]>NBA5195S0197-4580(98)00050-510.1016/S0197-4580(98)00050-5Elsevier Science Inc.Fig. 1Variation (mean ± SD) in NK cell cytotoxic activity (expressed as lytic units = L.U.) under basal conditions (spontaneous activity) and after cortisol incubation (10−6 M) in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 2Variation (mean ± SD) in NK cell cytotoxic activity (expressed as lytic units = 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).Fig. 3Variation (mean ± SD) in NK cell cytotoxic activity (expressed as lytic units = L.U.) under basal conditions (spontaneous activity) and after IFN-β incubation (650 IU/mL) in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 4Correlations between NK cytotoxic cell activity and Mini Mental State Examination (MMSE) score in patients with SDAT. rs = Spearman’s correlation coefficient.Fig. 5Calcium-dependent PKC activity (mean ± SD) after cortisol, IL-2, and IFN-β incubation in healthy elderly subjects (open bars) and in patients with SDAT (hatched bars).Fig. 6Representative Western blot analyses of PKC α and βII immunoreactivities in soluble NK cell fractions from healthy elderly subjects after 20 h exposure to solvent, cortisol, IFN-β, 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.Table 1Clinical and biochemical characteristics of healthy subjects and patients with SDAT (standard Deviation between brackets)Healthy subjectsPatients with SDATn1211men:women7:56:5age (years)78 (7)77 (6)BMI (kg/m2)22.5 (1.5)21.2 (1.7)Ht (%)44 (5)42 (3)Hb (g/dL)13.9 (1.4)13.7 (1.3)serum albumin (g/L)43 (3)41.7 (2.6)serum pre-albumin (g/L)0.33 (0.02)0.31 (0.02)transferrin (g/L)3.1 (0.2)3.0 (0.3)total lymphocytes (cells/mm3)1973 (51)1925 (51)Table 2Variations in NK cytotoxic activity evaluated in PBMC before immunomagnetic separationHealthy elderly subjectsPatients with SDATSpontaneous lysisL.U./107 cells19.1 ± 3.818.6 ± 3.7NK cytotoxic activity after cortisol (10−6 M)L.U./107 cells11.2 ± 2.714.1 ± 2.9percent inhibition (%)41.3 ± 6.024.1 ± 2.3aNK cytotoxic activity after IL-2 (100 IU)L.U./107 cells32.8 ± 5.637.6 ± 8percent stimulation (%)71.7 ± 3.7102.1 ± 7.3bNK cytotoxic activity after IFN-β (650 IU)L.U./107 cells34.2 ± 4.843.7 ± 9.6percent stimulation (%)79.1 ± 9.6134.9 ± 11.3bResults are mean ± SD (ap < 0.01,bp < 0.001 vs. healthy subjects).Table 3Effect of cortisol, IL-2, and INF-β on NK cell cytosolic PKC α and βII immunoreactivities in healthy elderly subjectsPKC αPKC βIIBasal0.328 ± 0.0410.470 ± 0.051Cortisol0.313 ± 0.048 (−5%)0.482 ± 0.068 (+2%)IL-20.099 ± 0.013 (−70%)a0.282 ± 0.034 (−40%)aIFN-β0.100 ± 0.027 (−70%)a0.157 ± 0.028 (−67%)aThe 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 ± SEM of three different assays.ap < 0.05 ANOVA and post hoc Dunnet’s test.Original ArticlesIncreased Natural Killer Cell Cytotoxicity in Alzheimer’s Disease May Involve Protein Kinase C DysregulationSolerteS.BSolerteA*FioravantiMFioravantiAPascaleAPascaleBFerrariEFerrariAGovoniSGovoniCBattainiFBattainiDADepartment of Internal Medicine, Geriatrics and Gerontology Clinic, University of Pavia, Pavia, ItalyBInstitute of Pharmacological Sciences, University of Milano, ItalyCInstitute of Pharmacology, University of Pavia and IRCCS S. Giovanni di Dio, Alzheimer Unit, Sacred Heart Hospital-FBF, Brescia, ItalyDDepartment of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Italy*Bruno Solerte, Department of Internal Medicine, University of Pavia, Ospedale S. Margherita, Piazza Borromeo, 2, 27100 Pavia, Italy.AbstractIncreased 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-β exposure) from SDAT patients. Our data demonstrate the presence of an increased cytotoxic response by NK cells to IL-2 (mean increase +102%) and IFN-β (mean increase +132%) in SDAT patients in comparison with healthy elderly subjects (+75% and +88% for IL-2 and IFN-β, respectively). A smaller suppression of NK cytotoxicity after cortisol was also observed in SDAT (mean decrease −24%) than in the control group (−44%). 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-β 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 α and βII 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.KeywordsAlzheimer’s diseaseDementiaAgingProtein kinase CNatural killerCytotoxicityCytokinesCortisolMagnetic cell sorterA 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 Ca2+-dependent mobilization of cytotoxic 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 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-β, and cortisol. Calcium-dependent PKC activity in soluble and particulate fractions of NK cells, and NK cytolytic function were also evaluated.1Methods1.1SubjectsThe 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 ± SD, 2.2 ± 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 ± SD = 10.3 ± 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 = 32.2 ± 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 = BMI) and biochemical parameters were demonstrated between healthy elderly subjects and patients with SDAT (Table 1).1.2ProceduresAfter an overnight fast, human peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood 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 μL buffer (per 107 total cells), containing PBS supplemented with 0.5% BSA, and incubated for 15 min. at 6°C with 20 μL 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 μL 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 non-magnetic 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, CD16+) 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 ± 1% for CD16, CD56+ 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 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 μg/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 > 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 μg/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 × 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-β 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-β (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 10−6 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 199+5% Albumin fraction V), 3 × 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-β and without the use of modulators, four different aliquots of NK cells were frozen on dry ice and stored at −80°C until the kinase assay (no more than 1 week). Cells were homogenized using a teflon glass homogenizer in 500 μL 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 μg/mL leupeptin. The homogenate was centrifuged at 100,000 × g for 1 h: the supernatant constituted the soluble fraction. The resulting pellet was treated with the same homogenization buffer containing 10 mg/mL CHAPS {3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate} for 20 min., sonicated, and centrifuged at 100,000 × 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 μg of soluble and particulate fractions were incubated at 37°C in 100 μL final volume of a buffer containing 3 μmol Tris-HCl pH 7.5, 0.8 μmol magnesium acetate, 0.1 μmol CaCl2, and 50 μg histone type III S as kinase substrate (basal activity). Stimulated activity was evaluated by introducing 40 μg phosphatidylserine and 4 μg diolein into the same incubation buffer. The reaction was started by adding 20 μM [γ−32P]-ATP (0.3 μCi/sample, S.A. 3000 Ci/mmole—Amersham, Italy) and stopped after 5 min. with 100 μL of 25% trichloroacetic acid and filtration through Schleicher and Schull ME 25 0.45 μm 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 α was diluted at 1:10000; PKC βI, βII, and γ 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-β. 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-β). 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-β (as dependent variables). p < 0.05 was accepted as the threshold of statistical significance (two-tailed). All analyses were run with the SPSS/PC+ V3.0 statistical package.2ResultsSpontaneous 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 ± 3.3 L.U./107 cells) and patients with SDAT (17.9 ± 3.6 L.U./107 cells) (Fig. 1  Fig. 2 Fig. 3   ). The data obtained in PBMC before and after MACS were superimposable (19.1 ± 3.8 vs. 18.6 ± 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 ± 3.4 L.U. 107 cells).A significantly (p < 0.01) reduced suppressive effect on cytotoxic activity (mean percent inhibition of −24%) was demonstrated in patients with SDAT as compared to healthy elderly subjects (mean percent inhibition of −44%) when NK cells and PBMC cells were cultured in the presence of hydrocortisone, 10−6 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 = 42.8 ± 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 +102%) than in healthy elderly subjects (mean percent stimulation of +75%) (p < 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 = 77.1 ± 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-β incubation (650 IU). Significantly increased NK and PBMC cell cytotoxic response to IFN-β was demonstrated in patients with SDAT (percent stimulation of +132%) in comparison with healthy elderly subjects (percent stimulation of +88%; p < 0.001). No variations in cytotoxic responses (during IFN-β 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-β on NK cytotoxic activity when healthy elderly subjects were compared to healthy young subjects (percent stimulation of NK cell activity after IFN-β = 86.4 ± 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 = 0.0208) and the percent stimulation of NK cytotoxicity after IL-2 (p = 0.0056) and IFN-β (p = 0.0044) and MMSE (Fig. 4  ); whereas cortisol did not correlate with MMSE (Fig. 4). On the 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 (−33%), even if the differences were not statistically significant (Fig. 5  ).Fig. 5 represents the effects of cortisol (10−6 M), IL-2 (100 IU/mL), and IFN-β (650 IU/mL) on cytosolic Ca2+-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-β significantly decreased cytosolic PKC activity in healthy elderly subjects (percent variation of −45% and −47%, respectively). This response was completely lacking in patients with SDAT, where PKC activity remained unchanged after exposure to IL-2 or IFN-β. 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 compared (0.35 ± 0.21 and 0.34 ± 0.21 nmoL/min./mg protein, respectively).The decreased PKC activity after IL-2 and IFN-β in healthy elderly subjects was associated with decreased levels of PKC α and βII isoforms (Fig. 6  ); whereas these isoforms were not influenced by cortisol. PKC βI immunoreactivity also migrated at 80 kDa and was of lower intensity than that for PKC βII, while PKC γ 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 PKCα and βII levels, IL-2 decreased cytosolic PKCα by 70% and PKCβII by 40% and IFN-β decreased PKCα by 70% and PKCβII by 67%.The basal levels of soluble PKCα and βII in NK cells of SDAT patients were not different from healthy subjects and cortisol, IL-2, and IFN-β treatment did not change isoform levels (data not shown).3DiscussionThe 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-β 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-β 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 cytotoxicity with IFN-β exposure is more pronounced by comparison to that found during IL-2 and IFN-γ [55, 56], suggesting that IFN-β 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-β, 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 PKCα and βII 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 α/β 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-β. 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 α and βII), 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 (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-β induced NK cytotoxic responses were inversely correlated with MMSE, suggesting a linkage between cytokine-mediated 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 immune-mediated 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.[58]AcknowledgementsWe wish to thank Dr. Silvia Severgnini for technical support with the immunological procedures; Dr. Teresa Ricciardi for clinical assistance in the Alzheimer’s Centre of our Department and Dr. W.C. 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