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Increased serum α1-antichymotrypsin in patients with probable Alzheimer's disease: an acute phase reactant without the peripheral acute phase response
Journal of Neuroimmunology
ELSEVIER
Journal of Neuroimmunology 57 (1995) 71-75
Increased serum qantichymotrypsin in patients with probable Alzheimer’s disease: an acute phase reactant without the peripheral acute phase response Federico Licastro
*, Maria
Cristina Morini, Elisabetta Polazzi, Lizabeth Jane Davis
Department of Experimental Pathology, University of Bologna, via S. Giacomo 14, I-40126 Bologna, Italy
Received 8 July 1994; revised 11 October 1994; accepted 12 October 1994
Abstract Serum levels of c-u,-antichymotrypsin (al-ACT) were measured in patients with early and late onset Alzheimer’s disease (e-AD, l-AD), patients with vascular dementia (VD) and healthy elderly. Patients with l-AD were divided into two groups, one had normal a,-ACT values and one had increased serum levels of cx,-ACT. Other acute phase proteins were also measured. The serum levels of a,-macroglobulin (a,-MG), n,-antitxypsin (al-AT), ceruloplasmin (CER), transferrin (TRSF) and cw,-acid glycoprotein (a,-ac.GL) were within the normal range. The C reactive protein (CRP) was occasionally detectable at low concentrations in e-AD, in both groups of I-AD patients and in VD patients. Low serum concentrations of interleukin-6 (IL-6) were found in a higher proportion of l-AD than in patients with e-AD or VD. These results indicated that increased levels of
a,-ACT Keywords:
along with occasional detection of IL-6 might be peripheral
markers of the ‘acute reaction’ in the brain.
Alzheimer’s disease; Interleukin-6; Acute phase proteins
1. Introduction
The dementia of Alzheimer’s type is a common cause of progressive and irreversible impairment of cognitive and intellectual functions (Katzman, 1986). The /3-amyloid deposition (Miyakawa et al., 1982) and the formation of paired helical filaments (Wisniewski et al., 1976) are the characteristic neuropathologic alterations associated with Alzheimer’s disease (AD). Since P-amyloid deposits also occur in the brain of non-demented elderly (Tomlinson et al., 19681, it has been hypothesized that the senescence of the brain might be accelerated in patients with AD. The @amyloid protein CAP) is the main component of the proteinaceous deposits in brain lesions of AD patients (Masters et al., 1985; Joachim et al., 1988) and derives from a proteolytic processing of a membrane protein named amyloid precursor protein (APPP) (Kang et al., 1987).
’ Corresponding 251315.
author. Phone (+39-51) 242084. Fax (+39-51)
Elsevier Science B.V. SSDI 0165-5728(94)00163-4
Other proteins, such as protease inhibitors (Abraham, 1992) and complement components (McGeer and McGeer, 1992), have been found in amyloid deposits and it has been proposed that an ‘acute reaction’ in the brain of patients with dementia of Alzheimer’s type could play a role in the pathogenesis of the disease (Abraham, 1992; Abraham et al., 1993). It is of interest that a serine protease inhibitor called cu,-antichymotrypsin (a,-ACT) is commonly found associated with AP in brain lesions (Abraham et al., 1988). Furthermore, this serpin has been suggested to be involved in the impaired proteolytic processing of ApPP which might lead to the formation of amyloidogenie AP and plaque deposition (Abraham, 1992). Levels of a,-ACT or its inhibitory activity have been reported to be specifically increased in CSF from AD patients (Matsubara et al., 1990; Licastro et al., 1994a). Furthermore, an increment of serum al-ACT has been also found in these patients (Matsubara et al., 1990; Brugge et al., 1992; Licastro et al., 1994b; Licastro et al., 1994~) and it has been suggested that this inhibitor could be used as a marker for a subgroup of patients with AD (Matsubara et al., 1990).
12
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
Generally speaking, the physiological function of serum (-u,-ACT is not well established, however this inhibitor suppresses the activity of some proteases, such as neutrophil cathepsin G and mast cell chymase (Travis and Salvesen, 1983). Serum a,-ACT is a glycoprotein with a molecular mass of 68 kDa; the compound belongs to the supergene family of serine protease inhibitors and is synthesized and secreted by the liver during the acute phase response in inflammation along with the other hepatic acute phase reactants (Travis and Salvesen, 1983). The serpin a,-ACT may be produced by other cells, since histiocytes, astrocytes, endothelial, epithelial and mast cells contain this protease inhibitor (Papadimitriou et al., 1980; Travis and Salvesen, 1983; Abraham, 1992). The origin and functions of serum (Y,-ACT in patients with AD are still unknown, and a possible role of this serpin in the pathogenesis of AD has not been defined. In the present report we have investigated whether in patients with different forms of dementia increased serum levels of al-ACT could be associated with the presence of serum IL-6 and other serum acute phase reactants.
2. Subjects and methods 2.1. Subjects
Eight patients with probable e-AD (mean age 6.5 + 2 years), 22 patients with I-AD (mean age 73 k 1 years) and 15 patients with VD (mean age 75 + 1 years) were studied. Clinical diagnosis of AD was performed according to the recommendations of DSM III-R and the NINCDS-ADRDA criteria (McKann et al., 1984). None of the demented patients had familial AD. The VD was defined according to the DSM-IJIR and the Hachinski ischemic score. Biochemical and metabolic parameters, ECG, chest X-ray, computerized brain tomography and hormonal status were evaluated to exclude other causes of dementia. Cognitive performances of demented patients were assessed using the Mini Mental State Examination (MMSE) (Folstein et al., 1975). None of the patients showed any clinical signs of inflammatory conditions. A case of l-AD with high body temperature and increased erythro-sedimentation rate was inserted as a positive case control. Controls consisted of 16 non-demented healthy elderly (mean age 76 + 1 years) and 8 young adults (mean age 37 + 2 years) without neurological or inflammatory alterations. 2.2. Serum collection Sera were obtained from blood samples collected in sterile tubes and centrifuged at 400 x g for 10 min.
2.3. a,-ACT determination Levels of (Y,-ACT in sera from patients and controls were measured by a competitive ELISA as previously described (Licastro et al., 1994b). Briefly, 96-well microtiter plates were coated with 100 ~1 of (Y,-ACT (Calbiochem) at a concentration of 1 pg/ml in 0.1 M bicarbonate/carbonate buffer, pH 9.6. After a overnight incubation at 4°C the antigen-coated plates were washed and blocked by adding 100 ~1 of Dulbecco’s buffer (DBSS, pH 7.4) containing 1% BSA for 30 min at room temperature. Twofold serial dilutions of a,-ACT or serum samples were then incubated with a rabbit anti-human a,-ACT antiserum (Calbiochem) for 90 min at 37°C. After washing three times, a horseradish peroxidase-conjugated anti-rabbit IgG (Boehringer-Mannheim) was added, the enzymatic reaction was developed for 15 min by adding the peroxidase’s substrate 2,2’-azino-di-[3-ethylbenz-thiazoline sulphonate] (ABTS, Boehringer-Mannheim) and the absorbance was measured at 405 nm. 2.4. Acute phase reactants measurement Serum levels of (u,-MG, cyi-AT, CER, CRP, TRSF and cu,-ac.GL were detected by commercial single radial immunodiffusion plates (Behring Institute, Italy). Briefly, 5 ~1 of standard serum (Behring Institute, Italy) or sera from demented and non demented subjects were plated and incubated at room temperature for 48 h. The calibration of each immunodiffusion plate was performed using a standard serum. The diameter of the precipitate was measured using a calibrated viewer and concentrations of acute phase reactants in serum samples were calculated according to the reference values from the manufacturer. 2.5. IL-6 detection Serum concentration of IL-6 was determined by a commercial ELISA (Inalco, Italy) specific for human IL-6. Recombinant IL-6 was used as a positive control in the standard curve.
3. Results The mean value of serum (w,-ACT from patients with l-AD (71 + 5 mg/dl) was statistically higher than that from both healthy elderly controls (39 f 5 mg/dl; P < 0.001) and the control disease, i.e. patients with VD (46 f 4 mg/dl; P < 0.001). Patients with e-AD also showed higher values of this inhibitor (68 f 7 mg/dl) than controls (P < 0.001) and patients with VD (P < 0.005). Two populations could be easily identified among l-AD patients where 9 patients out of 22 showed
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
73
Table 2 Serum levels of IL-6 from patients and healthy controls
I-AD B I-AD h e-AD VD Elderly controls Young controls
* _____ _________________ e-AD
IL-(i-positive
% of positivity
219 5/13
22 38 0 13 15 0
O/7 2/15 2/13 O/8
with increased serum levels of at-ACT ( > 67 mg/dl). with normal serum levels of ol,-ACT ( < 67 mg/dl).
VD
l-AD
Fig. 1. Serum levels of at-ACT in patients with different types of dementia. The area between the dotted lines represents the normal range calculated from 10 healthy elderly (mean values + 2 SD.).
serum levels of &,-ACT over the upper limit of the normal range; mean value 107 &-7 mg/dl (Fig. 1). Two patients with e-AD out of 8 and 1 VD patient out of 13 were outside the normal range for (u,-ACT. The normal range of the serpin (12-67 mg/dl) was defined by calculating 2 standard deviations of mean values obtained in 10 healthy elderly. Levels of c~,-MG, a,-AT, CER, TRSF and a,-ac.GL from l-AD patients with high serum level of CX,-ACTwere comparable to those from l-AD group with normal serum levels of cw,-ACT, patients with e-AD, VD and healthy elderly (Table 1). Serum CRP was sporadically detectable in patients with different forms of dementia (range values 0.9-2.9 mg/dl). One case of l-AD with clinical signs of acute inflammation showed increased serum a,-ACT (125 mg/dl), a,-MG, a,-AT, CRP and decreased TRSF, thus showing the classical peripheral acute phase response. IL-6 was detectable in none of the young controls and in two healthy elderly out of 13 where the maximum value was 7 pg/ml. Only demented patients with
1 phase
forms of dementia
I
01
Table Acute
a Patients b Patients
with different
proteins
in sera from patients
(Yz-MG (mg/dl) f f f +
23 10 28 53
I-AD a l-AD b e-AD VD
194 178 221 242
Elderly d I-AD ’
218 + 20 250
with different
at-AT (mg/dl) 191 195 201 178
+ + f +
13 18 21 30
209 + 16 440
forms of dementia
serum levels higher than 7 pg/ml were considered positive for serum IL-6 (Table 2). Patients with l-AD showed a higher percentage of positivity for this cytokine than patients with e-AD or VD. However, the levels of this cytokine were relatively low, i.e. 28-36 pg/ml in l-AD patients with high serum of a,-ACT and 12-56 pg/ml in the other group of l-AD patients and no statistical difference was present among different groups of patients according to the analysis of x2. Serum IL-6 was also measured in sera from young controls and was not detectable. No statistically significant correlation between age or sex and serum IL-6 levels was found in controls or patients with dementia (data not shown). The l-AD patient with clinical signs of infection and a peripheral acute phase response showed high levels of serum IL-6 (117 pg/ml).
4. Discussion Acute phase reactants belong to a heterogeneous family of proteins released from the liver into the circulation during acute inflammatory states (Laurel1 and Jeppsson, 1975). The protease inhibitor (Y,-ACT is one of these compounds and its serum levels increase
and healthy
CER (mg/dl)
CRP
25 22 26 25
i f F k
2 2 1 2
30 f 2 _
L Nine patients with increased serum levels of (Y,-ACT (> 67 mg/dl). Thirteen patients with normal serum levels of at-ACT (< 67 mg/dl). ’ Subjects with detectable serum CRP (range 0.9-2.9 mg/dl). d Data were obtained from 13 healthy elderly. ’ One I-AD patient with fever and high erythro-sedimentation rate. ’ mg/dl.
elderly
(%o)
TRSF (mg/dl)
a,-ac.GL (mg/dl)
3/9 c 4/13 2/8 3/7
(27) (31) (25) (43)
187 f 21 173 f 13 179 f 7 _
62+ 11 65 f 9 56 f 6 _
O/16 16 f
(0) _
191 f 10 59
65 f 5 _
74
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
rapidly during acute inflammation (Travis and Salvesen, 1983). The production and release of acute phase reactants have been shown to be regulated by cytokines released from cells of the immune system (Heinrich et al., 1990). In man, IL-6 appeared to be the major activator of the hepatic acute phase response, since this cytokine stimulates the production of CRP, serum amyloid A, haptoglobin, al-ACT, fibrinogen, a,-AT, a,-ac.GL, CER, and complement factor B (Andus et al., 1991). Moreover, IL-6 also inhibits the synthesis of albumin, TRSF, fibronectin, a-fetoprotein and complement factor 3 (Andus et al., 1991). Other cytokines such as interleukin-1 (IL-l), tumor necrosis factor-a and leukemia inhibitory factor have also been shown to affect the liver response during the acute phase of inflammation, but they appeared to act upon a restricted spectrum of acute phase proteins (Andus et al., 1991). Reactive astrocytes from virally infected brain (Frei et al., 1989) and those surrounding neuropathological lesions of AD patients (Bauer et al., 1992) have been shown to express the IL-6 mRNA and secrete the cytokine. Furthermore, endothelial cells could also synthesize IL-6 (Jirik et al., 1989). The production of IL-6 in both cell types was induced by IL-1 (Sironi et al., 1989 Bauer et al., 1992). Cells of extrahepatic origin have been shown to release a,-ACT. For instance, reactive astrocytes (Abraham et al., 1993) or endothelial cells (Papadimitriou et al., 1980) contained and released this serpin. In this study we confirmed previous observations showing increased serum levels of the serpin in sera from l-AD patients. However, only a proportion of these patients showed such an increment, as also reported by others (Matsubara et al., 1990; Brugge et al., 1992). Furthermore, our findings showed that an acute phase reaction was not detectable in l-AD patients with elevated serum a,-ACT. Present data and previously published ones (Licastro et al., 1994b) did not confirm those from Giometto et al. (1988) who showed an increment of serum CER and C3 and C4 complement factors. We found a sporadic positivity of sera from l-AD patients for CRP. However, this observation did not support the presence of a peripheral acute phase response, since the concentration of CRP was low. The presence of this compound was not specific, since a weak positivity was also present in a few l-AD patients with normal serum levels of a,-ACT and in VD patients. Moreover, the patient with l-AD showing clinical signs of inflammation had serum levels of CRP lo-times higher than those of the other l-AD patients and altered levels of the acute phase proteins. IL-6 was more often detectable in sera from l-AD
patients than in patients with e-AD or VD and in healthy elderly. However serum IL-6 levels were low and far below those detected during an acute inflammation, as we found in the l-AD patient with clinical signs of inflammation. These data partially paralleled those from a different study showing that mean serum levels of IL-6 in patients with AD were not elevated (Van Duijn et al., 1990). Serum IL-6 was not detectable in young healthy controls, while healthy elderly showed a slightly increased percentage of positivity for this cytokine. These findings are also partially in accordance with a recent report showing a small increase of serum IL-6 with advancing age in man (Ershler, 1993). It is of interest that serum IL-6 was not detectable in sera from patients with e-AD. Thus, an age factor might affect the presence of this cytokine in AD. However, we did not find any statistically significant correlation between serum IL-6 and age in demented patients or controls. It was unlikely that serum IL-6 in patients with dementia originated from cells of the immune system, since serum concentrations of this cytokine were low and an acute phase response was not present. This notion is reinforced by previous findings (Licastro et al., 1994~) showing that in AD patients cells of reticuloendothelial system were not activated, since normal serum levels of neopterin were found in these patients. In conclusion, our findings suggested that the hepatic origin of serum al-ACT in l-AD patients without clinical signs of peripheral acute inflammation was unlikely. The source of serum IL-6 also appeared to be outside of the peripheral immune system. Endothelial cells or perivascular microglial cells from vessels surrounding the senile amyloid plaques might be possible candidates. These cells may release both factors in response to the acute reaction localized around the cerebral amyloid deposits where cytokines (IL-l and IL-61, along with other acute phase reactants, are released by activated astrocytes and microglia (Rozemuller et al., 1992). Thus, increased serum levels of this protease inhibitor along with detectable IL-6 might represent peripheral signs of the hypothesized brain acute reaction in these patients. These peripheral markers might be helpful for monitoring the clinical history of the disease and useful for focusing therapeutical intervention. In fact, patients with l-AD showing increased serum (u,-ACT and IL-6 might benefit most of the anti-inflammatory treatment recently proposed (McGeer and Rogers, 1992).
Acknowledgements
This study was supported MURST 40 and 60%.
by grants from Italian
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
References Abraham, C.R., Selkoe, D.J. and Potter, H. (1988) Immunochemical identification of the serine protease inhibitor alpha-lantichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52, 487-501. Abraham, C.R. (1992) The role of acute-phase protein (pi- antichymotrypsin in brain dysfunction and injury. Res. Immunol. 143, 631-636. Abraham, CR., Kanemaru, K. and Mucke, L. (1993) Expression of cathepsin G-like and a,-antichymotrypsin-like protein in reactive astrocytes. Brain Res. 621, 222-232. Andus, T., Bauer. J. and Gerok W. (1991) Effects of cytokines on the liver. Hepatology 13, 364-375. Bauer, J., Ganter. U., Strauss, S., Stadtmuller, G., Frommberger, U., Bauer, H., Volk, B. and Berger, M. (1992) The participation of IL-6 in the pathogenesis of Alzheimer’s disease. Res. Immunol. 143, 650-657. Brugge, K., Katzman, R., Hill, L.R., Hansen, L.A. and Saitoh, T. (1992) Serological cY,-antichymotrypsin in Down’s syndrome and Alzheimer’s disease. Ann. Neurol. 32, 193-107 Ershler, W.B. (1993) Interleukin-6: a cytokine for gerontologist. J. Am. Geriatr. Sot. 41, 176-181. Folstein, M.F., Folstein, S.E. and McHugh, P.R. (1975) ‘Mini mental state’. A practical method for grading the cognitive state of patients for the clinician. J. Psychiatr. Res. 12, 189-198. Frei, K., Malipiero, U.V., Leist T.P., Zinkernagel R.M., Schwab, M.E. and Fontana A. (1989) On the cellular source of interleukin 6 produced in the central nervous system in viral disease. Eur. J. lmmunol. 19, 689-694. Giometto, B., Argentiero, V., Sanson, F., Ongaro, G. and Tavolato, B. (1988) Acute phase proteins in Alzheimer’s disease. Eur. Neurol. 28, 30-33. Heinrich, P.C.. Castell, J.V. and Andus, T. (1990) Interleukin-6 and the acute phase response. Biochem. J. 265, 621-636. Jirik, F.R., Podor, T.J., Hirano, T., Kishimoto, T., Loskutoff, D.J., Carson, D.A. and Lotz, M. (1989) Bacterial lipopolysaccharide and inflammatory mediators augment IL-6 secretion by human endothelial cells. J. Immunol. 142, 144-147. Joachim, C.L., Morris, J. and Selkoe, D.J. (1988) Clinically diagnosed Alzheimer’s disease: autopsy results in 150 cases. Ann. Neurol. 24,50-56. Kang, J.. Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and Muller, H.B. (1987) The precursor of Alzheimer’s disease A4 protein resembles a cell-surface receptor. Nature 325, 733-736. Katzman, R.N. (1986) Medical progress: Alzheimer’s disease. N. Engl. J. Med. 374, 964-973. Laurell, C.B. and Jeppsson, J.O. (1975) Protease inhibitor in plasma. In: The Plasma Proteins: Structure, Function and Genetic Control. Vol. 1, 2nd ed., (Putman, F.W., Ed.) Academic Press, NY, pp. 229-264. Licastro, F., Davis, L.J., Morini M.C., Cucinotta, D. and Savorani, G. (1994a) Cerebrospinal fluid of patients with senile dementia of
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Alzheimer’s type shows an increased inhibition of a-chymotrypsin. Alzheimer Dis. Assoc. Dis. 8, 241-249. Licastro, F., Morini M.C., Davis L.J., Malpassi P., Cucinotta, D., Parente, R., Melotti, C. and Savorani, G. (1994b) Increased chemiluminescence response of neutrophils from the peripheral blood of patients with senile dementia of Alzheimer’s type. J. Neuroimmunol. 51, 21-26. Licastro, F., Parnetti, L., Morini, M.C., Davis J.L., Cucinotta, D., Gaiti, A. and Senin, U. (1994~) The acute phase reactant cu,-antichymotrypsin is increased in cerebrospinal fluid and serum of patients with probable Alzheimer’s disease. Alzheimer Dis. Assoc. Dis. (in press). Masters, C.L., Simms, G., Weinmann, N.A., Multhaup, CT., McDonald, B.L. and Beyreuther, K. (1985). Amyloid plaque core protein in Alzheimer’s disease and Down’s syndrome. Proc. Natl. Acad. Sci. USA 82, 4245-4249. Matsubara, E., Hirai, S., Amari, M., Shoji. M., Yamaguchi, H. and Okamoto, K. (1990) Ishiguro K, Harigaya Y, Wakabayashi K. cu-1-Antichymotrypsin as a possible biochemical marker for Alzheimer-type dementia. Ann. Neurol. 28, 561-567. McGeer, P.L. and Rogers, J. (1992) Anti-inflammatory agents as a therapeutic approach to Alzheimer’s disease. Neurology 42, 447449. McGeer, P.L. and McGeer, E.G. (1992) Complement proteins and complement inhibitors in Alzheimer’s disease. Res. Immunol. 143, 621-624. McKann. G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E.M. (1984) Clinical diagnosis of Alzheimer’s disease: report of NINCDS-ADRDA work group under the auspices of Department of Health and Human Service Task Force on Alzheimer’s disease. Neurology 34, 939-944. Miyakawa, T.. Shimoji, A., Kuramoto, R. and Higuchi, Y. (1982) The relationship between senile plaques and cerebral blood vessels in Alzheimer’s disease and senile dementia. Virchows Arch. 40. 121-129. Papadimitriou, C.S., Stein, H. and Papacharalampous, N.X. (1980) Presence of u,-antichymotrypsin in hemopoietic and lymphoid tissue cells are revealed by immunoperoxidase method. Pathol. Res. Pratt. 169, 287-297. Rozemuller, J.M., van der Valk, P. and Eikelenboom, P. (1992) Activated microglia and cerebral amyloid deposits in Alzheimer’s disease. Res. Immunol. 143, 646-649. Sironi, M., Breviario, F., Proserpio. P.. Biondi, A., Vecchi, A., Damme, J.V., Dejane, E. and Mantovani, A. (19891 IL-l stimulates IL-6 production in endothelial cells. J. Immunol. 142, 549553. Tomlinson, B.E., Blessed, G. and Roth, M. (1968) Observations on the brains of non-demented old people. J. Neurol. Sci. 7,331-356. Travis, J. and Salvesen, G.S. (1983) Human plasma proteinase inhibitors Annu. Rev. Biochem. 52, 655-709. Van Duijn, C.M., Hofman, A. and Nagelkerken, L. (1990) Serum levels of interleukin-6 are not elevated in patients with Alzheimer’s disease. Neurosci. Lett. 108. 350-354. Wisniewski, H.M., Narang, H.K. and Terry. R.D. (19761 Neurofibrillary tangles of paired helical filaments. J. Neural. Sci. 27, 173-181.
ELSEVIER
Journal of Neuroimmunology 57 (1995) 71-75
Increased serum qantichymotrypsin in patients with probable Alzheimer’s disease: an acute phase reactant without the peripheral acute phase response Federico Licastro
*, Maria
Cristina Morini, Elisabetta Polazzi, Lizabeth Jane Davis
Department of Experimental Pathology, University of Bologna, via S. Giacomo 14, I-40126 Bologna, Italy
Received 8 July 1994; revised 11 October 1994; accepted 12 October 1994
Abstract Serum levels of c-u,-antichymotrypsin (al-ACT) were measured in patients with early and late onset Alzheimer’s disease (e-AD, l-AD), patients with vascular dementia (VD) and healthy elderly. Patients with l-AD were divided into two groups, one had normal a,-ACT values and one had increased serum levels of cx,-ACT. Other acute phase proteins were also measured. The serum levels of a,-macroglobulin (a,-MG), n,-antitxypsin (al-AT), ceruloplasmin (CER), transferrin (TRSF) and cw,-acid glycoprotein (a,-ac.GL) were within the normal range. The C reactive protein (CRP) was occasionally detectable at low concentrations in e-AD, in both groups of I-AD patients and in VD patients. Low serum concentrations of interleukin-6 (IL-6) were found in a higher proportion of l-AD than in patients with e-AD or VD. These results indicated that increased levels of
a,-ACT Keywords:
along with occasional detection of IL-6 might be peripheral
markers of the ‘acute reaction’ in the brain.
Alzheimer’s disease; Interleukin-6; Acute phase proteins
1. Introduction
The dementia of Alzheimer’s type is a common cause of progressive and irreversible impairment of cognitive and intellectual functions (Katzman, 1986). The /3-amyloid deposition (Miyakawa et al., 1982) and the formation of paired helical filaments (Wisniewski et al., 1976) are the characteristic neuropathologic alterations associated with Alzheimer’s disease (AD). Since P-amyloid deposits also occur in the brain of non-demented elderly (Tomlinson et al., 19681, it has been hypothesized that the senescence of the brain might be accelerated in patients with AD. The @amyloid protein CAP) is the main component of the proteinaceous deposits in brain lesions of AD patients (Masters et al., 1985; Joachim et al., 1988) and derives from a proteolytic processing of a membrane protein named amyloid precursor protein (APPP) (Kang et al., 1987).
’ Corresponding 251315.
author. Phone (+39-51) 242084. Fax (+39-51)
Elsevier Science B.V. SSDI 0165-5728(94)00163-4
Other proteins, such as protease inhibitors (Abraham, 1992) and complement components (McGeer and McGeer, 1992), have been found in amyloid deposits and it has been proposed that an ‘acute reaction’ in the brain of patients with dementia of Alzheimer’s type could play a role in the pathogenesis of the disease (Abraham, 1992; Abraham et al., 1993). It is of interest that a serine protease inhibitor called cu,-antichymotrypsin (a,-ACT) is commonly found associated with AP in brain lesions (Abraham et al., 1988). Furthermore, this serpin has been suggested to be involved in the impaired proteolytic processing of ApPP which might lead to the formation of amyloidogenie AP and plaque deposition (Abraham, 1992). Levels of a,-ACT or its inhibitory activity have been reported to be specifically increased in CSF from AD patients (Matsubara et al., 1990; Licastro et al., 1994a). Furthermore, an increment of serum al-ACT has been also found in these patients (Matsubara et al., 1990; Brugge et al., 1992; Licastro et al., 1994b; Licastro et al., 1994~) and it has been suggested that this inhibitor could be used as a marker for a subgroup of patients with AD (Matsubara et al., 1990).
12
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
Generally speaking, the physiological function of serum (-u,-ACT is not well established, however this inhibitor suppresses the activity of some proteases, such as neutrophil cathepsin G and mast cell chymase (Travis and Salvesen, 1983). Serum a,-ACT is a glycoprotein with a molecular mass of 68 kDa; the compound belongs to the supergene family of serine protease inhibitors and is synthesized and secreted by the liver during the acute phase response in inflammation along with the other hepatic acute phase reactants (Travis and Salvesen, 1983). The serpin a,-ACT may be produced by other cells, since histiocytes, astrocytes, endothelial, epithelial and mast cells contain this protease inhibitor (Papadimitriou et al., 1980; Travis and Salvesen, 1983; Abraham, 1992). The origin and functions of serum (Y,-ACT in patients with AD are still unknown, and a possible role of this serpin in the pathogenesis of AD has not been defined. In the present report we have investigated whether in patients with different forms of dementia increased serum levels of al-ACT could be associated with the presence of serum IL-6 and other serum acute phase reactants.
2. Subjects and methods 2.1. Subjects
Eight patients with probable e-AD (mean age 6.5 + 2 years), 22 patients with I-AD (mean age 73 k 1 years) and 15 patients with VD (mean age 75 + 1 years) were studied. Clinical diagnosis of AD was performed according to the recommendations of DSM III-R and the NINCDS-ADRDA criteria (McKann et al., 1984). None of the demented patients had familial AD. The VD was defined according to the DSM-IJIR and the Hachinski ischemic score. Biochemical and metabolic parameters, ECG, chest X-ray, computerized brain tomography and hormonal status were evaluated to exclude other causes of dementia. Cognitive performances of demented patients were assessed using the Mini Mental State Examination (MMSE) (Folstein et al., 1975). None of the patients showed any clinical signs of inflammatory conditions. A case of l-AD with high body temperature and increased erythro-sedimentation rate was inserted as a positive case control. Controls consisted of 16 non-demented healthy elderly (mean age 76 + 1 years) and 8 young adults (mean age 37 + 2 years) without neurological or inflammatory alterations. 2.2. Serum collection Sera were obtained from blood samples collected in sterile tubes and centrifuged at 400 x g for 10 min.
2.3. a,-ACT determination Levels of (Y,-ACT in sera from patients and controls were measured by a competitive ELISA as previously described (Licastro et al., 1994b). Briefly, 96-well microtiter plates were coated with 100 ~1 of (Y,-ACT (Calbiochem) at a concentration of 1 pg/ml in 0.1 M bicarbonate/carbonate buffer, pH 9.6. After a overnight incubation at 4°C the antigen-coated plates were washed and blocked by adding 100 ~1 of Dulbecco’s buffer (DBSS, pH 7.4) containing 1% BSA for 30 min at room temperature. Twofold serial dilutions of a,-ACT or serum samples were then incubated with a rabbit anti-human a,-ACT antiserum (Calbiochem) for 90 min at 37°C. After washing three times, a horseradish peroxidase-conjugated anti-rabbit IgG (Boehringer-Mannheim) was added, the enzymatic reaction was developed for 15 min by adding the peroxidase’s substrate 2,2’-azino-di-[3-ethylbenz-thiazoline sulphonate] (ABTS, Boehringer-Mannheim) and the absorbance was measured at 405 nm. 2.4. Acute phase reactants measurement Serum levels of (u,-MG, cyi-AT, CER, CRP, TRSF and cu,-ac.GL were detected by commercial single radial immunodiffusion plates (Behring Institute, Italy). Briefly, 5 ~1 of standard serum (Behring Institute, Italy) or sera from demented and non demented subjects were plated and incubated at room temperature for 48 h. The calibration of each immunodiffusion plate was performed using a standard serum. The diameter of the precipitate was measured using a calibrated viewer and concentrations of acute phase reactants in serum samples were calculated according to the reference values from the manufacturer. 2.5. IL-6 detection Serum concentration of IL-6 was determined by a commercial ELISA (Inalco, Italy) specific for human IL-6. Recombinant IL-6 was used as a positive control in the standard curve.
3. Results The mean value of serum (w,-ACT from patients with l-AD (71 + 5 mg/dl) was statistically higher than that from both healthy elderly controls (39 f 5 mg/dl; P < 0.001) and the control disease, i.e. patients with VD (46 f 4 mg/dl; P < 0.001). Patients with e-AD also showed higher values of this inhibitor (68 f 7 mg/dl) than controls (P < 0.001) and patients with VD (P < 0.005). Two populations could be easily identified among l-AD patients where 9 patients out of 22 showed
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
73
Table 2 Serum levels of IL-6 from patients and healthy controls
I-AD B I-AD h e-AD VD Elderly controls Young controls
* _____ _________________ e-AD
IL-(i-positive
% of positivity
219 5/13
22 38 0 13 15 0
O/7 2/15 2/13 O/8
with increased serum levels of at-ACT ( > 67 mg/dl). with normal serum levels of ol,-ACT ( < 67 mg/dl).
VD
l-AD
Fig. 1. Serum levels of at-ACT in patients with different types of dementia. The area between the dotted lines represents the normal range calculated from 10 healthy elderly (mean values + 2 SD.).
serum levels of &,-ACT over the upper limit of the normal range; mean value 107 &-7 mg/dl (Fig. 1). Two patients with e-AD out of 8 and 1 VD patient out of 13 were outside the normal range for (u,-ACT. The normal range of the serpin (12-67 mg/dl) was defined by calculating 2 standard deviations of mean values obtained in 10 healthy elderly. Levels of c~,-MG, a,-AT, CER, TRSF and a,-ac.GL from l-AD patients with high serum level of CX,-ACTwere comparable to those from l-AD group with normal serum levels of cw,-ACT, patients with e-AD, VD and healthy elderly (Table 1). Serum CRP was sporadically detectable in patients with different forms of dementia (range values 0.9-2.9 mg/dl). One case of l-AD with clinical signs of acute inflammation showed increased serum a,-ACT (125 mg/dl), a,-MG, a,-AT, CRP and decreased TRSF, thus showing the classical peripheral acute phase response. IL-6 was detectable in none of the young controls and in two healthy elderly out of 13 where the maximum value was 7 pg/ml. Only demented patients with
1 phase
forms of dementia
I
01
Table Acute
a Patients b Patients
with different
proteins
in sera from patients
(Yz-MG (mg/dl) f f f +
23 10 28 53
I-AD a l-AD b e-AD VD
194 178 221 242
Elderly d I-AD ’
218 + 20 250
with different
at-AT (mg/dl) 191 195 201 178
+ + f +
13 18 21 30
209 + 16 440
forms of dementia
serum levels higher than 7 pg/ml were considered positive for serum IL-6 (Table 2). Patients with l-AD showed a higher percentage of positivity for this cytokine than patients with e-AD or VD. However, the levels of this cytokine were relatively low, i.e. 28-36 pg/ml in l-AD patients with high serum of a,-ACT and 12-56 pg/ml in the other group of l-AD patients and no statistical difference was present among different groups of patients according to the analysis of x2. Serum IL-6 was also measured in sera from young controls and was not detectable. No statistically significant correlation between age or sex and serum IL-6 levels was found in controls or patients with dementia (data not shown). The l-AD patient with clinical signs of infection and a peripheral acute phase response showed high levels of serum IL-6 (117 pg/ml).
4. Discussion Acute phase reactants belong to a heterogeneous family of proteins released from the liver into the circulation during acute inflammatory states (Laurel1 and Jeppsson, 1975). The protease inhibitor (Y,-ACT is one of these compounds and its serum levels increase
and healthy
CER (mg/dl)
CRP
25 22 26 25
i f F k
2 2 1 2
30 f 2 _
L Nine patients with increased serum levels of (Y,-ACT (> 67 mg/dl). Thirteen patients with normal serum levels of at-ACT (< 67 mg/dl). ’ Subjects with detectable serum CRP (range 0.9-2.9 mg/dl). d Data were obtained from 13 healthy elderly. ’ One I-AD patient with fever and high erythro-sedimentation rate. ’ mg/dl.
elderly
(%o)
TRSF (mg/dl)
a,-ac.GL (mg/dl)
3/9 c 4/13 2/8 3/7
(27) (31) (25) (43)
187 f 21 173 f 13 179 f 7 _
62+ 11 65 f 9 56 f 6 _
O/16 16 f
(0) _
191 f 10 59
65 f 5 _
74
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
rapidly during acute inflammation (Travis and Salvesen, 1983). The production and release of acute phase reactants have been shown to be regulated by cytokines released from cells of the immune system (Heinrich et al., 1990). In man, IL-6 appeared to be the major activator of the hepatic acute phase response, since this cytokine stimulates the production of CRP, serum amyloid A, haptoglobin, al-ACT, fibrinogen, a,-AT, a,-ac.GL, CER, and complement factor B (Andus et al., 1991). Moreover, IL-6 also inhibits the synthesis of albumin, TRSF, fibronectin, a-fetoprotein and complement factor 3 (Andus et al., 1991). Other cytokines such as interleukin-1 (IL-l), tumor necrosis factor-a and leukemia inhibitory factor have also been shown to affect the liver response during the acute phase of inflammation, but they appeared to act upon a restricted spectrum of acute phase proteins (Andus et al., 1991). Reactive astrocytes from virally infected brain (Frei et al., 1989) and those surrounding neuropathological lesions of AD patients (Bauer et al., 1992) have been shown to express the IL-6 mRNA and secrete the cytokine. Furthermore, endothelial cells could also synthesize IL-6 (Jirik et al., 1989). The production of IL-6 in both cell types was induced by IL-1 (Sironi et al., 1989 Bauer et al., 1992). Cells of extrahepatic origin have been shown to release a,-ACT. For instance, reactive astrocytes (Abraham et al., 1993) or endothelial cells (Papadimitriou et al., 1980) contained and released this serpin. In this study we confirmed previous observations showing increased serum levels of the serpin in sera from l-AD patients. However, only a proportion of these patients showed such an increment, as also reported by others (Matsubara et al., 1990; Brugge et al., 1992). Furthermore, our findings showed that an acute phase reaction was not detectable in l-AD patients with elevated serum a,-ACT. Present data and previously published ones (Licastro et al., 1994b) did not confirm those from Giometto et al. (1988) who showed an increment of serum CER and C3 and C4 complement factors. We found a sporadic positivity of sera from l-AD patients for CRP. However, this observation did not support the presence of a peripheral acute phase response, since the concentration of CRP was low. The presence of this compound was not specific, since a weak positivity was also present in a few l-AD patients with normal serum levels of a,-ACT and in VD patients. Moreover, the patient with l-AD showing clinical signs of inflammation had serum levels of CRP lo-times higher than those of the other l-AD patients and altered levels of the acute phase proteins. IL-6 was more often detectable in sera from l-AD
patients than in patients with e-AD or VD and in healthy elderly. However serum IL-6 levels were low and far below those detected during an acute inflammation, as we found in the l-AD patient with clinical signs of inflammation. These data partially paralleled those from a different study showing that mean serum levels of IL-6 in patients with AD were not elevated (Van Duijn et al., 1990). Serum IL-6 was not detectable in young healthy controls, while healthy elderly showed a slightly increased percentage of positivity for this cytokine. These findings are also partially in accordance with a recent report showing a small increase of serum IL-6 with advancing age in man (Ershler, 1993). It is of interest that serum IL-6 was not detectable in sera from patients with e-AD. Thus, an age factor might affect the presence of this cytokine in AD. However, we did not find any statistically significant correlation between serum IL-6 and age in demented patients or controls. It was unlikely that serum IL-6 in patients with dementia originated from cells of the immune system, since serum concentrations of this cytokine were low and an acute phase response was not present. This notion is reinforced by previous findings (Licastro et al., 1994~) showing that in AD patients cells of reticuloendothelial system were not activated, since normal serum levels of neopterin were found in these patients. In conclusion, our findings suggested that the hepatic origin of serum al-ACT in l-AD patients without clinical signs of peripheral acute inflammation was unlikely. The source of serum IL-6 also appeared to be outside of the peripheral immune system. Endothelial cells or perivascular microglial cells from vessels surrounding the senile amyloid plaques might be possible candidates. These cells may release both factors in response to the acute reaction localized around the cerebral amyloid deposits where cytokines (IL-l and IL-61, along with other acute phase reactants, are released by activated astrocytes and microglia (Rozemuller et al., 1992). Thus, increased serum levels of this protease inhibitor along with detectable IL-6 might represent peripheral signs of the hypothesized brain acute reaction in these patients. These peripheral markers might be helpful for monitoring the clinical history of the disease and useful for focusing therapeutical intervention. In fact, patients with l-AD showing increased serum (u,-ACT and IL-6 might benefit most of the anti-inflammatory treatment recently proposed (McGeer and Rogers, 1992).
Acknowledgements
This study was supported MURST 40 and 60%.
by grants from Italian
F. Licastro et al. /Journal
of Neuroimmunology 57 (1995) 71-75
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