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Peripheral cytokine release in Alzheimer patients: correlation with disease severity
Neurobiology of Aging 24 (2003) 909–914
Peripheral cytokine release in Alzheimer patients: correlation with disease severity Gessica Sala a , Gloria Galimberti a,b , Carla Canevari a , Maria Elisabetta Raggi b , Valeria Isella a , Maurizio Facheris a , Ildebrando Appollonio a , Carlo Ferrarese a,b,∗ a
Department of Neurology, University of Milano-Bicocca, San Gerardo Hospital, via Donizetti, 106, Monza 20052 (MI), Italy b Scientific Institute “E. Medea”, Bosisio, Parini, Italy Received 4 April 2002; received in revised form 7 October 2002; accepted 12 December 2002
Abstract Various studies suggested that inflammation is involved in the pathogenesis of Alzheimer’s disease (AD). We investigated cytokine release from LPS-stimulated blood cells of 32 AD patients, with different disease severity, compared to 16 age-related controls. A significant decrease of IL-1beta and IL-6 secretion was observed in severely demented patients; TNF-alpha release was also decreased, but not significantly. By contrast, mild and moderate patients showed a cytokine release similar to controls. IL-1beta, IL-6 and TNF-alpha secretion was negatively correlated with the severity of dementia, quantified by the MMSE. Our data suggest that alterations of the immune profile are associated with AD progression. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer’s disease; Inflammation; Peripheral cytokine release; Disease severity
1. Introduction Among various factors involved in the pathogenesis of Alzheimer’s disease (AD), inflammatory-immunologic activation seems to play a major role. Several neuropathological studies showed a close association between neuritic plaques and local inflammatory response, as evidenced by the presence of acute phase proteins, cytokines, complement components and other inflammatory mediators next to amyloid beta (Abeta) deposits [1,20]. Moreover, microglia was found to be activated in the brain of AD patients by PET studies [5] suggesting its putative role in the mediation of amyloid toxicity. The presence of Abeta plaques may keep microglia persistently activated, leading to a condition of chronic inflammation at central nervous system level. Different studies performed in glial cultures and in macrophages revealed that Abeta stimulates both the synthesis and the release of pro-inflammatory cytokines such as interleukin (IL)-1beta [19], IL-6 [10] and TNF-alpha [21]. The same cytokines have been found over-expressed in the brain of AD patients [3,12,27].
∗ Corresponding author. Tel.: +39-039-233-3598; fax: +39-039-233-3586. E-mail address: [email protected] (C. Ferrarese).
Recent data indicate that genetic factors are involved in the pathogenesis of AD. Various genes have been investigated and some cytokine gene polymorphisms were found to be associated with AD onset. In fact, patients carrying the T/T polymorphism in the IL-1A gene promoter develop AD earlier than C/C carriers; by contrast, a weaker association was found between the T/T polymorphism of the IL-1B gene promoter and late onset AD [13,24]. Furthermore, an association of the C allele of the IL-6 genotype with a delayed initial onset and a reduced disease risk has been recently shown [25]. Quite interestingly, no association with the TNF-alpha or the TNF-beta gene polymorphism has been so far demonstrated [30]. As well as in brain and in cellular models, inflammatory markers were also investigated in cerebrospinal fluid (CSF) and serum of AD patients: IL-1beta [4] and TNF-alpha [29] were increased, whereas IL-6 was unaltered in CSF [14]. On the contrary, basal serum levels of IL-1beta, TNF-alpha, IL-6 and IL-10 were unchanged in one study [18], and increased in two other studies [9,17], although in all these studies they could be detected only in a minority of cases, either AD or controls. As a matter of fact, cytokine plasma levels are hard to detect at basal conditions, due to their short half-life; thus, cytokine release after stimulus was evaluated in some studies. Results have been somewhat controversial, but they all showed an activation of the peripheral immune status,
0197-4580/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0197-4580(03)00010-1
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G. Sala et al. / Neurobiology of Aging 24 (2003) 909–914
probably linked to the inflammatory condition present in AD brain. In particular, phytohemagglutinin (PHA)-stimulation of peripheral blood cells (PBC) evidenced an over-secretion of IL-2 and interferon (IFN)-gamma in AD respect to controls [15]. IL-1beta, IL-6, TNF-alpha and IL-10 release by lipopolysaccharide (LPS)-stimulated whole blood was found significantly higher in AD patients compared to controls [18]. In contrast to these studies, TNF-alpha release from blood cells was recently found significantly decreased in AD [6]. To further explore in AD patients cytokine release from peripheral blood cells after immune activation and to investigate if it might be related to disease severity, we studied the LPS-induced ex vivo release of IL-1beta, IL-6 and TNF-alpha from blood cells of AD patients, with different disease severity, compared to age-matched controls.
2. Patients and methods 2.1. Patients Thirty-two probable AD patients (10 men and 22 women) aged 53–92 years (mean ± S.D.: 72 ± 9 years) were selected for this study from the Department of Neurology of the University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy. The diagnosis of dementia was based on DMS-IV criteria; probable AD were selected according to the NINCDS-ADRDA criteria. Patient evaluation included medical history, physical and neurological examinations, and a neuroimaging study (computed tomography and/or magnetic resonance of the brain). Laboratory blood tests were performed to exclude metabolic causes of dementia (FT3, FT4, TSH, Vitamin B-12 and folic acid) and to assess the inflammatory status (erythrocyte sedimentation rate and C-reactive protein) of each recruited subject. Only subjects showing normal erythrocyte sedimentation rate and C-reactive protein values were selected for this study. In addition, all patients received a battery of neuropsychological tests, including the Mini-Mental State Examination (MMSE). Based on the MMSE, patients were classified as mild (MMSE score ≥ 21; mean ± S.D.: 23.5 ± 1.8), moderate (MMSE score: 11–20; mean ± S.D.: 16.4 ± 2.2) or severe (MMSE score ≤ 10; mean ± S.D.: 2.3 ± 3.0). These three subgroups were composed by 13, 7 and 12 patients, respectively. AD patients with concomitant neoplastic or hematological disorders, recent infections, abnormal white blood cell count or surgery, severe hepatic or renal insufficiency, myocardial infarction or cranial trauma in the previous 6 months or who had undergone antiplatelet, anti-inflammatory, antineoplastic, corticosteroid or immunosuppressive drug treatments in the preceding 2 weeks were excluded. Signs of malnutrition were excluded on the basis of physical examination and biochemical parameters (serum albumin, Vitamin B-12 and folic acid, mainly).
We also included 16 normal age-related controls. All control subjects underwent the same examinations and laboratory blood tests than AD patients. 2.2. Whole blood stimulation After informed consent, 5 ml of peripheral blood were drawn through the antecubital vein from AD patients and controls, collected in heparinized tubes and immediately processed. Blood was diluted 1:2 (vol/vol) in RPMI-1640 medium, containing l-glutamine 1% and antibiotics (penicillin 100 U/ml–streptomycin 100 ug/ml). Diluted blood was then divided in two aliquots and incubated for 4 h at 37 ◦ C and 5% CO2 , in presence of 100 ng/ml LPS (from Escherichia coli serotype 055:B5, Sigma) dissolved in sterile pyrogen-free saline or in presence of the same volume of sterile pyrogen-free saline. After incubation, blood was centrifuged at 400 × g for 10 min at room temperature, supernatants collected and stored at −20 ◦ C until assays. 2.3. Cytokine assay IL-1beta, IL-6 and TNF-alpha plasma levels and release were evaluated with IMMULITE System. Before assay, samples were thawed at room temperature and diluted with the sample diluents, specific for the different cytokines (IL-1beta 1:2, IL-6 1:40 and TNF-alpha 1:5), in order to obtain values lower then the upper limit of the calibration range (1000 pg/ml). The detection limit of the assay, defined as the concentration two standard deviations above the response at 0 dose, was approximately 1.5 pg/ml. 2.4. Statistical analysis All results are expressed as mean ± S.D. One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, was used to assess the significance of
Fig. 1. Dose–response curves for IL-1beta (䊏), IL-6 (䉱), TNF-alpha (䉲) release after whole blood cell stimulation with different LPS concentrations.
G. Sala et al. / Neurobiology of Aging 24 (2003) 909–914
differences between values of controls and patient subgroups. Correlation was computed with Pearson’s r test. 3. Results No difference in cytokine basal levels was found in plasma of AD patients and controls before stimulation (data not shown). To evaluate cytokine release from blood cells, pilot experiments on dose–response curves were performed using LPS concentrations ranging from 1 to 1000 ng/ml. Fig. 1 shows a typical dose–response curve for IL-1beta, IL-6 and TNF-alpha release from whole blood cells of normal control. Based on these results, our studies were performed using
Fig. 2. IL-1beta (a), IL-6 (b) and TNF-alpha (c) release by LPS-stimulated whole blood from normal controls (NC) and AD patients. MILD: mild AD patients, MOD: moderate AD patients; SEV: severe; AD patients. (a) ∗ P < 0.05 vs. NC, § P < 0.01 vs. MILD; (b) ◦ P < 0.05 vs. NC and MILD.
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100 ng/ml LPS concentration, which was found to be the minimal dose able to induce the optimal response. To verify consistency of the assay results, blood samples were taken from a subgroup of AD patients and controls after 2 month interval. Similar results were obtained from the same subjects, confirming the reliability of the assay. In AD patients a similar pattern of altered secretion was observed for all investigated cytokines, related to disease severity. While cytokine basal release was found similar in controls and in patient subgroups (data not shown), LPS-induced release was similar to controls in mild and moderate AD patients and reduced in severe demented patients. Fig. 2 shows the IL-1beta (a), IL-6 (b) and TNF-alpha (c) release by LPS-stimulated whole blood of normal controls and AD patient subgroups. A significant decrease of IL-1beta (P < 0.05) and IL-6 (P < 0.05) release was observed in severe AD patients. TNF-alpha release was also decreased in severe AD patients, but the difference did not reach significance. A significant correlation was observed between cytokine release and MMSE score of AD patients (r = 0.62, P < 0.0005 for IL-1beta; r = 0.53, P < 0.005 for IL-6; r = 0.41, P < 0.05 for TNF-alpha, Fig. 3). We also observed a
Fig. 3. Regression lines between MMSE score and cytokine release in AD patients. (a) r = 0.62, P < 0.0001; (b) r = 0.53, P < 0.005; (c) r = 0.41, P < 0.05.
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Fig. 4. Regression lines among cytokine release in AD patients. (a) r = 0.48, P < 0.01; (b) r = 0.17, p = ns; (c) r = 0.76, P < 0.0001.
high positive correlation among the cytokine secretions with the exception of TNF-alpha versus IL-1beta (Fig. 4). No correlation between white blood cell count and cytokine release was observed both in patients and controls. Since previous studies showed changes in hypothalamic– pituitary–adrenal axis function in AD patients, entailing increased cortisol levels which might be involved in immune depression [23], we evaluated cortisol circulating levels. Both patients and controls showed hormonal values in normal range.
4. Discussion In the present study we found, for the first time, a similar pattern of decreased IL-1beta, IL-6 and TNF-alpha release from LPS-stimulated PBC of severe AD patients, indicating the existence of a possible down-regulation of the peripheral immune response in the late stages of the disease. Although cytokine release in mild and moderate AD patients was similar to control values, the positive correlations observed in AD patients between IL-1beta, IL-6 and TNF-alpha release and MMSE scores suggest that cytokine production progressively decreases according to disease severity. To date, data on peripheral cytokines in AD patients are controversial, probably because each study used a different
methodological approach to evaluate cytokine release and different criteria to subgroup AD patients according to disease severity. Unaltered IL-1beta peripheral secretion in AD patients versus controls, after 6 h stimulation with 10 g/ml LPS, and a significant reduction of TNF-alpha release in moderate AD patients (MMSE score: 17±4) after 96 h incubation in the presence of 10 g/ml PHA were observed [15]. However, increased production of IL-1beta, TNF-alpha, IL-6 and IL-10 by LPS-stimulated whole blood of AD patients was also described [18]. These latter results seem in contrast with our findings, but, more recently, it has been shown that TNF-alpha release from blood cells is significantly decreased (−27%) in demented patients compared to controls [6]. Mechanisms involved in peripheral immune alterations in AD patients are presently under investigation. A peripheral immune alteration in AD patients might be triggered by brain immune activation, given the existence of inflammatory signaling pathway from the brain to the periphery [7]. Recently, intracerebroventricular injection of Abeta 1–42 in mice caused an IL-6 increase not only in brain, but also in plasma, demonstrating that centrally administered Abeta 1–42 effectively induces the systemic IL-6 response [27]. Thus, peripheral immune alterations might be linked to progressive deposition of Abeta 1–42 within the CNS [8]. Alternatively, circulating Abeta might directly affect immune functions. Although most authors described unchanged Abeta 1–42 levels in plasma of sporadic AD patients [28], recently, a longitudinal study on elderly controls showed that plasma Abeta generally rises with aging, suggesting that it may be elevated long before AD develops and subsequently decreases in AD patients [11]. Supporting this hypothesis, it is known that plasma Abeta declines when Abeta is deposited in brains of transgenic mouse model of AD [16]. Increased basal IL-6 levels in plasma have also been recently shown to be a risk for subsequent decline in cognitive functions [32]. Our findings, showing a significantly decreased release of the three major pro-inflammatory cytokines in severe AD patients, might reflect a decline in plasma Abeta levels or might be interpreted as a down-regulation of PBC, exposed to a chronic Abeta stimulus long before the onset of symptoms. While basal cytokine secretion was similar in patients and controls, a pro-inflammatory stimulus, such as LPS, was less effective in triggering cytokine release in severely demented patients. Accordingly, an association between the chronic expression of human amyloid precursor protein and a decrease in the immune response to human Abeta has been found in transgenic mice [22]. Chronic exposure of the immune system to Abeta in humans and in mouse models might lead to hyporesponsiveness in terms of cellular and humoral immune responses to Abeta itself, and this alteration could contribute to Abeta deposition responsible for AD. Furthermore, the mechanism of clearance of potentially amyloidogenic substance has been shown to be impaired in
G. Sala et al. / Neurobiology of Aging 24 (2003) 909–914
AD patients, which possess decreased lymphocyte autoreactivity against metabolic products of APP [31]. A strict link between hormonal pathway and immune system has also been established [33]. Some studies showed abnormalities at several levels of the hypothalamic– pituitary–adrenal (HPA) axis in patients with AD [23,26]. We investigated cortisol levels as one possible marker of HPA dysfunction and we did not find any alteration. Then, the impaired immune response found in severe AD patients could not be ascribed to hormonal dysregulation. In conclusion, the present study suggests the possibility to correlate impaired cytokine peripheral release and clinical conditions of AD patients, with a progressive impairment of immune functions in this disorder. This hypothesis might also explain, at least in part, some clinical data showing that severe AD patients have increased risk to be hospitalized because of infectious diseases independently of age, gender, education, comorbid medical conditions respect to both mild or moderate AD and controls [2]. In fact, a greater risk of medical complications that require hospital care, especially infections, appears to be characteristic of severe AD [2]. Finally, our study suggests the possibility to use peripheral cytokine release as a possible tool to further characterize immune dysfunction during AD progression.
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[11] Graff-Radford N, Ertekin-Taner N, Jadeja N, Younkin L, Petersen R, Younkin S, et al. Evidence that plasma amyloid beta protein may be useful as a premorbid biomarker for Alzheimer’s disease. Neurobiol Aging 2002;23(1):S384. [12] Griffin W, Stanley LC, Ling C, White L, Macleod V, Perrot L, et al. Brain interleukin-1 and S-100 immunoreactivity elevated in Down’s syndrome and Alzheimer’s disease. Proc Natl Acad Sci USA 1989;86:7611–5. [13] Grimaldi LM, Casadei VM, Ferri C, Veglia F, Licastro F, Annoni G, et al. Association of early-onset Alzheimer’s disease with an interleukin-1␣ gene polymorphism. Ann Neurol 2000;47(3):361–5. [14] Hampel H, Schoen D, Schwarz MJ, Kotter HU, Schneider C, Sunderland T, et al. Interleukin-6 is not altered in cerebrospinal fluid of first-degree relatives and patients with Alzheimer’s disease. Neurosci Lett 1997;228(3):143–6. [15] Huberman M, Shalit F, Roth-Deri I, Gutman B, Brodie C, Kott E, et al. Correlation of cytokine secretion by mononuclear cells of Alzheimer patients and their disease stage. J Neuroimmunol 1994;52:147–52. [16] Kawarabayashi T, Younkin LH, Saido TC, Shoji M, Ashe KH, Younkin SG. Age-dependent changes in brain, CSF and plasma amyloid () protein in the Tg2576 transgenic mouse model of Alzheimer’s disease. J Neurosci 2001;21(2):372–81. [17] Licastro F, Pedrini S, Caputo L, Annoni G, Davis LJ, Ferri C, et al. Increased plasma levels of interleukin-1, interleukin-6 and ␣-1-antichymotrypsin in patients with Alzheimer’s disease: peripheral inflammation or signals from the brain? J Neuroimmunol 2000;103:97–102. [18] Lombardi VR, Garcia M, Rey L, Cacabelos R. Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer’s disease (AD) individuals. J Neuroimmunol 1999;97(1/2):163–71. [19] Lorton D, Kocsis JM, King L, Madden K, Brunden KR. -Amyloid induces increased release of interleukin-1 from lipopolysaccharideactivated human monocytes. J Neuroimmunol 1996;67:21–9. [20] McGeer PL, Akiyama H, Itagaki S, McGeer EG. Activation of the classical complement pathway in brain tissue of Alzheimer patients. Neurosci Lett 1989;107:341–6. [21] Meda L, Cassatella MA, Szendrei GI, Otvos Jr L, Baron P, Villalba M, et al. Activation of microglial cells by -amyloid protein and interferon-␥. Nature 1995;374:647–50. [22] Monsonego A, Maron R, Zota V, Selkoe DJ, Weiner HL. Immune hyporesponsiveness to amyloid beta-peptide in amyloid precursor protein transgenic mice: implications for the pathogenesis and treatment of Alzheimer’s disease. PNAS 2001;98(18):10273–8. [23] Murialdo G, Barreca A, Nobili F, Rollero A, Timossi G, Gianelli MV, et al. Relationships between cortisol, dehydroepiandrosterone sulphate and insulin-like growth factor-I system in dementia. J Endocrinol Invest 2001;24(3):139–46. [24] Nicoll JA, Mrak RE, Graham DI, Stewart J, Wilcock G, MacGowan S, et al. Association of interleukin-1 gene polymorphism with Alzheimer’s disease. Ann Neurol 2000;47(3):365–8. [25] Papassotiropoulos A, Bagli M, Jessen F, Bayer TA, Maier W, Rao ML, et al. A genetic variation of the inflammatory cytokine interleukin-6 delays the initial onset and reduces the risk for sporadic Alzheimer’s disease. Ann Neurol 1999;45(5):666–8. [26] Rasmuson S, Nasman B, Carlstrom K, Olsson T. Increased levels of adrenocortical and gonadal hormones in mild to moderate Alzheimer’s disease. Dement Geriatr Cogn Disord 2002;13(2):74–9. [27] Song DK, Im YB, Jung JS, Cho J, Suh HW, Kim YH. Central beta-amyloid peptide-induced peripheral interleukin-6 responses in mice. J Neurochem 2001;76:1326–35. [28] Tamaoka A, Fukushima T, Sawamura N, Ishikawa K, Oguni E, Komatsuzaki Y, et al. Amyloid beta protein in plasma from patients with sporadic Alzheimer’s disease. J Neurol Sci 1996;141(12):65–8. [29] Tarkowski E, Blennow K, Wallin A, Tarkowski A. Intracerebral production of tumor necrosis factor-␣, a local neuroprotective agent
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in Alzheimer disease and vascular dementia. J Clin Immunol 1999;19:223–30. [30] Tarkowski E, Liljeroth AM, Nilsson A, Ricksten A, Davidsson P, Minthon L, et al. TNF gene polymorphism and its relation to intracerebral production of TNF␣ and TNF in AD. Neurology 2000;54:2077–81. [31] Trieb K, Ransmayr G, Sgonc R, Lassmann H, Grubeck-Loebenstein B. APP peptides stimulate lymphocyte proliferation in normals,
but not in patients with Alzheimer’s disease. Neurobiol Aging 1996;17(4):541–7. [32] Weaver JD, Huang MH, Albert M, Harris T, Rowe JW, Seeman TE. Interleukin-6 and risk of cognitive decline. Neurology 2002;59: 371–8. [33] Webster JI, Tonelli L, Sternberg EM. Neuroendocrine regulation of immunity. Annu Rev Immunol 2002;20:125–63.
Peripheral cytokine release in Alzheimer patients: correlation with disease severity Gessica Sala a , Gloria Galimberti a,b , Carla Canevari a , Maria Elisabetta Raggi b , Valeria Isella a , Maurizio Facheris a , Ildebrando Appollonio a , Carlo Ferrarese a,b,∗ a
Department of Neurology, University of Milano-Bicocca, San Gerardo Hospital, via Donizetti, 106, Monza 20052 (MI), Italy b Scientific Institute “E. Medea”, Bosisio, Parini, Italy Received 4 April 2002; received in revised form 7 October 2002; accepted 12 December 2002
Abstract Various studies suggested that inflammation is involved in the pathogenesis of Alzheimer’s disease (AD). We investigated cytokine release from LPS-stimulated blood cells of 32 AD patients, with different disease severity, compared to 16 age-related controls. A significant decrease of IL-1beta and IL-6 secretion was observed in severely demented patients; TNF-alpha release was also decreased, but not significantly. By contrast, mild and moderate patients showed a cytokine release similar to controls. IL-1beta, IL-6 and TNF-alpha secretion was negatively correlated with the severity of dementia, quantified by the MMSE. Our data suggest that alterations of the immune profile are associated with AD progression. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Alzheimer’s disease; Inflammation; Peripheral cytokine release; Disease severity
1. Introduction Among various factors involved in the pathogenesis of Alzheimer’s disease (AD), inflammatory-immunologic activation seems to play a major role. Several neuropathological studies showed a close association between neuritic plaques and local inflammatory response, as evidenced by the presence of acute phase proteins, cytokines, complement components and other inflammatory mediators next to amyloid beta (Abeta) deposits [1,20]. Moreover, microglia was found to be activated in the brain of AD patients by PET studies [5] suggesting its putative role in the mediation of amyloid toxicity. The presence of Abeta plaques may keep microglia persistently activated, leading to a condition of chronic inflammation at central nervous system level. Different studies performed in glial cultures and in macrophages revealed that Abeta stimulates both the synthesis and the release of pro-inflammatory cytokines such as interleukin (IL)-1beta [19], IL-6 [10] and TNF-alpha [21]. The same cytokines have been found over-expressed in the brain of AD patients [3,12,27].
∗ Corresponding author. Tel.: +39-039-233-3598; fax: +39-039-233-3586. E-mail address: [email protected] (C. Ferrarese).
Recent data indicate that genetic factors are involved in the pathogenesis of AD. Various genes have been investigated and some cytokine gene polymorphisms were found to be associated with AD onset. In fact, patients carrying the T/T polymorphism in the IL-1A gene promoter develop AD earlier than C/C carriers; by contrast, a weaker association was found between the T/T polymorphism of the IL-1B gene promoter and late onset AD [13,24]. Furthermore, an association of the C allele of the IL-6 genotype with a delayed initial onset and a reduced disease risk has been recently shown [25]. Quite interestingly, no association with the TNF-alpha or the TNF-beta gene polymorphism has been so far demonstrated [30]. As well as in brain and in cellular models, inflammatory markers were also investigated in cerebrospinal fluid (CSF) and serum of AD patients: IL-1beta [4] and TNF-alpha [29] were increased, whereas IL-6 was unaltered in CSF [14]. On the contrary, basal serum levels of IL-1beta, TNF-alpha, IL-6 and IL-10 were unchanged in one study [18], and increased in two other studies [9,17], although in all these studies they could be detected only in a minority of cases, either AD or controls. As a matter of fact, cytokine plasma levels are hard to detect at basal conditions, due to their short half-life; thus, cytokine release after stimulus was evaluated in some studies. Results have been somewhat controversial, but they all showed an activation of the peripheral immune status,
0197-4580/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0197-4580(03)00010-1
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G. Sala et al. / Neurobiology of Aging 24 (2003) 909–914
probably linked to the inflammatory condition present in AD brain. In particular, phytohemagglutinin (PHA)-stimulation of peripheral blood cells (PBC) evidenced an over-secretion of IL-2 and interferon (IFN)-gamma in AD respect to controls [15]. IL-1beta, IL-6, TNF-alpha and IL-10 release by lipopolysaccharide (LPS)-stimulated whole blood was found significantly higher in AD patients compared to controls [18]. In contrast to these studies, TNF-alpha release from blood cells was recently found significantly decreased in AD [6]. To further explore in AD patients cytokine release from peripheral blood cells after immune activation and to investigate if it might be related to disease severity, we studied the LPS-induced ex vivo release of IL-1beta, IL-6 and TNF-alpha from blood cells of AD patients, with different disease severity, compared to age-matched controls.
2. Patients and methods 2.1. Patients Thirty-two probable AD patients (10 men and 22 women) aged 53–92 years (mean ± S.D.: 72 ± 9 years) were selected for this study from the Department of Neurology of the University of Milano-Bicocca, San Gerardo Hospital, Monza, Italy. The diagnosis of dementia was based on DMS-IV criteria; probable AD were selected according to the NINCDS-ADRDA criteria. Patient evaluation included medical history, physical and neurological examinations, and a neuroimaging study (computed tomography and/or magnetic resonance of the brain). Laboratory blood tests were performed to exclude metabolic causes of dementia (FT3, FT4, TSH, Vitamin B-12 and folic acid) and to assess the inflammatory status (erythrocyte sedimentation rate and C-reactive protein) of each recruited subject. Only subjects showing normal erythrocyte sedimentation rate and C-reactive protein values were selected for this study. In addition, all patients received a battery of neuropsychological tests, including the Mini-Mental State Examination (MMSE). Based on the MMSE, patients were classified as mild (MMSE score ≥ 21; mean ± S.D.: 23.5 ± 1.8), moderate (MMSE score: 11–20; mean ± S.D.: 16.4 ± 2.2) or severe (MMSE score ≤ 10; mean ± S.D.: 2.3 ± 3.0). These three subgroups were composed by 13, 7 and 12 patients, respectively. AD patients with concomitant neoplastic or hematological disorders, recent infections, abnormal white blood cell count or surgery, severe hepatic or renal insufficiency, myocardial infarction or cranial trauma in the previous 6 months or who had undergone antiplatelet, anti-inflammatory, antineoplastic, corticosteroid or immunosuppressive drug treatments in the preceding 2 weeks were excluded. Signs of malnutrition were excluded on the basis of physical examination and biochemical parameters (serum albumin, Vitamin B-12 and folic acid, mainly).
We also included 16 normal age-related controls. All control subjects underwent the same examinations and laboratory blood tests than AD patients. 2.2. Whole blood stimulation After informed consent, 5 ml of peripheral blood were drawn through the antecubital vein from AD patients and controls, collected in heparinized tubes and immediately processed. Blood was diluted 1:2 (vol/vol) in RPMI-1640 medium, containing l-glutamine 1% and antibiotics (penicillin 100 U/ml–streptomycin 100 ug/ml). Diluted blood was then divided in two aliquots and incubated for 4 h at 37 ◦ C and 5% CO2 , in presence of 100 ng/ml LPS (from Escherichia coli serotype 055:B5, Sigma) dissolved in sterile pyrogen-free saline or in presence of the same volume of sterile pyrogen-free saline. After incubation, blood was centrifuged at 400 × g for 10 min at room temperature, supernatants collected and stored at −20 ◦ C until assays. 2.3. Cytokine assay IL-1beta, IL-6 and TNF-alpha plasma levels and release were evaluated with IMMULITE System. Before assay, samples were thawed at room temperature and diluted with the sample diluents, specific for the different cytokines (IL-1beta 1:2, IL-6 1:40 and TNF-alpha 1:5), in order to obtain values lower then the upper limit of the calibration range (1000 pg/ml). The detection limit of the assay, defined as the concentration two standard deviations above the response at 0 dose, was approximately 1.5 pg/ml. 2.4. Statistical analysis All results are expressed as mean ± S.D. One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, was used to assess the significance of
Fig. 1. Dose–response curves for IL-1beta (䊏), IL-6 (䉱), TNF-alpha (䉲) release after whole blood cell stimulation with different LPS concentrations.
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differences between values of controls and patient subgroups. Correlation was computed with Pearson’s r test. 3. Results No difference in cytokine basal levels was found in plasma of AD patients and controls before stimulation (data not shown). To evaluate cytokine release from blood cells, pilot experiments on dose–response curves were performed using LPS concentrations ranging from 1 to 1000 ng/ml. Fig. 1 shows a typical dose–response curve for IL-1beta, IL-6 and TNF-alpha release from whole blood cells of normal control. Based on these results, our studies were performed using
Fig. 2. IL-1beta (a), IL-6 (b) and TNF-alpha (c) release by LPS-stimulated whole blood from normal controls (NC) and AD patients. MILD: mild AD patients, MOD: moderate AD patients; SEV: severe; AD patients. (a) ∗ P < 0.05 vs. NC, § P < 0.01 vs. MILD; (b) ◦ P < 0.05 vs. NC and MILD.
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100 ng/ml LPS concentration, which was found to be the minimal dose able to induce the optimal response. To verify consistency of the assay results, blood samples were taken from a subgroup of AD patients and controls after 2 month interval. Similar results were obtained from the same subjects, confirming the reliability of the assay. In AD patients a similar pattern of altered secretion was observed for all investigated cytokines, related to disease severity. While cytokine basal release was found similar in controls and in patient subgroups (data not shown), LPS-induced release was similar to controls in mild and moderate AD patients and reduced in severe demented patients. Fig. 2 shows the IL-1beta (a), IL-6 (b) and TNF-alpha (c) release by LPS-stimulated whole blood of normal controls and AD patient subgroups. A significant decrease of IL-1beta (P < 0.05) and IL-6 (P < 0.05) release was observed in severe AD patients. TNF-alpha release was also decreased in severe AD patients, but the difference did not reach significance. A significant correlation was observed between cytokine release and MMSE score of AD patients (r = 0.62, P < 0.0005 for IL-1beta; r = 0.53, P < 0.005 for IL-6; r = 0.41, P < 0.05 for TNF-alpha, Fig. 3). We also observed a
Fig. 3. Regression lines between MMSE score and cytokine release in AD patients. (a) r = 0.62, P < 0.0001; (b) r = 0.53, P < 0.005; (c) r = 0.41, P < 0.05.
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Fig. 4. Regression lines among cytokine release in AD patients. (a) r = 0.48, P < 0.01; (b) r = 0.17, p = ns; (c) r = 0.76, P < 0.0001.
high positive correlation among the cytokine secretions with the exception of TNF-alpha versus IL-1beta (Fig. 4). No correlation between white blood cell count and cytokine release was observed both in patients and controls. Since previous studies showed changes in hypothalamic– pituitary–adrenal axis function in AD patients, entailing increased cortisol levels which might be involved in immune depression [23], we evaluated cortisol circulating levels. Both patients and controls showed hormonal values in normal range.
4. Discussion In the present study we found, for the first time, a similar pattern of decreased IL-1beta, IL-6 and TNF-alpha release from LPS-stimulated PBC of severe AD patients, indicating the existence of a possible down-regulation of the peripheral immune response in the late stages of the disease. Although cytokine release in mild and moderate AD patients was similar to control values, the positive correlations observed in AD patients between IL-1beta, IL-6 and TNF-alpha release and MMSE scores suggest that cytokine production progressively decreases according to disease severity. To date, data on peripheral cytokines in AD patients are controversial, probably because each study used a different
methodological approach to evaluate cytokine release and different criteria to subgroup AD patients according to disease severity. Unaltered IL-1beta peripheral secretion in AD patients versus controls, after 6 h stimulation with 10 g/ml LPS, and a significant reduction of TNF-alpha release in moderate AD patients (MMSE score: 17±4) after 96 h incubation in the presence of 10 g/ml PHA were observed [15]. However, increased production of IL-1beta, TNF-alpha, IL-6 and IL-10 by LPS-stimulated whole blood of AD patients was also described [18]. These latter results seem in contrast with our findings, but, more recently, it has been shown that TNF-alpha release from blood cells is significantly decreased (−27%) in demented patients compared to controls [6]. Mechanisms involved in peripheral immune alterations in AD patients are presently under investigation. A peripheral immune alteration in AD patients might be triggered by brain immune activation, given the existence of inflammatory signaling pathway from the brain to the periphery [7]. Recently, intracerebroventricular injection of Abeta 1–42 in mice caused an IL-6 increase not only in brain, but also in plasma, demonstrating that centrally administered Abeta 1–42 effectively induces the systemic IL-6 response [27]. Thus, peripheral immune alterations might be linked to progressive deposition of Abeta 1–42 within the CNS [8]. Alternatively, circulating Abeta might directly affect immune functions. Although most authors described unchanged Abeta 1–42 levels in plasma of sporadic AD patients [28], recently, a longitudinal study on elderly controls showed that plasma Abeta generally rises with aging, suggesting that it may be elevated long before AD develops and subsequently decreases in AD patients [11]. Supporting this hypothesis, it is known that plasma Abeta declines when Abeta is deposited in brains of transgenic mouse model of AD [16]. Increased basal IL-6 levels in plasma have also been recently shown to be a risk for subsequent decline in cognitive functions [32]. Our findings, showing a significantly decreased release of the three major pro-inflammatory cytokines in severe AD patients, might reflect a decline in plasma Abeta levels or might be interpreted as a down-regulation of PBC, exposed to a chronic Abeta stimulus long before the onset of symptoms. While basal cytokine secretion was similar in patients and controls, a pro-inflammatory stimulus, such as LPS, was less effective in triggering cytokine release in severely demented patients. Accordingly, an association between the chronic expression of human amyloid precursor protein and a decrease in the immune response to human Abeta has been found in transgenic mice [22]. Chronic exposure of the immune system to Abeta in humans and in mouse models might lead to hyporesponsiveness in terms of cellular and humoral immune responses to Abeta itself, and this alteration could contribute to Abeta deposition responsible for AD. Furthermore, the mechanism of clearance of potentially amyloidogenic substance has been shown to be impaired in
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AD patients, which possess decreased lymphocyte autoreactivity against metabolic products of APP [31]. A strict link between hormonal pathway and immune system has also been established [33]. Some studies showed abnormalities at several levels of the hypothalamic– pituitary–adrenal (HPA) axis in patients with AD [23,26]. We investigated cortisol levels as one possible marker of HPA dysfunction and we did not find any alteration. Then, the impaired immune response found in severe AD patients could not be ascribed to hormonal dysregulation. In conclusion, the present study suggests the possibility to correlate impaired cytokine peripheral release and clinical conditions of AD patients, with a progressive impairment of immune functions in this disorder. This hypothesis might also explain, at least in part, some clinical data showing that severe AD patients have increased risk to be hospitalized because of infectious diseases independently of age, gender, education, comorbid medical conditions respect to both mild or moderate AD and controls [2]. In fact, a greater risk of medical complications that require hospital care, especially infections, appears to be characteristic of severe AD [2]. Finally, our study suggests the possibility to use peripheral cytokine release as a possible tool to further characterize immune dysfunction during AD progression.
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