318
?veurosciem't, LetI~p ~, 129 ( 1991 ) .31,~ 32t~ ~?~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91 $ 03.5() ,4 DONIS 030439409100468K
NSL 07981
Elevated circulating tumor necrosis factor levels in Alzheimer's disease H o w a r d Fillit, W a n h o n g Ding, Luc Buee, Jill K a l m a n , Larry Altstiel, Brian Lawlor and Giselle Wolf-Klein The Ritter Department of Geriatrics and Adult Development, and the Departments of Medicine, Psychiatry, the Fishberg Center for Neurobiology. and the Alzheimer's Disease Research Center, Mount Sinai Medical Center, New York, N Y 10029-6574 (U.S.A .), the Parker Jewish Geriatric Center, New Hyde Park, N Y (U.S.A.) andlNSERM Unit 156, Lille (France) (Received 27 February 1991; Revised version received 20 May 1991; Accepted 2t May 1991)
Key words: Alzheimer's disease; Cytokine; Tumor necrosis factor; Immunopathology Recent investigations have demonstrated a local inflammatory response in Alzheimer's disease (AD), including microglia and cytokines. Levels of the cytokine tumor necrosis factor a (TNF-a) in sera from patients with AD and age-matched controls were measured by an enzyme-linked immunoassay and a cytotoxicity bioassay. Significantly elevated levels of TNF were found in AD sera compared to controls. Elevated circulating TNF may be derived from the local CNS inflammatory reaction in AD, and may account for some systemic manifestations of AD such as weight loss. Future studies may determine if, in the absence of complicating disorders which may elevate TNF, circulating TNF could be a marker of AD inflammatory activity.
Substantial evidence has accumulated that Alzheimer's disease (AD) is associated with a local inflammatory reaction in senile plaques [9] which may be immune-mediated [3], and includes extensive microglial invasion [13], lymphocytic infiltration, complement and cytokine deposition, including interleukin-1 [5]. Tumor necrosis factor ~ (TNF-~) [6] is a cytokine which plays an important immuno-enhancing role in the local acute and chronic inflammatory response in response to a variety of stimuli. Brain microglia elaborate both interleukin-1 and TNF [4]. TNF and other cytokines such as interleukin-1 may also have deleterious systemic effects, such as the induction of acute shock or chronic cachexia in animals [17], and have been related to weight loss and cachexia in a variety of human disorders [2, 6, 7]. We hypothesized that some patients with AD, without other illnesses, would have elevated serum TNF which resulted from an AD-associated inflammatory reaction. Sera were obtained from 3 groups of AD patients and age-matched controls, including patients from the Rockefeller University Hospital (RUH); the Parker Jewish Geriatric Center (PJGC); and the Atzheimer's Disease Research Center at Mount Sinai Medical Center (ADRC). All AD patients met NINCDS criteria for probable AD [l 1] and did not suffer from concurrent maCorrespondence: H. Fillit, Department of Geriatrics, Box 1070, Mt. Sinai Medical Center, New York, NY 10029~5574, U.S.A. Fax: (l) (212) 8609737.
jor diseases which might have contributed to cognitive dysfunction and potentially to elevated TNF levels. All of the patients were ambulatory and seen as outpatients in Clinics at the 3 sites; none were bedridden nursing home or home care patients with pressure sores, and none of the patients had active infections which also might contribute to elevated TNF levels. Controls included healthy elderly individuals and non-demented patients with neurologic and psychiatric disorders, including affective disorders, schizophrenia, and Parkinson's disease. All sera were obtained in an aseptic fashion and stored at -70°C prior to assay. Informed consent was obtained in all cases. Initially, TNF in sera from RUH subjects (24 AD patients and 9 age-matched elderly controls) was measured by an enzyme-linked immunoassay (ELISA) capture assay [12]. Only two elderly controls had elevated TNF levels (88 and 110 pg/ml). Ten of 24 AD subjects had high TNF levels (> 100 pg/ml), and 4 AD patients had TNF levels which were above 1000 pg/ml. Mean TNF levels in the controls was 36 + 36 pg/ml; mean TNF in AD patients was 348 + 590 pg/ml. Despite the high degree of variation in the AD groups (with some patients having normal values and some with markedly elevated values), differences between groups were significant It-test for independent samples, two-tailed; P -0.017). We sought to confirm the initial ELISA studies by a more sensitive and specific bioassay in a separate AD
319
population [12]. T N F was measured essentially as previously described [7]. Briefly, L929 cells were seeded at a density of 4.5 x 104 in 96 well flat bottom plates and cultured in Minimal Essential Medium (Gibco) containing 5% fetal calf serum and 1% penicillin-streptomycin. Ten microliter serum samples and standards of human recombinant T N F (Genzyme Corp.) were added in triplicate to L929 cell cultures and incubated in the presence of I /~g of actinomycin D (Sigma) per ml of culture for 18 h. Cytotoxicity was measured by a colorimetric method employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Ten microliters of fresh MTT were added to each microwell and the plates were incubated for 4 h at 3T~C. Acid-isopropanol (100/~1) and distilled water (100 /tl) are then added and the plates read in a microtiter plate reader (Flow Laboratories, Multiskan Plus II) with a test wavelength of 570 nm. Cytotoxicity was determined according to the standard formula: E - B/M - B x 100%, where E is the experimental result (optical density at 570 nm) in the presence of serum, B is the background result in the absence of serum, and M is the maximum cytotoxicity determined by the addition of 1% Triton X-100. As a control, we demonstrated that addition of T N F (human recombinant) (hrTNF) alone resulted in >80% cytotoxicity. h r T N F cytotoxicity could be blocked by the concomitant addition of 3/tl of a rabbit antiserum to TNF-:~. PJGC and ADRC sera were employed for T N F bioassay studies. All sera were heat-inactivated by incubation at 5 6 C for 30 rain in a water bath prior to study to eliminate endogenous complement activity. To demonstrate that L929 cytotoxicity by human serum was due to TNF-~, serum samples were incubated in both the presence and absence of 10 ILl of rabbit anti-human TNF-:~ serum (Genzyme Corp.) capable of neutralizing 4 x 10 3 units of T N F per ml. Addition of rabbit antihuman T N F serum blocked >80% of cytotoxicity induced by patient serum. T N F levels in AD patients were significantly elevated compared to controls (Fig. 1). Mean cytotoxicity for the controls (n = 21) was 24 + 26%. Mean cytotoxicity for AD patients (u = 72) was 51 + 28%. Mean cytotoxicity for Alzheimer's patients and the controls were significantly different (t-test for independent samples (twotailed analysis; P = 0.00017). Thirty-six of 72 AD patients (50%) had cytotoxicity values greater than one standard deviation above the mean for controls. The prevalence of high cytotoxicity values > 30% was significantly greater in the AD group (Fisher's exact test, twotailed; P = 0.0000009). Recent data clearly indicate an inflammatory component to the pathogenesis of AD [9]. The origin of circulating T N F in AD may be from inflammatory lesions
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o |~
i
~ 40 ~" 20
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Alzheimer
Control
Fig. 1. Scattergram of TNF levels in Alzheimer's disease and control sera as determined by L929 cytotoxicity.
associated with microglia surrounding and invading senile plaques. Local alterations in blood brain barrier (BBB) function in AD [14] may facilitate the passage of T N F from plaques into the systemic circulation. Alternatively, elevated levels of T N F could result from a systemic reaction in AD. Serum levels of other acute phase reactants such as :d-antichymotrypsin (ACT) are also elevated in patients with AD [8]. However, ACT and other acute phase reactants may also originate in senile plaques [1, 16]. Our data further support the hypothesis that antiinflammatory treatment [10], such as prostaglandin inhibitors and other agents which reduce T N F and other cytokine activity [15], might be beneficial in preventing the progression of AD regardless of the etiology of AD. Blockade of systemic toxic T N F effects might also prevent systemic complications of AD such as cachexia, contributing to immunodeficiency and increased susceptibility to infection. Measures of circulating T N F and other acute phase reactants may be useful in monitoring the activity of the inflammatory component of AD. This work was supported in part by the Florence J. Gould Foundation, and NIH Grant AG05138. The authors would also like to thank Drs. Robert Butler, Andre Delacourte, Myron Miller, and Felix Silverstone for their support. l Abraham, C.R., Selkoe, D.J. and Potter, H., Immunochemical identification of the serine protease inhibitor alpha-one antichymotrypsin in the brain anayloid deposits of Alzheimer's disease, Cell, 52 (1988) 487 501. 2 Beutler, B., The presence of cachectin/tumor necrosis factor in human disease states, Am. J. Med., 85 (1988) 287 288. 3 Fillit, H.M., Foley, P., Bradford, H.F., Bucht, G., Winblad, B., McEwen, B., Luine, V.N. and Hardy, J., Autoimmunity to cholinergic-specific antigens of the brain in senile dementia of the Alzbeimer's type, Drug Dev. Res., 15 (1988) 143 151. 4 Frei, K., Siepl, C., Groscurth, P., Bodmer, S. and Fontana, A., lmmunobiology of microglial cells, Ann. N.Y. Acad. Sci., 540 (1988} 218 227.
320 5 Griffin, W.S.T., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White, C.L. and Araoz, C., Brain interleukin 1 and S100 immunoreactivity are elevated in Down syndrome and Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7611-7615. 6 Grunfeld, C. and Palladino, M.A., Tumor necrosis factor: immunologic, antitumor, metabolic, and cardiovascular activities, Adv. Intern. Med., 35 (1990) 45-72. 7 Levine, B., Kalman, J., Mayer, L., Fillit, H.M. and Packer, M., Elevated circulating levels of tumor necrosis factor in severe chronic congestive heart failure, N. Engl. J. Med., 323 (1990) 236 241. 8 Matsubara, E., Hirai, S., Amari, M., Shoji, M., Yamaguchi, H., Okamoto, K., Ishiguro, K., Harigaya, Y. and Wakabayashi, K., Alpha-one antichymotrypsin as a possible biochemical marker for Alzheimer type dementia, Ann. Neurol., 28 (1990) 56l 567, 9 McGeer, P.L., Akiyama, H., Itagaki, S. and McGeer, E.G., Immune system response in Alzheimer's disease, Can. J. Neurol. Sci., 16 (1989) 516-527. 10 McGeer, P.L., McGeer, E., Rogers, J. and Sibley, J., Anti-inflammatory drugs and Alzheimer disease, Lancet, 335 (1990) 1037. 11 McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E.M., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of
12
13
14
15 16
17
the Department of Health and Human Services Task Force on Alzheimer's Disease, Neurology, 34 (1984) 939 944. Meager, A., Leung, H. and Woolley, J., Assays for lumour necrosis factor and related cytokines, J. Immunol. Methods, 116 (1989) I 17. Perlmutter, L.S., Barron, E. and Chui, H.C., Morphologic association between microglia and senile plaque amyloid in Alzheimer's disease, Neurosci. Lett., 119 (1990) 32-36. Perlmutter, L.S. and Chui, H.C., Microangiopathy, the vascular basement membrane and Alzheimer's disease: a review, Brain Res. Bull., 24 (1990) 677~86. Remick, D.G. and Kunkel, S.L., Editorial: toxic effects of cytokines in vivo, Lab. Invest., 60 (1989) 317-319. Rozemuller, J.M., Stam, F.C. and Eikelenboom, P., Acute phase reactants are present in amorphous plaques in the cerebral but not in the cerebellar cortex of patients with Alzheimer's disease, Neurosci. Lett., 119 (1990) 75 78. Tracey, K.J., Wei, J., Manogue, K.R., Fong, U., Hesse, D.G., Nguyen, H.T., Kuo, G.C., Beutler, B., Cotran, R.S., Cerami, A. and Lowry, S.F., Cachectin/tumor necrosis factor induces cachexia, anemia and inflammation, J. Exp. Med., 167 (1988) 1211 1227.
?veurosciem't, LetI~p ~, 129 ( 1991 ) .31,~ 32t~ ~?~ 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91 $ 03.5() ,4 DONIS 030439409100468K
NSL 07981
Elevated circulating tumor necrosis factor levels in Alzheimer's disease H o w a r d Fillit, W a n h o n g Ding, Luc Buee, Jill K a l m a n , Larry Altstiel, Brian Lawlor and Giselle Wolf-Klein The Ritter Department of Geriatrics and Adult Development, and the Departments of Medicine, Psychiatry, the Fishberg Center for Neurobiology. and the Alzheimer's Disease Research Center, Mount Sinai Medical Center, New York, N Y 10029-6574 (U.S.A .), the Parker Jewish Geriatric Center, New Hyde Park, N Y (U.S.A.) andlNSERM Unit 156, Lille (France) (Received 27 February 1991; Revised version received 20 May 1991; Accepted 2t May 1991)
Key words: Alzheimer's disease; Cytokine; Tumor necrosis factor; Immunopathology Recent investigations have demonstrated a local inflammatory response in Alzheimer's disease (AD), including microglia and cytokines. Levels of the cytokine tumor necrosis factor a (TNF-a) in sera from patients with AD and age-matched controls were measured by an enzyme-linked immunoassay and a cytotoxicity bioassay. Significantly elevated levels of TNF were found in AD sera compared to controls. Elevated circulating TNF may be derived from the local CNS inflammatory reaction in AD, and may account for some systemic manifestations of AD such as weight loss. Future studies may determine if, in the absence of complicating disorders which may elevate TNF, circulating TNF could be a marker of AD inflammatory activity.
Substantial evidence has accumulated that Alzheimer's disease (AD) is associated with a local inflammatory reaction in senile plaques [9] which may be immune-mediated [3], and includes extensive microglial invasion [13], lymphocytic infiltration, complement and cytokine deposition, including interleukin-1 [5]. Tumor necrosis factor ~ (TNF-~) [6] is a cytokine which plays an important immuno-enhancing role in the local acute and chronic inflammatory response in response to a variety of stimuli. Brain microglia elaborate both interleukin-1 and TNF [4]. TNF and other cytokines such as interleukin-1 may also have deleterious systemic effects, such as the induction of acute shock or chronic cachexia in animals [17], and have been related to weight loss and cachexia in a variety of human disorders [2, 6, 7]. We hypothesized that some patients with AD, without other illnesses, would have elevated serum TNF which resulted from an AD-associated inflammatory reaction. Sera were obtained from 3 groups of AD patients and age-matched controls, including patients from the Rockefeller University Hospital (RUH); the Parker Jewish Geriatric Center (PJGC); and the Atzheimer's Disease Research Center at Mount Sinai Medical Center (ADRC). All AD patients met NINCDS criteria for probable AD [l 1] and did not suffer from concurrent maCorrespondence: H. Fillit, Department of Geriatrics, Box 1070, Mt. Sinai Medical Center, New York, NY 10029~5574, U.S.A. Fax: (l) (212) 8609737.
jor diseases which might have contributed to cognitive dysfunction and potentially to elevated TNF levels. All of the patients were ambulatory and seen as outpatients in Clinics at the 3 sites; none were bedridden nursing home or home care patients with pressure sores, and none of the patients had active infections which also might contribute to elevated TNF levels. Controls included healthy elderly individuals and non-demented patients with neurologic and psychiatric disorders, including affective disorders, schizophrenia, and Parkinson's disease. All sera were obtained in an aseptic fashion and stored at -70°C prior to assay. Informed consent was obtained in all cases. Initially, TNF in sera from RUH subjects (24 AD patients and 9 age-matched elderly controls) was measured by an enzyme-linked immunoassay (ELISA) capture assay [12]. Only two elderly controls had elevated TNF levels (88 and 110 pg/ml). Ten of 24 AD subjects had high TNF levels (> 100 pg/ml), and 4 AD patients had TNF levels which were above 1000 pg/ml. Mean TNF levels in the controls was 36 + 36 pg/ml; mean TNF in AD patients was 348 + 590 pg/ml. Despite the high degree of variation in the AD groups (with some patients having normal values and some with markedly elevated values), differences between groups were significant It-test for independent samples, two-tailed; P -0.017). We sought to confirm the initial ELISA studies by a more sensitive and specific bioassay in a separate AD
319
population [12]. T N F was measured essentially as previously described [7]. Briefly, L929 cells were seeded at a density of 4.5 x 104 in 96 well flat bottom plates and cultured in Minimal Essential Medium (Gibco) containing 5% fetal calf serum and 1% penicillin-streptomycin. Ten microliter serum samples and standards of human recombinant T N F (Genzyme Corp.) were added in triplicate to L929 cell cultures and incubated in the presence of I /~g of actinomycin D (Sigma) per ml of culture for 18 h. Cytotoxicity was measured by a colorimetric method employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Ten microliters of fresh MTT were added to each microwell and the plates were incubated for 4 h at 3T~C. Acid-isopropanol (100/~1) and distilled water (100 /tl) are then added and the plates read in a microtiter plate reader (Flow Laboratories, Multiskan Plus II) with a test wavelength of 570 nm. Cytotoxicity was determined according to the standard formula: E - B/M - B x 100%, where E is the experimental result (optical density at 570 nm) in the presence of serum, B is the background result in the absence of serum, and M is the maximum cytotoxicity determined by the addition of 1% Triton X-100. As a control, we demonstrated that addition of T N F (human recombinant) (hrTNF) alone resulted in >80% cytotoxicity. h r T N F cytotoxicity could be blocked by the concomitant addition of 3/tl of a rabbit antiserum to TNF-:~. PJGC and ADRC sera were employed for T N F bioassay studies. All sera were heat-inactivated by incubation at 5 6 C for 30 rain in a water bath prior to study to eliminate endogenous complement activity. To demonstrate that L929 cytotoxicity by human serum was due to TNF-~, serum samples were incubated in both the presence and absence of 10 ILl of rabbit anti-human TNF-:~ serum (Genzyme Corp.) capable of neutralizing 4 x 10 3 units of T N F per ml. Addition of rabbit antihuman T N F serum blocked >80% of cytotoxicity induced by patient serum. T N F levels in AD patients were significantly elevated compared to controls (Fig. 1). Mean cytotoxicity for the controls (n = 21) was 24 + 26%. Mean cytotoxicity for AD patients (u = 72) was 51 + 28%. Mean cytotoxicity for Alzheimer's patients and the controls were significantly different (t-test for independent samples (twotailed analysis; P = 0.00017). Thirty-six of 72 AD patients (50%) had cytotoxicity values greater than one standard deviation above the mean for controls. The prevalence of high cytotoxicity values > 30% was significantly greater in the AD group (Fisher's exact test, twotailed; P = 0.0000009). Recent data clearly indicate an inflammatory component to the pathogenesis of AD [9]. The origin of circulating T N F in AD may be from inflammatory lesions
lOO
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°~ 60
o |~
i
~ 40 ~" 20
! °
|
•
$
Alzheimer
Control
Fig. 1. Scattergram of TNF levels in Alzheimer's disease and control sera as determined by L929 cytotoxicity.
associated with microglia surrounding and invading senile plaques. Local alterations in blood brain barrier (BBB) function in AD [14] may facilitate the passage of T N F from plaques into the systemic circulation. Alternatively, elevated levels of T N F could result from a systemic reaction in AD. Serum levels of other acute phase reactants such as :d-antichymotrypsin (ACT) are also elevated in patients with AD [8]. However, ACT and other acute phase reactants may also originate in senile plaques [1, 16]. Our data further support the hypothesis that antiinflammatory treatment [10], such as prostaglandin inhibitors and other agents which reduce T N F and other cytokine activity [15], might be beneficial in preventing the progression of AD regardless of the etiology of AD. Blockade of systemic toxic T N F effects might also prevent systemic complications of AD such as cachexia, contributing to immunodeficiency and increased susceptibility to infection. Measures of circulating T N F and other acute phase reactants may be useful in monitoring the activity of the inflammatory component of AD. This work was supported in part by the Florence J. Gould Foundation, and NIH Grant AG05138. The authors would also like to thank Drs. Robert Butler, Andre Delacourte, Myron Miller, and Felix Silverstone for their support. l Abraham, C.R., Selkoe, D.J. and Potter, H., Immunochemical identification of the serine protease inhibitor alpha-one antichymotrypsin in the brain anayloid deposits of Alzheimer's disease, Cell, 52 (1988) 487 501. 2 Beutler, B., The presence of cachectin/tumor necrosis factor in human disease states, Am. J. Med., 85 (1988) 287 288. 3 Fillit, H.M., Foley, P., Bradford, H.F., Bucht, G., Winblad, B., McEwen, B., Luine, V.N. and Hardy, J., Autoimmunity to cholinergic-specific antigens of the brain in senile dementia of the Alzbeimer's type, Drug Dev. Res., 15 (1988) 143 151. 4 Frei, K., Siepl, C., Groscurth, P., Bodmer, S. and Fontana, A., lmmunobiology of microglial cells, Ann. N.Y. Acad. Sci., 540 (1988} 218 227.
320 5 Griffin, W.S.T., Stanley, L.C., Ling, C., White, L., MacLeod, V., Perrot, L.J., White, C.L. and Araoz, C., Brain interleukin 1 and S100 immunoreactivity are elevated in Down syndrome and Alzheimer disease, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 7611-7615. 6 Grunfeld, C. and Palladino, M.A., Tumor necrosis factor: immunologic, antitumor, metabolic, and cardiovascular activities, Adv. Intern. Med., 35 (1990) 45-72. 7 Levine, B., Kalman, J., Mayer, L., Fillit, H.M. and Packer, M., Elevated circulating levels of tumor necrosis factor in severe chronic congestive heart failure, N. Engl. J. Med., 323 (1990) 236 241. 8 Matsubara, E., Hirai, S., Amari, M., Shoji, M., Yamaguchi, H., Okamoto, K., Ishiguro, K., Harigaya, Y. and Wakabayashi, K., Alpha-one antichymotrypsin as a possible biochemical marker for Alzheimer type dementia, Ann. Neurol., 28 (1990) 56l 567, 9 McGeer, P.L., Akiyama, H., Itagaki, S. and McGeer, E.G., Immune system response in Alzheimer's disease, Can. J. Neurol. Sci., 16 (1989) 516-527. 10 McGeer, P.L., McGeer, E., Rogers, J. and Sibley, J., Anti-inflammatory drugs and Alzheimer disease, Lancet, 335 (1990) 1037. 11 McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E.M., Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA work group under the auspices of
12
13
14
15 16
17
the Department of Health and Human Services Task Force on Alzheimer's Disease, Neurology, 34 (1984) 939 944. Meager, A., Leung, H. and Woolley, J., Assays for lumour necrosis factor and related cytokines, J. Immunol. Methods, 116 (1989) I 17. Perlmutter, L.S., Barron, E. and Chui, H.C., Morphologic association between microglia and senile plaque amyloid in Alzheimer's disease, Neurosci. Lett., 119 (1990) 32-36. Perlmutter, L.S. and Chui, H.C., Microangiopathy, the vascular basement membrane and Alzheimer's disease: a review, Brain Res. Bull., 24 (1990) 677~86. Remick, D.G. and Kunkel, S.L., Editorial: toxic effects of cytokines in vivo, Lab. Invest., 60 (1989) 317-319. Rozemuller, J.M., Stam, F.C. and Eikelenboom, P., Acute phase reactants are present in amorphous plaques in the cerebral but not in the cerebellar cortex of patients with Alzheimer's disease, Neurosci. Lett., 119 (1990) 75 78. Tracey, K.J., Wei, J., Manogue, K.R., Fong, U., Hesse, D.G., Nguyen, H.T., Kuo, G.C., Beutler, B., Cotran, R.S., Cerami, A. and Lowry, S.F., Cachectin/tumor necrosis factor induces cachexia, anemia and inflammation, J. Exp. Med., 167 (1988) 1211 1227.
