ORIGINAL CONTRIBUTION Cytokine Gene Expression as a Function of the Clinical Progression of Alzheimer Disease Dementia James D. Luterman, PhD; Vahram Haroutunian, PhD; Shrishailam Yemul, PhD; Lap Ho, PhD; Dushyant Purohit, MD; Paul S. Aisen, MD; Richard Mohs, MD; Giulio Maria Pasinetti, MD, PhD Background: Inflammatory cytokines have been linked gyrus (P,.01). When stratified by the Consortium to to Alzheimer disease (AD) neurodegeneration, but little Establish a Registry for Alzheimer’s Disease (CERAD) is known about the temporal control of their expression neuropathological criteria, IL-6 mRNA expression in in relationship to clinical measurements of AD demen- both the entorhinal cortex (P,.05) and superior tem- tia progression. poral gyrus (P,.01) correlated with the level of neuro- fibrillary tangles but not neuritic plaques. However, in Design and Main Outcome Measures: We mea- the entorhinal cortex, TGF-b1 mRNA did not correlate sured inflammatory cytokine messenger RNA (mRNA) with the level of either neurofibrillary tangles or neu- expression in postmortem brain specimens of elderly sub- ritic plaques. Interestingly, in the superior temporal jects at different clinical stages of dementia and neuro- gyrus, TGF-b1 mRNA expression negatively correlated pathological dysfunction. with neurofibrillary tangles (P,.01) and showed no relationship to the pathological features of neuritic Setting and Patients: Postmortem study of nursing plaques. home patients. Conclusions: The data are consistent with the hypoth- Results: In brains of cognitively normal control sub- esis that cytokine expression may differentially contrib- jects, higher interleukin 6 (IL-6) and transforming ute to the vulnerability of independent cortical regions growth factor b1 (TGF-b1) mRNA expression was during the clinical progression of AD and suggest that observed in the entorhinal cortex and superior temporal an inflammatory cytokine response to the pathological gyrus compared with the occipital cortex. Compared effects of AD does not occur until the late stages of the with age-matched controls, subjects with severe/ disease. These findings have implications for the design terminal dementia, but not subjects at earlier disease of anti-inflammatory treatment strategies. stages, had higher IL-6 and TGF-b1 mRNA expression in the entorhinal cortex (P,.01) and superior temporal Arch Neurol. 2000;57:1153-1160 EVERAL CYTOKINES have been (mRNA) level is increased in postmor- associated with Alzheimer tem AD brains10 and correlates with amy- disease (AD) neuropathol- loid deposition in cerebral blood vessels. ogy. The level of the proin- However, TGF-b1 may also have nonin- flammatory cytokine inter- flammatory functions and may play an im- From the Neuroinflammation leukin 6 (IL-6) is increased in the brain, portant role in the growth and survival of S 11-13 Research Laboratories, blood, and cerebrospinal fluid of patients neurons in the AD brain. The expres- 1-6 Department of Psychiatry with AD, and IL-6 has been implicated sion of the cytokine tumor necrosis fac- (Drs Luterman, Yemul, Ho, in the transformation of diffuse to neu- tor a (TNF-a) is decreased in the frontal and Pasinetti), Department of ritic plaques in the AD brain.7 Interleu- cortex, superior temporal gyrus, and en- Pathology (Dr Purohit), and kin 1 (IL-1) has also been linked to amy- torhinal cortex of AD patients compared Psychiatry (Dr Mohs), Mount loid plaque transition from the diffuse to with non-AD controls14 and has both pro- Sinai School of Medicine, New dense core stage8 and the propagation of tective and destructive functions.15 York, NY; Department of the inflammatory signal through the in- Although cytokine expression has been Psychiatry, Bronx Veterans duction of S100b.4,9 Transforming growth shown to be regulated in the AD brain, pre- Affairs Medical Center, Bronx, NY (Dr Haroutunain); and factor b1 (TGF-b1) has been shown to vious studies evaluated postmortem tissue Department of Neurology, promote b-amyloid deposition in trans- from severely affected patients at the end 10 Georgetown University School genic mouse models and therefore may stages of AD. There is presently little infor- of Medicine, Washington, DC exacerbate amyloidogenic pathology. In mation on the dynamic expression of in- (Dr Aisen). addition, the TGF-b1 messenger RNA flammatory cytokines relative to the clini- (REPRINTED) ARCH NEUROL / VOL 57, AUG 2000 WWW.ARCHNEUROL.COM 1153 ©2000 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 METHODS complementary DNA (cDNA) templates for the following human cytokine genes was used: IL-6, TGF-b1, IL-1b, PATIENT SELECTION CRITERIA TNF-b, TNF-a, IL-1a, IL-1 receptor antagonist (IL-1Ra), and interferon gamma (IFN-g). The probe set also in- Human postmortem brains from AD and age-matched cluded the housekeeping genes L32 and glyceralde- non-AD cases were obtained from the Alzheimer’s Disease hyde-39 phosphate dehydrogenase (GAPDH) for normal- Research Center (ADRC) of the Mount Sinai School of Medi- ization of assay conditions. Details on the generation of cine, New York, NY. The cases selected had either no sig- phosphate P 32–labeled antisense RNA probes and condi- nificant neuropathological features or only neuropatho- tions of the RNase protection assay (RPA) are provided by logical features associated with AD.17,18 A multistep approach the manufacturer. The quantity of radioactively labeled based on cognitive and functional status during the last 6 RNase protection fragments was determined using a Mo- months of life was applied to the assignment of Clinical De- lecular Dynamics Storm 860 Phosphor Screen Scanner with mentia Rating (CDR) scores, as previously reported.17,18 Sub- the ImageQuant software package (Molecular Dynamics, jects were divided into groups on the basis of their CDR Sunnyvale, Calif). Each RPA analysis was conducted with scores as follows: 0, nondemented; 0.5, questionable de- 10 µg of total RNA, according to A260 values. Data are ex- mentia; 1, mild dementia; 2, moderate dementia; and 4 to pressed as a ratio of the specific mRNA of interest normal- 5, very severe dementia. ized to the constitutively expressed GAPDH mRNA. Nor- The extent of neurofibrillary tangle and b-amyloid neu- malization of cytokine mRNA signals to L32 mRNA did not ritic plaque deposition was assessed in accord with the Con- change the outcome results (not shown). sortium to Establish a Registry for Alzheimer’s Disease (CERAD) neuropathological battery.17-19 Multiple (5 in gen- RNA SAMPLE QUALITY AND EXCLUSION eral) high-power fields (3200, 0.5 mm2) were examined CRITERIA FOR RPA in each histological slide, containing specimens obtained from multiple brain regions, according to the CERAD re- Initial quality control consisted of agarose gel assessment of gional sampling scheme. The density of neurofibrillary ethidium bromide staining of both 18–Svedberg flotation unit tangles and neuritic plaques was rated on a 4-point scale (Sf) and 28-Sf ribosomal RNA (rRNA) integrity as well as as follows: 0, absent; 1, sparse; 3, moderate; and 5, severe. detection of low-molecular-weight “smearing” of degraded Plaques were visualized following either Bielschowsky sil- mRNA. All samples were subjected to RPA, regardless of ini- ver or thioflavine S staining.20,21 The investigators were blind tial quality control assessment. Only samples with nonde- to the clinical diagnosis of each case until all quantitative graded RNA determined by both ethidium bromide and RPA analyses were completed and values were assigned to each analysis were included in the study. For the entorhinal cor- specimen. tex, 19 (24%) of the 78 subjects were removed from the study because of poor RNA quality; for the superior temporal gy- RNA PREPARATION rus, 16 (20%) of 79 patients were removed; and for the oc- cipital cortex, 9 (36%) of 25 patients were removed. In each Total RNA was prepared with the Ultraspec RNA Isola- brain region examined, RNA exclusion was similarly dis- tion System (Biotecx Laboratories, Houston, Tex), based tributed among the different CDR categories. on the acid guanidinium thiocyanate-phenol-chloroform method.22 STATISTICS RIBONUCLEASE PROTECTION ASSAY Data were analyzed by analysis of variance (ANOVA) com- bined with a post hoc Dunnett multiple comparison test Total RNA was assayed with the RiboQuant Multiprobe to compare each experimental group with the control group. RNase (ribonuclease) Protection Assay System (PharMin- Correlation analysis was performed by calculating the Pear- gen, San Diego, Calif). A custom probe set containing son R coefficient. cal progression of AD dementia. The current study is the RESULTS first to examine the mRNA expression patterns of several inflammatory cytokines in different brain regions as a func- tion of the clinical progression of AD. PATIENT POPULATION It has been suggested that the neuropathological fea- tures of AD occur in different brain areas in a predict- Patient information for this study is summarized in able temporal pattern during the course of the disease Table 1. Extraneous factors that might influence cyto- progression.16 Therefore, cytokines were measured in the kine gene expression were similar across all groups. Cy- entorhinal cortex (Brodmann area [BM] 36/38) and su- tokine mRNA degradation is a concern with long post- perior temporal gyrus (BM 22), 2 brain areas affected in mortem delays. The ANOVA indicated that there were AD, and in the occipital cortex (BM 17), an area that is no significant differences (P..05) among the different less affected in AD. In this study, we determined the pat- CDR groups with respect to postmortem intervals (data tern of cytokine gene expression as a function of AD clini- not shown). Furthermore, all groups had a statistically cal dementia and the neuropathological features of AD similar (P..05) age at death (data not shown). Cyto- and discussed the relevance of these findings to the de- kine expression can also be impacted by antemortem sign of anti-inflammatory drug trials in AD. inflammatory conditions that may require anti- (REPRINTED) ARCH NEUROL / VOL 57, AUG 2000 WWW.ARCHNEUROL.COM 1154 ©2000 American Medical Association.