 mM phenylmethylsulfonylfluoride, and 20 ␮g/mL leupeptin. The homogenate was
centrifuged at 100,000 ϫ g for 1 h: the supernatant constituted the

193
soluble fraction. The resulting pellet was treated with the same
homogenization buffer containing 10 mg/mL CHAPS {3-[(3Cholamidopropyl) dimethylammonio]-1-propanesulfonate} for 20
min., sonicated, and centrifuged at 100,000 ϫ g for 1 h: the
supernatant represented the solubilized particulate fraction.
Calcium-dependent PKC activity was assessed as previously
described and the antibodies against PKC isoforms were provided
by W.C. Wetsel (7). Briefly, 4 ␮g of soluble and particulate
fractions were incubated at 37°C in 100 ␮L final volume of a
buffer containing 3 ␮mol Tris-HCl pH 7.5, 0.8 ␮mol magnesium
acetate, 0.1 ␮mol CaCl2, and 50 ␮g histone type III S as kinase
substrate (basal activity). Stimulated activity was evaluated by
introducing 40 ␮g phosphatidylserine and 4 ␮g diolein into the
same incubation buffer. The reaction was started by adding 20 ␮M
[␥Ϫ32P]-ATP (0.3 ␮Ci/sample, S.A. 3000 Ci/mmole—Amersham,
Italy) and stopped after 5 min. with 100 ␮L of 25% trichloroacetic
acid and filtration through Schleicher and Schull ME 25 0.45 ␮m
filters under vacuum. Samples were washed 3 times with a solution
containing 10% trichloroacetic acid, 10 mM NaH2PO4, and 5 mM
EGTA. Radioactivity retained by filters was determined by liquid
scintillation using Formula 989 (NEN, Cologno Monzese, Italy).
Protein content was measured by the Bradford’s micromethod
(12). Specific PKC activity was evaluated as the difference
between stimulated and basal activities and expressed as nmol/
min./mg protein.
Cytosolic fractions were prepared from NK cells as previously
described.
Sample aliquots were diluted with sample buffer, boiled for 5
min., and processed as described (7). Antibody to PKC ␣ was
diluted at 1:10000; PKC ␤I, ␤II, and ␥ at 1:5000. Western blot was
developed using enhanced chemiluminescence according to the
manufacturer’s instructions (Amersham, Milano, Italy). With regard to the densitometric analysis, the image of the Western blot
was acquired with a Nikon CCD video camera module. The optical
density of the immunoreactive bands was calculated and the peak
area of a given band was analyzed by means of the Image 1.47
program for digital image processing (Wayne Rasband, NIH,
Research Services Branch, NIMH, Bethesda, MD, USA).
One-way analysis of variance (ANOVA F-test) was employed
to measure differences in clinical and biochemical parameters
among healthy subjects and patients with SDAT. ANOVA was
also used to evaluate differences in the cytotoxicity and PKC
activity of NK cells recorded under basal conditions (spontaneous
lysis) and after incubation with cortisol, IL-2, or IFN-␤. Tukey’s
and Dunnet’s post hoc tests were used when ANOVA proved
significant. Non-parametric Spearman’s correlation coefficient
was employed to evaluate the correlation between MMSE score
and NK cytotoxic activity (spontaneous and after cortisol, IL-2,
and IFN-␤). Multiple regression analysis was performed to assess
the relationship between clinical and biochemical parameters (as
independent variables) with the spontaneous NK cytotoxicity and
the percent of variation of NK cell activity after cortisol, IL-2, and
IFN-␤ (as dependent variables). p Ͻ 0.05 was accepted as the
threshold of statistical significance (two-tailed). All analyses were
run with the SPSS/PCϩ V3.0 statistical package.
RESULTS

Spontaneous NK cytotoxic activity, evaluated in the entire
PBMC cell population before the immunomagnetic separation
(MACS) and in NK cells after MACS, were compared in healthy
young and elderly subjects and in patients with SDAT. No
differences in spontaneous cytotoxicity were found between
healthy elderly subjects (17.5 Ϯ 3.3 L.U./107 cells) and patients
with SDAT (17.9 Ϯ 3.6 L.U./107 cells) (Figs. 1-3). The data

194

SOLERTE ET AL.

FIG. 1. Variation (mean Ϯ SD) in NK cell cytotoxic activity (expressed as lytic units ϭ L.U.) under basal conditions
(spontaneous activity) and after cortisol incubation (10Ϫ6 M) in healthy elderly subjects (open bars) and in patients with
SDAT (hatched bars).

obtained in PBMC before and after MACS were superimposable
(19.1 Ϯ 3.8 vs. 18.6 Ϯ 3.7 L.U./107 cells) (Table 2). The
spontaneous NK cytotoxicity of both groups was also similar to
that found in healthy young subjects (17.7 Ϯ 3.4 L.U. 107 cells).
A significantly (p Ͻ 0.01) reduced suppressive effect on
cytotoxic activity (mean percent inhibition of Ϫ24%) was demonstrated in patients with SDAT as compared to healthy elderly
subjects (mean percent inhibition of Ϫ44%) when NK cells and
PBMC cells were cultured in the presence of hydrocortisone, 10Ϫ6
M (Fig. 1, Table 2). The results obtained in NK and PBMC cells
were comparable (Table 2). Moreover, a similar pattern of NK
cytotoxic activity (during cortisol incubation), was demonstrated
when healthy elders were compared to healthy young subjects
(percent inhibition of NK cell activity after cortisol ϭ 42.8 Ϯ
5.1%).
NK cytotoxic activity, evaluated in NK cells (Fig. 2) and in the
whole PBMC cell population (Table 2), was analyzed in healthy
elderly subjects and in patients with SDAT during IL-2 incubation
(100 IU/mL/cells). Higher NK and PBMC cell cytotoxic response
to IL-2 was found in patients with SDAT (mean percent stimulation of ϩ102%) than in healthy elderly subjects (mean percent
stimulation of ϩ75%) (p Ͻ 0.001). No difference in cytolytic
response during IL-2 incubation was demonstrated when NK cells
(Fig. 2) were compared to PBMC cells (Table 2). The modulatory

effect of IL-2 was also similar when healthy elderly subjects and
healthy young subjects were compared (percent stimulation on NK
cell activity after IL-2 ϭ 77.1 Ϯ 3.7%).
NK cytotoxic activity measured in NK cells (Fig. 3) and in the
entire PBMC cell population (Table 2) was also evaluated in
healthy elderly subjects and in patients with SDAT during IFN-␤
incubation (650 IU). Significantly increased NK and PBMC cell
cytotoxic response to IFN-␤ was demonstrated in patients with
SDAT (percent stimulation of ϩ132%) in comparison with healthy
elderly subjects (percent stimulation of ϩ88%; p Ͻ 0.001). No
variations in cytotoxic responses (during IFN-␤ incubation) were
demonstrated when NK (Fig. 3) and PBMC cells (Table 2) were
compared. There were also no differences in the modulatory
effects of IFN-␤ on NK cytotoxic activity when healthy elderly
subjects were compared to healthy young subjects (percent stimulation of NK cell activity after IFN-␤ ϭ 86.4 Ϯ 9.3%).
Non-parametric Spearman’s correlation coefficient (rs) analysis
demonstrated several inverse correlations between NK cell cytotoxic activity and the impairment of cognitive function (evaluated
by MMSE) in patients with SDAT. In these patients, there were
significant correlations between spontaneous NK cytotoxicity (p ϭ
0.0208) and the percent stimulation of NK cytotoxicity after IL-2
(p ϭ 0.0056) and IFN-␤ (p ϭ 0.0044) and MMSE (Fig. 4);
whereas cortisol did not correlate with MMSE (Fig. 4). On the

FIG. 2. Variation (mean Ϯ SD) in NK cell cytotoxic activity (expressed as lytic units ϭ L.U.) under basal conditions
(spontaneous activity) and after IL-2 incubation (100 IU/mL) in healthy elderly subjects (open bars) and in patients with
SDAT (hatched bars).

NATURAL KILLER CELLS AND PROTEIN KINASE C IN AD

195

FIG. 3. Variation (mean Ϯ SD) in NK cell cytotoxic activity (expressed as lytic units ϭ L.U.) under basal conditions
(spontaneous activity) and after IFN-␤ incubation (650 IU/mL) in healthy elderly subjects (open bars) and in patients with
SDAT (hatched bars).

contrary, no correlations were demonstrated between NK activity
and the other clinical and biochemical parameters incorporated as
independent variables into the multiple regression analysis.
Lower basal values of PKC activity of NK cells were found in
SDAT patients than in healthy elderly subjects (Ϫ33%), even if the
differences were not statistically significant (Fig. 5).
Figure 5 represents the effects of cortisol (10Ϫ6 M), IL-2 (100
IU/mL), and IFN-␤ (650 IU/mL) on cytosolic Ca2ϩ-dependent
PKC activity in healthy elderly subjects and in patients with
SDAT. The PKC data are presented as specific activity in
nmoL/min./mg protein. No significant variations in PKC activity
were measured after cortisol incubation in healthy elderly subjects
and in patients with SDAT. On the contrary, IL-2 and IFN-␤
significantly decreased cytosolic PKC activity in healthy elderly
subjects (percent variation of Ϫ45% and Ϫ47%, respectively).
This response was completely lacking in patients with SDAT,
where PKC activity remained unchanged after exposure to IL-2 or
IFN-␤. The enzyme activity in the membrane compartment was
unmodified in basal conditions and throughout all treatments (data
not shown) when healthy elders and SDAT patients were com-

TABLE 2
VARIATIONS IN NK CYTOTOXIC ACTIVITY EVALUATED IN PBMC
BEFORE IMMUNOMAGNETIC SEPARATION
Healthy elderly
subjects

Spontaneous lysis
L.U./107 cells
NK cytotoxic activity after
cortisol (10Ϫ6 M)
L.U./107 cells
percent inhibition (%)
NK cytotoxic activity after
IL-2 (100 IU)
L.U./107 cells
percent stimulation (%)
NK cytotoxic activity after
IFN-␤ (650 IU)
L.U./107 cells
percent stimulation (%)

Patients with
SDAT

19.1 Ϯ 3.8

18.6 Ϯ 3.7

11.2 Ϯ 2.7
41.3 Ϯ 6.0

14.1 Ϯ 2.9
24.1 Ϯ 2.3**

32.8 Ϯ 5.6
71.7 Ϯ 3.7

37.6 Ϯ 8
102.1 Ϯ 7.3***

34.2 Ϯ 4.8
79.1 Ϯ 9.6

43.7 Ϯ 9.6
134.9 Ϯ 11.3***

Results are mean Ϯ SD (**p Ͻ 0.01, ***p Ͻ 0.001 vs. healthy subjects).

pared (0.35 Ϯ 0.21 and 0.34 Ϯ 0.21 nmoL/min./mg protein,
respectively).
The decreased PKC activity after IL-2 and IFN-␤ in healthy
elderly subjects was associated with decreased levels of PKC ␣
and ␤II isoforms (Fig. 6); whereas these isoforms were not
influenced by cortisol. PKC ␤I immunoreactivity also migrated at
80 kDa and was of lower intensity than that for PKC ␤II, while
PKC ␥ was not expressed in NK cells (data not shown). The
quantification of the PKC bands in healthy elderly subjects is
reported in Table 3. While cortisol did not modify PKC␣ and ␤II
levels, IL-2 decreased cytosolic PKC␣ by 70% and PKC␤II by
40% and IFN-␤ decreased PKC␣ by 70% and PKC␤II by 67%.
The basal levels of soluble PKC␣ and ␤II in NK cells of SDAT
patients were not different from healthy subjects and cortisol, IL-2,
and IFN-␤ treatment did not change isoform levels (data not
shown).
DISCUSSION

The results of the present study indicate that NK cells of
patients with SDAT were more sensitive to stimulation of cytotoxic activity during IL-2 and IFN-␤ exposure than NK cells of
healthy elderly subjects. In the latter group, the functional activity
of NK cells was similar to that of healthy young subjects. The
increased cytokine-mediated NK cytotoxicity in SDAT was also
associated with an impaired suppression of NK cell activity by
cortisol. These effects on functional activity were evident in both
NK and PBMC cells. In fact, with our experimental design we
were able clearly to demonstrate that cytotoxic activity, as expressed by immunoseparated NK cells (under basal conditions and
after the use of modulators), was similar to that observed in the
whole population of PBMC cells. In agreement with previous data
(55,56), the increase in NK cytotoxic response to IL-2 and IFN-␤
would seem to be specific to SDAT patients, as NK cells exhibited
a normal response to cytokines in healthy elders, as well as in
patients with dementia of multi-infarct origin. The reduction in the
suppressive effect of cortisol on NK cytotoxicity may contribute
further to activating the NK cellular compartment in SDAT, and in
addition compromise any capacity for physiological suppression of
excessive activation. These results reveal some interesting new
insights into the involvement of the NK cellular compartment in
the neuroimmune hypothesis of SDAT (27,39,54 –56). Moreover,
the specificity of NK cell dysregulation in SDAT might provide a
new biological marker of the disease.
In healthy subjects and SDAT patients, the increase in NK cell

196

SOLERTE ET AL.

FIG. 4. Correlations between NK cytotoxic cell activity and Mini Mental State Examination (MMSE) score in patients with
SDAT. rs ϭ Spearman’s correlation coefficient.

cytotoxicity with IFN-␤ exposure is more pronounced by comparison to that found during IL-2 and IFN-␥ (55,56), suggesting that
IFN-␤ should be used in clinical models investigating NK cytotoxic activity (33,37).
In addition to supporting the hypothesis of a linkage between
immunity and neuropathology in SDAT (27,39,54 –56), the evidence of specific NK functional disorders in our patients with
SDAT may be of clinical relevance. These observations may
provide a useful aid in the early diagnosis and in the understanding
of pathogenetic mechanisms, and also introduce new perspectives
for therapeutic strategies (38).
We investigated the activity and levels of PKC in the NK cells,
as this is a specific enzyme involved in the regulation of NK
cytotoxic activity toward tumor cells (9). The basal cytosolic PKC
activity was reduced in NK cells derived from patients with SDAT,
although the effect was not statistically significant. This observation agrees qualitatively with the reported data of decreased PKC
activity in fibroblasts and platelets of SDAT patients (6,10,22,65).
In spite of the reduction in PKC activity, the spontaneous cytotoxic
activity of NK cells was similar to that found in healthy elders,
suggesting no effect of PKC on the regulation of spontaneous
cytotoxic function. On the contrary, PKC seems to be associated
mainly with NK activation by cytokines. Following 20-h treatment
with IL-2 and IFN-␤, the cytosolic PKC activity of NK cells was
decreased in healthy elderly subjects but not in SDAT patients, and
this decrease was associated with diminished levels of both PKC␣
and ␤II isoforms.
As mentioned, the activation of PKC leads to exocytosis of

preformed cytotoxic granules. It is tempting to speculate that after
20 h of incubation PKC is physiologically down-regulated in
healthy old subjects, thus contributing to the extinction of the NK
function. On the other hand, this mechanism is suggested to be
lacking in SDAT. The down-regulatory process might be altered in
the NK cells derived from these patients (and therefore not yet
detectable at the end of incubation period selected in our experiments). Another control mechanism might also be envisaged.
Experimental data have shown that PKC activation can inhibit
IL-2 responses in NK cells and IFN ␣/␤ responses in monocytes
(52). This control mechanism may be operant in our system and we
might speculate that, in order to activate feed-back inhibition, PKC
must be activated and subsequently, metabolized. This could result
in decreased PKC activity in healthy elders after long-term
exposure to IL-2 and IFN-␤. In SDAT patients, where the
mechanism of PKC activation of NK might be deficient, as shown
in other tissues (6,10,22,65,66), PKC cannot feed-back cytokine
activity efficiently, thereby leading to permanent NK activation. In
support of these suggestions, the cytokine-mediated decrease in
PKC activity that was observed in healthy elderly subjects may be
related to decreased levels of the major calcium-dependent isoforms present (i.e., PKC ␣ and ␤II), as shown by Western blot
analysis. It remains to be established which steps are involved in
coupling cytokine activation to PKC signal transduction in NK
cells. Obviously it should be recalled that the PKC signal transduction system is not the only one that regulates NK cell activity
toward different targets. Other kinases (9,45,60) and G protein

NATURAL KILLER CELLS AND PROTEIN KINASE C IN AD

197

FIG. 5. Calcium-dependent PKC activity (mean Ϯ SD) after cortisol, IL-2, and IFN-␤
incubation in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).

(PKC-independent) signals (19,61) may mediate the regulatory
action of cytokines.
The biochemical events leading to cytokine-dependent mechanisms of NK overactivation in SDAT may be related to the
clinical course of the disease and in particular to the development
of cognitive derangement. In this context, the spontaneous and
IL-2 and IFN-␤ induced NK cytotoxic responses were inversely

correlated with MMSE, suggesting a linkage between cytokinemediated NK activity and the deterioration of cognitive status in
these patients. Moreover, it is well known that IL-2 negatively
influences cognitive processes in a dose-dependent manner by
reducing cholinergic control of learning and memory (4,15).
Finally, with regard to the neuroimmune hypothesis of SDAT
(27,38,39,54), it is tempting to speculate that the NK overactivation (55,56) might play a role in the development of immunemediated brain damage in this disease, by means of the interaction
of these cells with astrocytes and microglia, as these cells also
directly express cytokine receptors (41). In this context, the
neurobiological basis for immune effects on neuropathology and
cognitive derangement in SDAT may provide important information for creating strategies to delay the clinical progression of the
disease.
ACKNOWLEDGEMENTS

We wish to thank Dr. Silvia Severgnini for technical support with the
immunological procedures; Dr. Teresa Ricciardi for clinical assistance in

TABLE 3
EFFECT OF CORTISOL, IL-2, AND INF-〉 ON NK CELL CYTOSOLIC PKC 〈
AND 〉II IMMUNOREACTIVITIES IN HEALTHY ELDERLY SUBJECTS
PKC ␣

FIG. 6. Representative Western blot analyses of PKC ␣ and ␤II immunoreactivities in soluble NK cell fractions from healthy elderly subjects
after 20 h exposure to solvent, cortisol, IFN-␤, and IL-2. Five micrograms
of protein were loaded and diffused by electrophoresis on the gels. The
arrowheads on the left denote the size of the specific immunoreactive band
for each PKC isoform, whereas those on the right show the migration
positions of the molecular size standards.

Basal
Cortisol
IL-2
IFN-␤

PKC ␤II

0.328 Ϯ 0.041
0.313 Ϯ 0.048 (Ϫ5%)
0.099 Ϯ 0.013 (Ϫ70%)*
0.100 Ϯ 0.027 (Ϫ70%)*

0.470 Ϯ 0.051
0.482 Ϯ 0.068 (ϩ2%)
0.282 Ϯ 0.034 (Ϫ40%)*
0.157 Ϯ 0.028 (Ϫ67%)*

The densitometry of each immunoreactive band was normalized to the
immunoreactivity of a rat brain cerebellum cytosolic fraction, utilized as
internal positive control in each blot (ratio of NK/cerebellum PKC
immunoreactivity). Values are mean Ϯ SEM of three different assays.
*p Ͻ 0.05 ANOVA and post hoc Dunnet’s test.

198

SOLERTE ET AL.

the Alzheimer’s Centre of our Department and Dr. W.C. Wetsel for the
antibodies against calcium-dependent PKC isoforms. The work was

supported in part by a grant from M.U.R.S.T. (University of Pavia) and
from the National Research Council (C.N.R.).

REFERENCES
1. Albright, J. W.; Albright, J. F. Age-associated decline in natural killer
(NK) activity reflects primarily a defect in function of NK cells. Mech.
Ageing Dev. 31:295–306; 1985.
2. American Psychiatric Association Diagnostic and Statistical Manual
of Mental Disorders. Third Edition. Revised. Washington DC: American Psychiatric Association; 1987.
3. Araga, S.; Kagimoto, H.; Funamoko, K.; Takahashi, K. Reduced
natural killer cell activity in patients with dementia of the Alzheimer
Type. Acta Neurol. Scand. 84:259 –263; 1991.
4. Araujo, D. M.; Lapchak, P. A.; Collier, B.; Quirion, R. Localization of
interleukin-2 (IL-2) receptors in the rat brain: Interaction with the
cholinergic system. Brain Res. 498:257–266; 1989.
5. Auernhammer, C. J.; Strasburger, C. J. Effects of growth hormone and
insulin-like growth factor I on the immune system. Eur. J. Endocrinol.
133:635– 645; 1995.
6. Baker, A. C.; Ko, L. W.; Blass, J. P. Systemic manifestations of
Alzheimer’s disease. Age 11:60 – 65; 1988.
7. Battaini, F.; Pascale, A.; Lucchi, L.; Racchi, M.; Bergamaschi, S.;
Parenti, M.; Wetsel, W. C.; Govoni, S.; Trabucchi, M. Expression and
regulation of calcium-independent protein kinase C in NG 108 –15 cell
differentiation. Biochem. Biophys. Res. Commun. 203:1423–1431;
1994.
8. Bender, B. S.; Chrest, F. J.; Adler, W. H. Phenotypic expression of
natural killer cell association membrane antigens and cytolytic function of peripheral blood cells from different aged humans. J. Clin. Lab.
Immunol. 21:31–36; 1986.
9. Bonnema, J. D.; Karnitz, L. M.; Schoon, R. A.; Abraham, R. T.;
Leibson, P. J. Fc receptor stimulation of phosphatidylinositol 3-kinase
in natural killer cells is associated with protein kinase C-independent
granule release and cell-mediated cytotoxicity. J. Exp. Med. 180:
1427–1435; 1994.
10. Bosman, G. J. C. G. M.; Schuurmans Stekhoven, J. H.; Melenhorst,
J. J.; Van Zuylen, A. J.; Bartholomeus, I. G. P.; Van Kalmthout,
P. J. C.; De Grip, W. J. Are thrombocyte membranes altered in
Alzheimer’s disease? A morphometric and biochemical study. Neurobiol. Aging 13:711–716; 1992.
11. Boyum, A. Isolation of mononuclear cells and granulocytes from
human blood. Scand. Clin. Lab. Invest. 21(Suppl.):31–50; 1968.
12. Bradford, M. M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein dye
binding. Anal. Biochem. 72:248 –254; 1976.
13. Carpen, O.; Saksela, E. Directed extocytosis in the NK-cell-mediated
cytotoxicity. A review. Nat. Immun. Cell Growth Regul. 7:1–12; 1988.
14. Cole, G.; Dobkins, K. R.; Hansen, L. A.; Terry, R. D.; Saitoh, T.
Decreased levels of protein kinase C in Alzheimer brain. Brain Res.
452:165–174; 1988.
15. Denikoff, K. D.; Rubinow, D. R.; Papa, M. Z.; Simpson, C.; Seipp,
C. A.; Lotze, M. T.; Chang, A. E.; Rosenstein, D.; Rosenberg, D. A.
The neuropsychiatric effects of treatment with IL-2 and lymphokine
activated killer cells. Ann. Intern. Med. 107:293–300; 1987.
16. Folstein, M.; Folstein, S.; McHugh, P. R. Mini Mental State. A
practical method for grading the cognitive state of patients for the
clinician. J. Psychiatr. Res. 12:189 –198; 1975.
17. Gandy, S.; Czernik, A. N.; Greengard, P. Phosphorylation of Alzheimer’s disease amyloid precursor peptide by protein kinase C. Proc.
Natl. Acad. Sci. USA 85:6218 – 6221; 1988.
18. Gatti, G.; Cavallo, R.; Sartori, M. L.; Del Ponte, D.; Masera, R.;
Salvadori, A.; Carignola, A.; Angeli, A. Inhibition by cortisol of
human natural killer (NK) cell activity. J. Steroid Biochem. 26:49 –58;
1987.
19. Gauperts, B. D. GE: A GTP-binding protein mediating exocytosis.
Ann. Rev. Physiol. 52:591– 606; 1990.
20. Gidlund, M.; Orn, A.; Wigzell, H.; Senik, A.; Gresser, I. Enhanced NK
cell activity in mice injected with interferon and interferon inducers.
Nature 273:759 –761; 1978.

21. Gillespie, S. L.; Golde, T. E.; Younkin, S. G. Secretory processing of
the Alzheimer amyloid ␤/A4 protein precursor is increased by protein
phosphorylation. Biochem. Biophys. Res. Commun. 187:1285–1290;
1992.
22. Govoni, S.; Bergamaschi, S.; Racchi, M.; Battaini, F.; Binetti, G.;
Bianchetti, A.; Trabucchi, M. Cytosol protein kinase C down regulation in fibroblasts from Alzheimer’s disease patients. Neurology
43:2581–2586; 1993.
23. Hachinski, V. C.; Lassen, N. A.; Marshall, J. Multi-infarct dementia:
A cause of mental deterioration in the elderly. Lancet ii:207–210;
1974.
24. Henney, C. S.; Kuribayashi, K.; Kern, D. E.; Gillis, S. IL-2 augments
natural killer cells activity. Nature 291:335–337; 1981.
25. Hercend, T.; Schmidt, R. E. Characteristics and uses of natural killer
cells. Immunol. Today 9:291–293; 1988.
26. Holbrook, N. J.; Cox, W. I.; Horner, H. C. Direct suppression of
natural killer activity in human peripheral blood leucocyte cultures by
glucocorticoids and its modulation by interferon. Cancer Res. 43:
4019 – 4025; 1983.
27. Huberman, M.; Shalit, F.; Roth-Deri, I.; Gutman, B.; Brodie, C.; Ko,
H. E.; Sredini, B. Correlation of cytokine secretion by mononuclear
cells of Alzheimer patients and their disease stage. J. Neuroimmunol.
52:147–152; 1994.
28. Julius, M. H.; Simpson, E.; Herzenberg, L. A. A rapid method for
isolation of functional thymus-derived murine lymphocytes. Eur.
J. Immunol. 3:645– 649; 1973.
29. Korzeniewski, C.; Callewaert, D. M. An enzyme-release assay for
natural cytotoxicity. J. Immunol. Methods. 64:313–320; 1983.
30. Krishnaray, R.; Blandford, G. Age-associated alterations in human
natural killer cells. 1. Increased activity as perconventional and kinetic
analysis. Cell Immunol. Immunopathol. 45:268 –285; 1987.
31. Krishnaray, R.; Blandford, G. Age-associated alterations in human
natural killer cells. 2. Increased frequency of selective NK subsets.
Cell Immunol. Immunopathol. 114:137–148; 1988.
32. Leibson, P. J.; Midthun, D. E.; Windebank, K. P.; Abraham, R. T.
Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation. J. Immunol. 145:
1498 –1504; 1990.
33. Liberati, A. M., Schippa, M.; Partuesi, M. G.; Grazia-Proietti, M.; De
Angelis, V.; Ferragoli, A.; Civieri, S.; Di Clemente, F.; Palmisano, L.;
Berruto, P. IFN-␤ induced biochemical and immunological modifications in hairy cell leukemia patients. Hematologica 76:375–382; 1991.
34. Ligthart, G. J.; Corberard, J. X.; Geertzen, H. G.; Meinders, A. E.;
Knoox, D. L.; Hijmans, W. Necessity of the assessment of health
status in human immunogerontological studies: evaluation of the
SENIEUR protocol. Mech. Ageing Dev. 55:89 –105; 1990.
35. Ligthart, G. J.; Schmidt, H. R.; Hijmans, W. Natural killer cell function
is not diminished in the healthy aged and is proportional to the number
of NK cells in the peripheral blood. Immunology 68:396 – 402; 1989.
36. Lotzova, E.; Ades, E. W. Natural killer cells: Definition, heterogeneity, lytic mechanism, function and clinical application. Nat. Immun.
Cell Growth Regul. 8:1–9; 1989.
37. Malto, L.; Carballido, J.; Mauzano, L.; Lapena, P.; Alvarez-Mon, M.
`
Interferon-␤ enhances the natural killer activity of patients with
bladder carcinoma. Cancer Immunol. Immunother. 38:406 – 410; 1994.
38. McGeer, P. L.; Rogers, J. Anti-inflammatory agents as a therapeutic
approach to Alzheimer’s disease. Neurology 42:447– 449; 1992.
39. McGeer, P. L.; Rogers, J.; McGeer, E. G. Neuroimmune mechanisms
in Alzheimer’s disease pathogenesis. Alzheimer Dis. Assoc. Disord.
8:149 –158; 1994.
40. McKhann, G.; Drachman, D.; Folstein, M.; Katzman, R.; Price, D.;
Stadlan, E. M. Clinical diagnosis of Alzheimer’s disease: Report of the
NINCDS-ADRDA Workgroup under the auspices of Department of
Health and Human Services Task Force on Alzheimer’s Disease.
Neurology 34:939 –944; 1984.

NATURAL KILLER CELLS AND PROTEIN KINASE C IN AD
41. McRae, A.; Dahlstrom, A.; Ling, E. A. Microglial in neurodegenerative disorders: Emphasis on Alzheimer’s disease. Gerontology 43:95–
108; 1997.
42. Miltenyi, S.; M 129 ller, W.; Weichel, W.; Radbruch, A. High gradient
magnetic cell separation with MACS. Cytometry 11:231–238; 1990.
43. Mysliwska, J.; Mysliwski, A.; Witkowski J. Age-dependent decline of
natural killer and antibody-dependent cell mediated cytotoxicity of
human lymphocytes is connected with decrease of their acid phosphatase activity. Mech. Ageing Dev. 31:1–11; 1985.
44. Nitsch, R. M.; Slack, B. E.; Wurtman, R. J.; Growdon, J. H. Release
of Alzheimer amyloid precursor derivatives stimulated by activation of
muscarinic aceylcholine receptors. Science 258:304 –307; 1992.
45. O’Shea, J. J.; Weissman, A. M.; Kennedy, I. C. S.; Ortaldo, J. R.
Engagement of the natural killer cell IgG Fc receptor results in tyrosine
phosphorylation of the zeta chain. Proc. Natl. Acad. Sci. USA
88:350 –354; 1991.
46. Ortaldo, J. R.; Herberman, R. B. Heterogeneity of natural killer cells.
Ann. Rev. Immunol. 2:359 –373; 1984.
47. Pflueger, E.; Mueller, E. A.; Anderer, F. A. Preservation of cytotoxic
function during multi-cycle immunomagnetic cell separations of human NK cells using a new type of magnetic bead. J. Immunol.
Methods 129:165–173; 1990.
48. Pross, H.; Baynes, M. Spontaneous human lymphocyte mediated
cytotoxicity against tumor target cells. A short review. Cancer Immunol. Immunother. 3:75– 85; 1977.
49. Pross, H. F.; Baynes, M. G.; Rubin, P.; Shragge, P.; Patterson, M. S.
Spontaneous human lymphocyte-mediated cytotoxicity against tumor
target cells. IX. The quantification of natural killer cell activity. J. Clin.
Immunol. 1:51– 63; 1981.
50. Robertson, M. J.; Ritz, J. Biology and clinical relevance of human
natural killer cells. Blood 76:2421–2438; 1990.
51. Saitoh, T.; Masliah, E.; Jin, L. W.; Cole, G. M.; Wieloch, T.; Shapiro,
I. P. Protein kinases and protein phosphorylation in neurologic
disorders and cell death. Lab. Invest. 64:596 – 616; 1991.
52. Sandberg, K.; Eloranta, M. L.; Gobl, A. E.; Alm, G. V. Phorbol
ester-mediated inhibition of IFN-␣/beta gene transcription in blood
mononuclear leukocytes. J. Immunol. 147:3116 –3121; 1991.
53. Schimdt, R. E.; McDernott, R. P.; Bartley, G.; Bertovich, M.; Amato,
D. A.; Austen, K. F.; Schlossman, S. F.; Stevens, R. L.; Ritz, J.
Specific release of proteoglycans from human natural killer cells
during target lysis. Nature 318:289 –291; 1985.
54. Singh, V. K. Neuroautoimmunity: Pathogenetic implications for Alzheimer’s disease. Gerontology 43:79 –94; 1997.
55. Solerte, S. B.; Fioravanti, M.; Severgnini, S.; Cerutti, N.; Pezza, N.;

199

56.

57.

58.

59.

60.

61.

62.
63.
64.

65.

66.

67.

Locatelli, M.; Terenzi, F.; Ferrari, E. Excitatory pattern of ␥-interferon
on natural killer cell activity in senile dementia of the Alzheimer type.
Dementia Geriatr. Cogn. Disord. 8:308 –313; 1997.
Solerte, S. B.; Fioravanti, M.; Severgnini, S.; Locatelli, M.; Renzullo,
M.; Pezza, N.; Cerutti, N.; Ferrari, E. Enhanced cytotoxic response of
natural killer cells to IL-2 in Alzheimer’s disease. Dementia 7:343–
348; 1996.
Thompson, J. S.; Wekstein, D. R.; Rhoades, J. L.; Kirkpatrick, C.;
Brown, S. A.; Roszman, T.; Straus, R.; Tietz, N. The immune status of
healthy centenarians. J. Am. Geriatr. Soc. 2:274 –281; 1984.
Timonen, T.; Ranki, A.; Saksele, E.; Hayry, P. Fractionation, morphological and functional characterization of effector cells responsible for
human natural killer activity against cell-line targets. Cell Immunol.
48:133–139; 1979.
Ting, A. T.; Einspahr, K. E.; Abraham, R. T.; Leibson P. J. Fc gamma
receptor signal transduction in natural killer cells. Coupling to phospholipase C via a G protein-independent, but Tyrosine kinase-dependent pathway. J. Immunol. 147:3122–3127; 1991.
Ting, A. T.; Karnitz, L. M.; Schoon, R. A.; Abraham, R. T.; Leibson,
J. P. Fc ␥-receptor activation induces the tyrosine phosphorylation of
both phospholipase C (PLC)-␥1 and PLC-␥2 in natural killer cells. J.
Exp. Med. 176:1751–1757; 1992.
Ting, A. T.; Schoon, R. A.; Abraham, R. T.; Leisbon, P. J. Interaction
between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis. J. Biol.
Chem. 267:23957–23962; 1992.
Trinchieri, G. Biology of natural killer cells. Adv. Immunol. 47:187–
376; 1989.
Trinchieri, G.; Santoli, D. Enhancement of human natural killer cell
activity by interferon. J. Immunol. 120:1845–1850; 1978.
Trinchieri, G.; Matsumoto-Kobayashi, M.; Clark, S. C.; Seehra, J.;
London, L.; Perussia, B. Response of resting human peripheral blood
natural killer cells to interleukin 2. J. Exp. Med. 160:1147–1151; 1984.
Van Huynh, T.; Cole, G.; Katzman, R.; Huang, K. P.; Saitoh, T.
Reduced protein kinase C immunoreactivity and altered protein
phosphorylation in Alzheimer’s disease fibroblasts. Arch. Neurol.
46:1195–1199; 1989.
Wang, H-Y.; Pisano, M. R.; Friedman, E. Attenuated protein kinase C
activity and translocation in Alzheimer’s disease brain. Neurobiol.
Aging 15:293–298; 1994.
Young, J. DE.; Cohn, Z. A. Cellular and humoral mechanisms of
cytotoxicity: Structural and functional analogies. Adv. Immunol.
41:269 –332; 1987.

