Induction of immune system mediators in the hippocampal formation in Alzheimer's and Parkinson's diseases: Selective effects on specific interleukins and interleukin receptors

NeuroscienceVol. 61, No. 4, pp. 745-154,

Pergamon

0306-4522(94)E0013-I

1994 Elsevier Science Ltd Copyright IQ 1994 IBRO Printed in Great Britain. All rights reserved 0306-4522/94 $7.00 + 0.00

INDUCTION OF IMMUNE SYSTEM MEDIATORS IN THE HIPPOCAMPAL FORMATION IN ALZHEIMER’S AND PARKINSON’S DISEASES: SELECTIVE EFFECTS ON SPECIFIC INTERLEUKINS AND INTERLEUKIN RECEPTORS D. M. ARAUJO* and P. A. LAPCHAK University of Southern California. Dept. of Neurogerontology,

Andrus Gerontology CA 90089-0191. U.S.A.

Center, Los Angeles,

Abstract-The present study determined whether molecules normally associated with immune signalling processes, specifically the lymphokines interleukin-I/?, -2, -3 and -6, can be detected in the human hippocampal formation, and whether their levels are altered in neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Interleukin-I/?. -2, -3 and -6 were measured in posrmortem tissues from I4 control neurologically normal subjects, 24 patients with Alzheimer’s disease and 17 patients with Parkinson’s disease. In order to assess the extent of the cholinergic deficit in the Alzheimer’s disease brains, choline acetyltransferase activity in the hippocampal formation was first determined. In the Alzheimer’s disease tissues, choline acetyltransferase activity was significantly reduced (by 58%) compared to the control hippocampi, whereas that in the Parkinson’s disease hippocampi was not significantly different from control. Using radioimmunoassays with antisera specific for the respective interleukin, marked increases in the content of immunoreactive interleukin-l/l (99%), interleukin-2 (129%) and interleukin-3 (64%) could be detected in the Alzheimer’s, but not the Parkinson’s disease hippocampi. Interleukin-6 levels were not significantly different in either group, compared to the control hippocampi. Since striking elevations in various interleukins were detected in the Alzheimer’s disease hippocampi. the possibility that concomitant alterations in interleukin receptor sites also occurred was investigated. Using radioligand binding to hippocampal membranes. low levels of interleukin binding were measured in the control hippocampi. In the Alzheimer’s tissues, significant elevations in [‘2sI]interleukin-I,!l (by 65%) and [‘ZSI]interIeukin-2 (by 69%) binding were noted. In contrast, [‘ZI]interleukin-3 binding was not different in the AIzheimer’s disease compared to the control tissues. In the hippocampal formation of Parkinson’s disease brains, only [“‘I]interleukin-2 binding was significantly increased (by 80%). In summary, the present results indicate that there is pronounced activation of immune system function, particularly specific immune mediators such as the interleukins, in the hippocampal formation in Alzheimer’s disease, and further suggest that stimulation of immune function may be an integral component of the pathological changes that occur in this disease.

Recent studies have shown that a variety of classical immune system antigens and mediators are present in the adult mammalian brain,“~~-42~52~56~~~~~76 where they are thought to be involved in immune-related phenomena. In the CNS, these immune mediators and antigens appear to be associated with a wide variety of intrinsic cell types such as neurons, astrocytes, oligodendrocytes, and particularly microglia. ‘6.35,s2-56 During instances of CNS injury29-32and in disease states 11,17,41.42,52~5/8,61.63,65.66 both microglia and astrocytes tend to proliferate around the area of primary insult. Moreover, in neurodegenerative diseases such as Alzheimer’s disease (AD), there appears

*To whom correspondence

should be addressed at: P.O. Box 2061, West Chester, PA 19380, U.S.A. Abbmiatiom: AD, Alzbeimer’s disease; p-APP, /?-amyloid precursor protein; BSA, bovine serum albumin; ChAT, choline acetyltransferase; IL, interleukin; PD, Parkinson’s disease; RIA, radioimmunoassay; TPB, tetraphenyl boron. 745

to be an early involvement of microglia in the pathoThe proliferation of microgenesis of plaques. 35,4’,63 glia that occurs around senile plaques creates a local microenvironment where the synthesis and release of immune mediators may increase dramatically. For example, several studies have indicated that mediators of the classical complement pathway are present in the AD brain, in regions exhibiting the highest degree of pathology.20~4’~s2~54.M66.75.76 This elevated expression of components of the complement cascade and other immune mediators in the vicinity of senile plaques appears to be the consequence mainly of enhanced synthesis and secretion by cells intrinsic to the brain.“~4’~52~56~58~6’~63 66However, contribution by other cell types that may infiltrate a compromised blood-brain barrier cannot be completely excluded. Thus, the presence of macrophages, T-lymphocytes and B-lymphocytes, cells that are normally found only in the periphery, has been reported in the AD brain.40*65,66,‘6

746

D. M. Araujo and P. A. Lapchak

The increased densities of immune mediators in the brain following trauma or during the course of neurodegenerative diseases suggests that these molecules may be integral components of a CNS derense system. However, it is also possible that in the intact CNS, these cytokines may participate directly in the normal development of various cell types (for a review, see Ref. 3). For instance, interleukin-1 (IL-l), a cytokine that appears to be involved in the wound healing and repair that follows CNS trauma,30.3’ has also been detected in the intact mammalian CNS.i’.‘4.7’ Similarly, other IL peptides and mRNA, as well as their receptors, including IL-2,‘.” IL-3:’ IL-668.79and IL-g,‘” have also been identified in rat brain cells. Since IL-l has been found in elevated concentrations around senile plaques in AD,34 this cytokine has been implicated in the progressive pathogenesis of the disease. Further support for this has been obtained from studies showing that IL-I stimulates the production of the /j’-amyloid precursor protein (p-APP) in vitro. I4‘c3’ Howcvcr, unlike IL- 1, the status, and consequently the potential involvement of other ILs in the pathogenesis of AD, has not been conclusively demonstrated. Thus, the main purpose of the present study was to determine whether ILs (IL-I& IL-2, IL-3 and IL-6) and IL receptors, which have been identified in the mammalian CNS (see above), can be detected in human post mortem brain tissue and whether their levels are altered in AD. For this, radioimmunoassay (RIA) or immunoassay systems and radioligand binding assays specific for each of the ILs were used to assess IL levels and [“‘I]IL binding in the hippocampal formation, a region that is known to be most susceptible to the subsequent neurodegeneration pathology and observed in AD (for a review, see Ref. IO). As a comparison. IL and IL receptor levels were also measured in post nmrtem tissues from patients with

Parkinson’s disease (PD), in which hippocampal pathology is not a critical factor in the progression of the disease. EXPERIMENTAL PROCEDURES

IL-lp, IL-2 and IL-6 RIA kits, as well as [“‘I]IL-I[] (2300 Ci/mmol), [‘251]1L-2 (673 Ci/mmol) and [‘251]IL-3 (380 Ci/mmol), were purchased from Amersham Corp. (Arlington Heights, IL). Quantikine immunoassay kits for the determination of IL-3 were obtained from R&D Systems (Minneapolis, MN). Unlabeled ILs (recombinant human) were bought from Upstate Biotechnology Inc. (Lake Placid. NY). [‘H]Acetyl-CoA (200 mCi/mmol) and Aquasolwere from New England Nuclear (Boston, MA). Physostigmine sulfate, choline chloride, bovine serum albumin (BSA), Triton X-100, tetraphenylboron (TPB) and 3-heptanone were purchased from Sigma Chemical Co. (St Louis, MO) Human bran tissues Hippocampal lissues from 14 control, 24 AD and 17 PD patients were generously donated by the following brain banks: Harvard Medical School, Belmont, MA (Dr Edward D. Bird); UCLA, Los Angeles, CA (Dr W. W. Tourtellotte): University of Southern California, Los Angeles, CA (Drs Carol A. Miller and Jenny K. Tang); Douglas Hospital Research Center, Montreal, Canada (Dr Remi Qmrion), The clinical and neuropathological characteristics available for the cases used in this study are summarized in Table I. All AD cases were judged to have moderate to severe pathology, based on the distribution of senile plaques ( > 40!mm2) and neurofibrillary tangles?,*’ In addition. the PD cases were determined as moderate to severe, as characterized by neuronal loss, Lewy body accumulation and gliosis in the substantia nigra, and classical clinical features such as resting tremor, akincsia and other extrapyramidal symptoms. Normal control brain tissues were characterized as those obtained from age-matched subjects that were fret of clinically-assessed neurological or psychiatric symptoms and whose plaque indices were less than IO/mm’. As shown in Table 1, the average age at time of death. mean posr morrem delay and other measures in the AD and PD groups were not significantly different from the control group.

Table 1. f+sr mortem data of the human hippocampal study

AD

PD

24 to/14

17 16/l

68.2 t 4.2 44493

74.3 + I .I 56-89

71.2 k 1.8 6Om85

10.1 + 1.2 5520.5

8.2 +2.1 2-23

8.5 + 1.7 2.5-23.5

1238 i 8.5 9x0- 1500 7

1031 + 77 81&1430 I1

Control Number Gender (M/F) Age at death (years) Mean & S.E.M. Range Post mortem delay (h) Mean) S.E.M. Range Fresh brain weight (g) Mean + S.E.M. Range-

NA

tissues used in the present

14 836

1312f92 960.-1490 9

Post mortem delay is expressed as the time interval between death and placement of the sectioned tissues at -70’C or on dry ice. All of the AD cases were judged to have moderate to severe pathology (see Experimental Procedures). NA, data not available for the indicated number of cases. The immediate causes or major contributory factors of death were not available for 95% of the cases. There were no statistically signficant differences (P z 0.70) between the groups for any of the data (age at death, postmortemdelay, brain weight) listed.

lnterleukins

in Alzheimer’s and Parkinson’s

741

diseases

50 r

Preparation of human brain tissues All hippocampal tissues were kept at - 70°C or on dry ice prior to being processed for experimental measures. Tissues were homogenized using a Brinkmann polytron (setting 5, 30 s) in 1.5ml of Tris (50 mM)-buffered saline @H 7.4) containing 1% Triton X-100. Aliquots of these samples were further processed, as indicated below, for determination of IL levels, IL binding and choline acetyltransferase (ChAT) activity. Assay for choline acetyltransferase activity Hippocampal ChAT activity was determined as described previously.* Briefly, aliquots (35~1) of the hippocampal homogenates described above were incubated (38”C, 30 min) in medium (15 al) containing unlabeled acetyl-CoA and [‘H]acetyl-CoA (to a final concentration of 0.25 mM, or approximately 200,000 d.p.m. per tube), choline chloride (12.5 mM), physostigmine sulfate (0.2 mM), NaCl (300 mM), sodium phosphate buffer (28 mM, pH 7.4) and Triton X-100 (0.35%). ChAT activity was calculated by determining the amount of [3H]acetylcholine formed from the substrate [‘Hjacetyl-CoA, by quantitating the sample [3H]acetylcholine that was extracted into the top organic phase with TPEheptanone (15 mg/ml). [sH] in an aliquot of the organic phase from each sample was assessed with approximately 40% efficiency by liquid scintillation spectrometry using Aquasol- as the solvent. Proteins were measured using BSA as the standard.15

0

’ CONTROL

AD

PD

Fig. I. ChAT activity in hippocampal homogenates from control (n = 14) AD (n = 24) and PD (n = 17) brains. Results are presented as the individual values for each control (open circles), AD (filled triangles) and PD (open diamonds) hippocampus and are the average of triplicate measures. Horizontal lines correspond to the mean ChAT activity in each group. ACh, acetylcholine.

calculated as the difference in radioactivity bound in the absence compared to that in the presence of unlabeled IL, and represented approximately 40% of the total binding.

Assay for hippocampal interleukins

Statistical analyses

IL levels in the hippocampal formation of control, AD and PD brains were assessed in extracts of hippocampal homogenates, as described previously.’ IL-l/?, IL-2 and IL-6 immunoreactive peptides were measured using RIA kits that contained recombinant human [‘zsI]IL-I/l, [‘251]lL-2 or [‘251]lL-6 as the tracer. The antisera used for each of these RIA kits were directed against human ILs and were specific for the respective IL. Cross-reactivity with other ILs was negligible (<0.5%). Magnetic separation of antibodybound [‘2sl]lL from free [“?]lL was done by incubating samples with a secondary antisera/Amerlex-M conjugate. After decanting the supernatants, the “‘1 in the resulting pellets was determined by counting in a gamma-counter (greater than 98% efficiency for ‘*‘I). The limit of detection of the RlAs ranged from 0.05 to 2.5 fmol/tube. IL-3 in the extracts of hippocampal homogenates was detected using an immunoassay kit containing microtiter plates coated with murine antisera against IL-3, a horseradish peroxidase/lL-3 antiserum conjugate, recombinant human IL-3 standards, hydrogen peroxide, antisera directed against human IL-3 and a chromogen (tetramethylbenzidine). The cross-reactivity of the IL-3 antisera with human IL-3 was lOO%, whereas that with other ILs and other cytokines was less than 0.09%. Tissue IL-3 was determined using a spectrophotometer, by measuring the optical density (450 nm) of the final reaction product. The minimum detectable amount of IL-3 that is detected using this assay system ranges from 0. I to 2 fmol/tube.

Each experimental measure presented in the figures corresponds to an individual tissue sample. In the tables, results are expressed as the meansf S.E.M. of the number of

Assaysfor hippocampal interleukin binding

Assays to determine hippocampal IL receptor binding for each IL were done as described previously.’ Briefly, [‘251]lL1fi, [‘*‘l]lL-2 and [‘2’l]lL-3 binding sites were measured by incubating aliquots (500 fig of protein) of the hippocampal homogenates (see above) for 2 hat 22°C in 50 mM Tris-HCI buffer (PH 7.4; final volume 0.5 ml) containing 0.1% BSA and 0.2 nM of the respective radioligand. Following the incubation, the receptor-bound [‘251]lLs were separated from free [‘*sl]lLs by centrifugation (5000 rpm, 15 min) in a microfuge. The supernatants were discarded and [r2’l] in the pellets was quantitated. Non-specific binding was determined in the presence of a 500-fold excess of the respective unlabeled IL (0.1 PM). Specific binding was

tissues indicated. Statistical significance was first assessed using one-way ANOVA, followed by an assessment of linear contrasts according to Scheffe and Student’s unpaired I-test, using the Number Cruncher Statistical System. In addition, Pearson correlation analyses were done to determine whether significant correlations (P < 0.05) between hippocampal IL levels and ChAT activity were evident. RESULTS Hippocampal choline acetyltransferase activity

As described ergic

deficit

previously,8

the extent of the cholin-

in AD can bc assessed by measuring

ChAT activity in hippocampal tissues. Thus, ChAT activity in hippocampal homogenates prepared from AD tissues was compared with that in control tissues. As shown in Fig. 1, there was significant scatter in ChAT activity measured in both the control and the PD. but not in the AD, hippocampi. Although there was some overlap between the two groups, the mean ChAT activity in the AD hippocampi was significantly (P i 0.001) reduced (by 58%) compared to the controls (Table 2). In contrast, the mean ChAT activity in the PD hippocampi was not different from that in the control tissues (P > 0.90) (Fig. 1, Table 2). As demonstrated in a previous report,’ this indicates an absence of significant dementia in the PD cases used in this study, which is consistent with the clinical data (see also Experimental Procedures). Hippocampal interleukin levels

This series of experiments first determined whether measurable levels of the cytokines IL-l/I, IL-2, IL-3 and IL-6 could be detected in human post mortem

748

D. M. Araujo and P. A. Lapchak

Table 2. Hippocampal

choline acetyltransferase

activity

Table 3. lnterleukin

ChAT Activity (nmol acetylcholine formed/mg protein/h) Controls AD PD

18.56+3.77 7.80 i_ 0.56 (58%)* 17.23 f 2.44 (7.2%)

Results are expressed as the mean 2 S.E.M. of the number of tissues in each group (14 controls, 24 AD and 17 PD hippocampi). Numbers in parentheses indicate the percent decrease in ChAT activity observed in the AD and PD hi~p~ampal tissues compared to the control tissues. ChAT activity in the PD hippocampi was not significantly different from the control group (P > 0.50). *Significantly decreased compared to the control hippocampi (P < 0.001).

tissues. The results show that low levels of all four ILs, as measured either with RIA (IL-I& IL-2, IL-B) or immunoassay kits (IL-3), were present in hippocampal homogenates from control brains (Fig. 2, Table 3). However, significant (P < 0.001) increases in the mean content of IL-Ifl (by 99%), IL-2 (by 129%) and IL-3 (by 64%) in the AD, compared to the control hippocampi, were evident (Fig. 2, Table 3). In contrast, although two values for IL-6 content were higher in the AD compared to the

levels in the hippocampal

Control IL-HI IL-2 IL-3 IL-6

6.15 5.41 0.97 2.29

f 2 * +

formation

IL Content (fmol/mg protein) AD

0.97 1.03 0.07 0.53

12.27 12.41 t .59 2.84

+ I .04* f 0.94* + 0.09* t 0.58

PD 7.56 5.58 1.32 2.38

+ L = -

Results are expressed as the mean 1_ S.E.M. of 14 control, 24 AD and 17 PD hippocampi and are the amount of IL detected per mg protein in each separate tissue sample. IL levels in the PD hippocampi were not signi~cantly different from the control tissues (P > 0.40). *IL-I& IL-2 and IL-3 levels in the AD hippocampi were significantly increased compared to the control hippocampi (P < O.OOl), whereas IL-6 levels were not significantly altered (P zs 0.65).

control hippocampi (Fig. ID), the mean IL-6 levels in the AD hippocampi were not markedly different (P > 0.65) from the control or the PD groups (Fig. 2D, Table 3). In addition, there were no notable alterations in the content of ILs in the PD hippocampi; mean IL-lb, IL-2, IL-3 and IL-6 levels in the PD hippo~mp~ were not statisticalIy different (P > 0.40) from those measured in the control tissues (Fig. 2. Table 3). C

2.5

r

.

CONTROL

AD

CDNTROL

AD

0

PD

0.55 0.63 0.10 0.24

CONTROL

AD

PD

CONTROL

AD

PD

*

Fig. 2. IL content in hippocampal homogenates of control (open circles), AD (filled tnangies) and PD (open diamonds) tissues. RIAs or immunoassays (see Experimental Procedures) of tissue extracts were done to quantitate levels ofimmunoreactive IL-IS (A), IL-2 (B), IL-3 (C) and IL-6(D) in the hippocampal homogenates. Each value plotted is the average of duplicate measures for each individual tissue sample. Horizontal bars correspond to the mean value of IL content for each of the three groups.

Interleukins in Alzheimer’s and Parkinson’s diseases Hippocampal interleukin receptor binding

To determine whether modifications in the levels of endogenous ligands for IL receptor sites are reflected in concomitant alterations in receptor densities, as shown for the peripheral immune system,‘~“~74IL binding in the AD hippocampi was assessed and compared to that in the control tissues. Since only limited quantities of tissues were available, single point binding in hippocampal homogenates was used. Therefore, the binding of [‘251]ILsto hippocampal homogenates was assessed using a single concentration (0.2 nM) of the radioligands that was within the range of Kd values reported for these ILs in the peripheral immune system.‘,‘4 The results presented in Table 4 show that using 0.2 nM of the respective radioligand, low levels of [‘251]ILbinding could be detected in the control hippocampi. Moreover, ["'I]IL-1 fi and [“‘I]IL-2 binding in the AD tissues was significantly enhanced compared to the control group, by 65% and 69%, respectively (Table 4). However, despite the increase in IL-3 levels in the AD hippocampi (see above), [‘*‘I]IL-3 binding was not comparably elevated (Table 4). Interestingly, [“51]IL-2 binding was significantly (P < 0.001) enhanced (by 80%) in the PD hippocampi (Table 4) whereas no obvious changes in IL-2 peptide could be detected (see above). In contrast, neither [‘251]IL-l/? nor [‘251]IL-3 binding were significantly (P > 0.50) altered in the PD hippocampi (Table 4).

149

correlation between decreased ChAT activity and enhanced IL content in the AD hippocampal formation (Fig. 3). This was the case whether hippocampal IL-lp (Fig. 3A), IL-2 (Fig. 3B), IL-3 (Fig. 3C) or IL-6 (Fig. 3D) levels were compared to ChAT activity in the same tissues, with the correlation coefficients (P) corresponding to 0.328, 0.496, 0.584, and 0.995 (with r values that ranged from -0.208 to 0.118; d.f. = 22), respectively. In addition, significant correlations between IL levels and ChAT activity in the control (P > 0.75, d.f. = 12) and PD (P > 0.66, d.f. = 15) hippocampi were not observed (data not shown). IS there a cvrrelativn between increases in interleukinl/3 levels and other interleukins in the Alzheimer’s disease hippocampi?

In the peripheral immune system, IL-l/l is known to stimulate the release of IL-2 from immune cell~.‘.“,‘~ Similarly, in the CNS, IL-l/l appears to enhance IL-2 release from microglia* and IL-3 release from astroglia.27,2P,32Thus, potential correlations between hippocampal IL-lb levels and these other ILs in the AD brains were assessed. The results shown in Fig. 4 do not indicate a significant correlation (d.f. = 22) between the content of IL-lfl and IL-2 (Fig. 4A; P > 0.77) or between IL-lb and IL-3 (Fig. 4B; P > 0.67) in the AD hippocampi. DISCUSSION

Is there a correlation between decreased choline acetyltransferase activity and elevated interleukin levels in the Alzheimer’s disease hippocampi?

In order to determine whether there was a correlation between induction of immune mediators and the cholinergic deficit observed in AD, in the tissues used in this study, values for IL content and ChAT activity in the same hippocampi were plotted (Fig. 3). The results demonstrate that there was no clear Table 4. [i2SI]Interleukin binding

in the hippocampal

formauon

Control IL-lb IL-2 IL-3

3.05 f 0.42 0.63 + 0.06 I .70 + 0.40

[‘*‘I]lL bound (fmol/mg protein) AD 5.03 * 0.41*

1.06f 0.09t 1.59+0.10

PD 3.15 + 0.53 1.12*0.09t 1.95 + 0.29

Values are expressed as the mean + S.E.M. of the number of tissues in each group (14 controls, 24 AD and 17 PD brains) and are the amount (fmol/mg protein) of [‘*‘I]IL specifically bound (total minus non-specific binding) to hippocampal homogenates (see Experimental Procedures). *Significantly increased compared to [‘251]IL-lB binding in the control tissues (P < 0.01); tsignificantly increased compared to [‘2sI]IL-2 binding in the control hippocampi (P < 0.001). [tz51]IL-l/I binding was not different in the PD comoared to the control tissues (P > 0.85). [‘2’I]IL-3 binding was not significantly (P z 0.50) altered in either AD or PD hippocampi, compared to the controls.

The main purpose of the present study was to determine whether ILs and IL receptors in the hippocampal formation are differentially affected in neurodegenerative diseases such as AD and PD, which are characterized by markedly different etiologies (cf. Introduction). The detection of several ILs and IL receptor binding in the control hippocampal tissues indicates that many of the critical components required for a rudimentary “immune system” are present in the normal brain. Moreover, using RIAs specific for the detection of IL-Ij, IL-2, IL-3 and IL-6, clear distinctions between the AD and PD hippocampi were discernible. Thus, marked elevations in IL-l/?, IL-2 and IL-3 were measured in the AD, but not the PD, hippocampi. In addition, [‘2’I]IL-lfi and [“‘I]IL-2 binding were augmented in the AD hippocampi. In the PD hippocampi, a selective enhancement in [‘*“I]IL-2 binding was apparent, whereas [‘2’I]IL-ljI and [“‘I]IL-3 binding were not noticeably altered compared to the control tissues. These results indicate that induction of hippocampal immune mediators in the hippocampal formation may be manifest only with the onset of specific pathology, such as that which occurs in AD. Interleukin- l/l and

interleukin-1fi receptors in the

hippocampal formation

Elevated densities of IL-l immunoreactive in cortical tissues from AD brains have

cells been

2

4

6

8

10

12

0

2

ChAT ACTIVITY fnmol ACh/mg protein/h1 D

if: c)

t5

-

10

-

Z

0

*

tu2 LE -=

10

12

A

‘+

i

l-e ZP

E

8

;a 8-

3

LO

00

6

12 10

g ,f

4

ChAT ACTIVITY lnmol ACh/mg protoin/hl

&A

SE”

*

a$

A 5

-

LE -=

A

6

-

4

-

A

2-

A

"*','.'.',J 0

2

4

6

8

ChAT ACTIVITY (nmol ACh/mg protein/h1

10

12

0

63

&A% &%A

h 0

A

Q A

A&

A'

","s""l' 0

2

4

6

8

10

12

ChAT ACTIVITY (nmol ACh/mg protein/h1

Fig. 3. Analysis of potential correlations between ChAT activity and IL content in hip~ampai homogenates from AR brains (n = 24). Levels of immunoreactive IL-lj? (A), IL-2 (B), IL-3 (C) and IL-6 (D) were plotted against the corresponding value for ChAT activity in the same tissue. The lines represent a computer-generated least-squares linear regression plot. No significant correlations were detected (P > 0.32). ACh, acetylcholine.

d~umcnted previously.34 However, results from the that may be further susceptible to the elevated levels present work provide the first quantitative evidence of IL-18 secreted by microglia. Consequently, this for enhanced IL-lb levels in the hippocampal formicroenvironment that is enriched with immune sysmation in AD. Since microglia have been shown to tem mediators and cells responsive to them, may contribute further to activation of other mechanisms be the main IL-l-producing cells of the CNSy9 increases in IL- 1s in the AD hippocampi are a likely involved in the pathogenesis of AD. consequence of microglial proliferation. Indeed, overEvidence that IL-l stimulates fi-APP expression expression of activated microglia in the vicinity of in ~:irro,‘~,‘~~~~ suggests that this cytokine may play a senile plaques has been reported in AD. ~~~~~~~~~~~~~~~~~~~~ significant role in /?-APP processing and in the subAlthough the consequences of this elevated microsequent synthesis and plaque deposition of bamyloid. glial-derived IL-lb in close proximity to senile fi-amyloid, in turn, stimulates microglial proliferation and secretion of IL-l4 and other immune system plaques are not entirely clear, it has been suggested mediators.” Since /?-amyloid has been shown to be that IL-1 may trigger a cascade of events that toxic to hippocampal neurons in vitru,6297.‘8the conultimately contributes to the pathogenesis of AD. tinuous cycle of IL-I induction of fi-amyloid synFollowing CNS injury, for instance, IL-1 and other thesis, and vice versa, may compromise neuronal cytokines released from microglia stimulate astroglial function. Further detrimental effects to neuronal proliferation and neovascularization in proximity to viability may also be mediated directly by IL-l, the initial insult.30~32@Similarly, in AD, IL-l-induced particularly since IL-1 receptor sites appear to be gliosis may result in the release of astroglial-derived widespread in regions enriched with neurons.1823.24 cytokines that subsequently reactivate microglial proFor instance, long-term exposure to high concenliferation and re-stimulate IL-1 release.4m6 Moreover, trations of IL- 1 has been shown to reduce hippocamthis proliferation of plaque-associated astroglia may pal neuronal survival.2 in uivo administration of IL- 1 account, at least in part, for the enhanced IL-l/? has been reported to exacerbate neuronal death binding measured in the AD hippocampal homogeninduced by ischemia. 67In addition, although IL-1 has ates. Thus, there is an increase in the number of cells

Interleukins in Atzheimer’s and Parkinson’s diseases

been shown to enhance nerve growth factor mRNA levels in the CNS70and has been implicated in the promotion of cholinergic sprouting:’ other potentially det~mental effects of IL-1 administration on neurotrophic factors, such as the attenuation of hippocampal brain-derived neurotrophic factor expression,48have been observed. Therefore, a continuous cycle of glial activation that results in release of IL-I and other cytokines may contribute both directly and indirectly to overall plaque pathogenesis and neurodegeneration in AD. In the PD hippocampi, no significant changes in IL-18 levels or [‘“‘flIL-l/l binding were evident. This suggests that induction of immune system mediators such as IL-lfi may be associated with specific pathology. Thus, whereas AD hippocampi are characterized by marked decrements in ChAT activityF9 the presence of numerous plaques’“,~‘,35,6’~63 and microglia (see above), PD hippocampi are not.4z~5s These hallmarks of pathology are evident in PD hippocampi only when there is marked dementia associated with the disease.9,55In PD cases in which no prominent

01

’

0

c , 5

’

’

10

15

s

1

20

25

.

’

IL-13 CONTENT

(fmol/mg protein)

OJ . 0

’ 5

’

( 10

IL-le

*

’

’

15

20

’

’

3

25

CONTENT

(fmol/mg protein)

Fig. 4. Analysisof correlationbetweenIL-IF content and that ofeither IL-2 (A) or IL-3 {B).Results are plotted as the individu~ values for each tissue. IL-l/Z levels for each individual tissue were plotted against the corresponding IL-2 (A) or IL-3 (B) value for the same tissue. The lines represent a computer-generated least-squares linear regression plot. No significant correlations were detected (P > 0.67).

751

dementia is noted, deficits in ChAT activity,’ as well as proliferation of microglia,42~5s with the subsequent enhancement of immune system mediators, appear to be restricted to the major site of pathology, the nigrostriatai pathway. Nevertheless, the tack of any significant correlation between reductions in ChAT activity and elevations in IL-l/l levels (and other ILs) in the AD hippocampi suggests that the two phenomena may be unrelated events. Thus, whereas induction of immune system mediators may correlate directly with mi~roglial proliferation and subsequent plaque deposition (see above), decrements in ChAT activity may be triggered, at least in some cases, by other mechanisms. Consistent with this, McGeer and colleaguesS5 reported that increases in microglial markers did not necessarily coincide with deficits in ChAT activity in the hipp~ampa1 formation in AD. Itlterleukin-2 and interleukin-2 receptors in the hippocampal formation

Until recently, the presence of IL-2 and its receptors in the intact CNS’,” had not been conclusively demonstrated. Although both appear to be elevated following CNS injury@ or lesions,? their status in neurodegenerative diseases has been investigated only recently. The present study demonstrates that measurable levels of IL-2 can be detected in the normal human brain (controls) and that these levels are significantly elevated in AD. Furthermore, although IL-1 has been shown to stimulate IL-2 synthesis in the immune system?‘3*74the lack of correlation between IL-l/l and IL-2 levels in the AD hippocampi suggests that the two may be independent phenomena. The primary sources of IL-2 in the human brain, particularly in AD and other neurological diseases, appear to be T-lympho~~es~“*~~66 Nevertheless, identi~cation of IL-2 in the intact rat brain’s49indicates that this lymphokine may also be localized to cells intrinsic to the CNS. Moreover, many of these IL-Zpositive cells also appear to express IL-2 receptors.49 Previous studies have reported increases in IL-2 receptor immunor~ctivity in AD brains,‘Z*Mbut no changes in soluble IL-2 receptors in eerebrospinal fluid.4SThe present study demonstrates that IL-2 binding is clearly increased in the hippocampal formation in AD. Interestingly, IL-2 binding was also enhanced in the PD hippocampi, even though IL-2 levels were not significantly changed. The reasons for the discrepancy between levels of the endogenous ligand and its receptor in the PD hippocampi are not known, However, since IL-2 receptors are localized to a variety of cell types that do not necessarily express IL-2 itself,‘~‘3*74.76 it is possible that an early involvement of immune cells other than microglia and T-lymph~ytes may occur in the PD hippocampal formation. Although the consequences of IL-2 and IL-2 receptor up-regulation to CNS function are not clear (for

reviews, see Refs 3 and 59), it is likely that various

152

D. M. Araujo and P. A. Lapchak

neuronal populations may be differentially altered. For example, IL-Z appears to reduce hippocampal neuronal survival,2 whereas it has been reported to enhance cortical neuronal viability.69 In addition, IL-2 decreases hippocampal acetylcholine7.19 and hypothalamic noradrenergic4’ neurotransmission, but it stimulates striatal dopaminergic46 and hypothalamic opiatergi?’ function.

studied, IL-6 may not be a key component in the “immune response” mounted in AD. This is supported by our finding that IL-6 levels were not different in the AD compared to the control hippocampal tissues.

Interleukin -3 and interleukin-6: peptide leveLs and receptor binding in the hippocampal formation

In summary, the present study implicates several ILs and their receptors in the pathogenesis of AD. The cascade of events resulting from activation of immune system mediators may ultimately contribute both directly and indirectly to the degeneration of cholinergic neurons, as well as other neuronal populations, that occurs in AD. Thus, direct effects on neuronal plasticity and neurotransmission are likely, as well as indirect neurotoxic effects mediated by /I-amyloid production and deposition into plaques. Since immune system activation was not evident in the PD hippocampi, the continuous cycle of glialneuronal interactions that escalate into hippocampal neuronal death are dependent on the presence of specific pathological insults, such as plaque formation. In conclusion, future potential therapeutic interventions in AD may consider focusing not only on the modulation of immune system activation, but also on the inhibition of mechanisms of immune system-mediated induction of plaque components.

IL-3” and IL-668~79 mRNAs have been detected in the CNS. Both factors appear to have similar effects on basal forebrain cholinergic3’~“~” and hippocampal6 neurons in vitro. Moreover, IL-3 secreted by astroglia appears to be a significant stimulus for the proliferation of microglia. 26.27The subsequent release of microglial-derived mitogens such as IL-l may result in astrogliosis, such as has been observed in AD (cf. Introduction). Thus, the increased levels of hippocampal IL-3 measured in the present study may be related to the proliferation of IL-3-positive astroglia. However, the lack of correlation between hippocampal IL-lb and IL-3 levels in the AD brains suggests that microglial and astroglial activation may be temporally separated events. Whereas IL-3 stimulates microglial proliferation. IL-6 in the CNS appears to function mainly as an autocrine factor for astroglia.‘8,79 In addition, IL-6 also stimulates nerve growth factor production by astroglia28 and has been implicated as an important factor in the tissue repair that follows CNS injury. Thus, IL-6 levels are known to be increased significantly following CNS trauma.‘* In contrast, the status of this lymphokine in neurodegenerative diseases has not been thoroughly investigated. Although IL-6 immunoreactivity has been demonstrated in the vicinity of senile plaques,” serum levels of the lymphokine do not appear to be elevated in AD patients. ‘l This suggests that unlike the other ILs

CONCLUSIONS

4cknow[edgernenrs_This work was supported by NIH Grant NS22933 and the National Parkinson Foundation. P.A.L. was supported by a grant from the French Foundation for Alzheimer Research. The Harvard Medical School Brain Tissue Resource Center was sunported in Dart by PHS grant MH/NS 31862. The Nationa
REFERENCES I. Arai K., Lee F., Miyajima

2. 3. 4. 5. 6. 7. 8. 9.

A., Miyatake S.. Arai N. and Yokota T. (1990) Cytokines: coordinators of immune and inflammatory responses. A. Rev. Biochem. 59, 783-836. Araujo D. M. (1992) Contrasting effects of specific lymphokines on the survival of hippocampal neurons in culture Adv, behav. Biol. 40, 113-122. Araujo D. M., Chabot J.-G. and Quirion R. (1990) Potential neurotrophic factors in the mammalian central nervous system: functional significance in the developing and aging brain. Int. Rev. Neurobiol. 32, 141-174. Araujo D. M. and Cotman C. W. (1992) b-Amyloid stimulates glial cells in vi&o to produce growth factors that accumulate in senile plaques in Alzheimer’s disease. Brain Res. 569, 141-145. Araujo D. M. and Cotman C. W. (1992) Basic FGF in astroglial, microglial, and neuronal cultures: characterization of binding sites and modulation of release by lymphokines and trophic factors. ./. Neurosci. 12, 1668-1678. Araujo D. M. and Cotman C. W. (1993) Trophic effects of interleukin-4, -7, and -8 on hippocampal neuronal cultures: potential involvement of glial-derived factors. Brain Rex. 600, 49-55. Araujo D. M., Lapchak P. A., Collier B. and Quirion R. (1989) Localization of interleukin-2 immunoreactivity and interleukin-2 receptors in the rat brain: interaction with the cholinergic system. Brain Res. 498, 257-266. Araujo D. M., Lapchak P. A., Robitaille Y., Gauthier S. and Quirion R. (1988) Differential alteration of various cholinergic markers in cortical and subcortical regions of human brain in Alzheimer’s disease. J. Neurochem. 51, 292-299. Aubert I., Araujo D. M., Cecyre D., Robitaille Y., Gauthier S. and Quirion R. (1992) Comparative alterations of nicotinic and muscarinic binding sites in Alzheimer’s and Parkinson’s diseases. J. Neurochem. 58, 529-541.

Interleukins in Alzheimer’s and Parkinson’s diseases

753

10. Ball M. (1985) Hippocampal histopathology: a clinical substrate for dementia of the Alzheimer type. In Interdisciplinary Topics in Gerontology (ed. von Hahn H. P.), pp. 16-37. S. Karger Press, Base], Switzerland. If . Baumann N., Baron Van Evercooren A., Jacque C. and Zalc B. (1993) Glial biology and disorders. Curr. @in. Neurol. Neurosurg. 6, 27-33. 12. Beaudet A.. Arauio D. M., Ouirion R. and LaDchak P. A. (1990) Immunoautaradio~~~c t~ti~tion of interleukin-2 receptors (Tat antigen) in rat and human b&n. Sot. A&o&~. Absrr. 16, 499. 15 _ 13. Blatteis C. M. (L990) Neuromodulative actions of cvtokines. Yule J. Bioi. Med. 63, 133-146. 14. Blume A. J. ani Vi&k M. P. (1989) Focusing on IL-i promotion of B-amyloid pre&or protein synthesis as an early event in Alzheimer’s disease. Neurobiol. Aging. 19, 406-408. 15. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of prolein utilizing the principle of protein-dye binding. Anafyf. 5iochem. 72, 248-253. t6. Cras P., Kawai M., Siedlak S. and Perry G. (1991) Microglia are associated with the extracellular neurofib~itary tangles of Alzheimer’s disease. Brain Res, 558, 312-314. 17. Dickson D. W., Lee S. C., Mattiace L. A.. Yen S. C. and Brosnan C. (1993) Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer’s disease. GIia 7, 75-83. IS. Dinarello C. A., Clark B. D., Ikejima T., Puren A. J., Savage N. and Rosff P. M. (1990) Interleukin 1 receptors and biological responses. Yale J. Biol. Med. 63, 87-93. 19. Donnelly R. J., Friedhoff A. J., Beer B., Blume A. J. and Vitek M. P. (1990) Interleukin-L stimulates the Beta-amyloid precursor protein promoter. Cell molec, Neurobiol. IO, 485-495. 20. Eikelenboom P., Hack C. E., Rozemuller J. M. and Stam F. C. (1989) Complement activation in amyloid plaques in Alzheimer’s disease. VircfiQws Archiv. B Cetf Parhol. 56, 259-262. 21. Etienne P., Robitalfle Y., Wood P., Gauthier S., Nair N. P. V. and Quirion R. (1986) Nucleus basalis neuronal Ioss, neuritic plaques and choline acetyltransferase activity in advanced Alzheimer’s disease. Neuroscience 19, 1279-1291. 22. Fagan A. M. and Gage F. H. (1990) Cholinergic sprouting in the hippocampus: a proposed role for IL-l. Expi Neural. 110, 105-120. 23. Farrar W. L., Hill J. M., Harel-Belan A. and Vinocur M. (1987) The immune logical brain. ~mmun. Reo. 100,361-378. 24. Farrar W. L., Kilian P. L., Ruff M. R., Hill J. M. and Pert C. B. (1987) Visualization and characterization of interleukin 1 receptors in the brain. .I. Immun. 139, 459-468. 25. Farrar W. L., Vinocur M. and Hill J. M. (1989) In situ histochemistry to~tization of interteukin-3 mRNA in mouse brain. Blood 73, 137-140. 26. Fontana A., Bodmer S. and Frei K. (1987) Immunoregulatory factors secreted by astrocytes and glioblastoma cells. Lymphokines 14, 91-122. 27. Frei K. and Fontana A. (1986) Astrocvte-derived IL-3 as a growth factor for micronlia and oeritoneal macroohaaes. . u 3. hmun. 11, 3521-3527. . 28 Frel K., Malipiero U. V., Leist T. P., Zinkernagel R. M., Schwab M. E. and Fontana A. (1989) On the cellular sour= and function of interieukin 6 produced in the central nervous system in viral diseases. Eur. J. immun. 19, 689-694. 29. Giulian D. (1987) Amoeboid microglia as effecters of inflammation in the central nervous system. J. Nct4rosei. Rex 1% 155-171. 30. Giulian D. and Lachman L. B. (1985) Interleukin-I stimulation of astrogliai proliferation after brain injury. Science 228, 497499. 31. Giulian D., Woodward J., Young D. J., Krebs F. and Lachman B. (1988) Interleukin-I injected into mammalian brain stimulates astro@iosis and neovascularization. J. Neurosci. 8, 2485-2490. 32. Giulian D., Young D. J., Woodward J., Brown D. C. and Lachman B. (1988) Interleukin-I is an astrogfial growth factor in the developing brain. J. Neurosci. 8, 709-714. 33. Goldga~r D., Harris H. W., Hia T., Maciag T., Donnelly R. J., Jacobsen J. S., Vitek M. P. and Gajdusek D. C. (1989) Interleukin 1 regulates synthesis of amyloid b-protein precursor mRNA in human endotheliat cells. Proc. natn. Acad. Sci. U.S.A. 86, 7606-7610. 34. Griffin W. S. T., Stanley L. C., Ling C., White L., Macleod V., Perrot L. J., White C. L. and Araoz C. (1989) Brain interleukin-1 and S-100 immunoreactivity are elevated in Down syndrome and Alzheimer disease. Proc. nutn. Acad. Sci. U.S.A. 86, 7611-7615. 3s. Haga S., Akai K. and Ishii T. (1989) Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain. Acta neuro~i~o~. 77, 569-575. 36. Haga S., Ikeda K., Sato M. and Ishii T. (1993) Synthetic Aizheimer amyloid @iA4 peptides enhance production of complement C3 component by cultured microglial cells. Bruin Res. 601, 88-94. 31. Hama T., Kushima Y., Miyamoto M., Kubota M., Takei M. and Hatanaka H. (1991) Interleukin-6 improves the survival of mesencephalic catecholaminergic and septal cholinergic neurons from postnatal, two-week-old rats in cultures. Neuroscience 40, 44-452. 38. Hama T., Miyamoto M., Tsukui H., Nishio C. and Hatanaka H. (1989) Interleukin-6 as a neurotrophic factor for promoting the survival ofcultured basal forebrain cholinergic neurons from postnatal rats. Neurosci. Left. 104,340-344. 39. Hanisch U.-K., Seto D. and Quirion R. (1993) M~utation of hipp~ampat a~tytchotin~ release: a potent central action of interleukin-2. J. Neurosci. i3, 3368-3374. 40. ftagaki S., McGeer P. L. and Akiyama H. (1988) Presence of T-cytotoxic suppressor and leucocyte common antigen positive cells in Alzheimer’s disease brain tissue. Neurosci. Letf. 91, 259-264. 41. Itagaki S., McGeer P. L., Akiyama H., Zhu S. and Selkoe D. (1989) Relationship of microglia and astrocytes to amyloid deposits in Alzheimer’s disease. J. Neuroimmun. 24, 173-182. 42. Itagaki S., McGeer P. L. and McGeer E. G. (1987) HLA-DR reactive microglia in Parkinson’s disease. J. Neuroimmun. 16, 81-89. 43. Johnson S., ~rn~rt-Et~he~]s M., Pasinetti G. M., Rozovsky I. and Finch C. E. (1992) Complement mRNA in the mammalian brain: responses to Alzheimer’s disease and ex~~mental brain lesioning. ~e~r~jol, Aging 13, 641-648. 44. Kamegai M., Niijima K., Kunishita T., Nishizawa M., Ogawa M., Araki M., Ueki A., Konishi Y. and Tabira T. (1990) Interleukin 3 as a trophic factor for central choline@ neurons in vitro and in r;iuo.Neuron 2, 429-436. 4s. Kittur 8. D., Klttur D. S., Soncrant T. T., Rapoport S. I., Tourtelotte W. W., Nagel J. E. and Adler W. H. (1990) Soluble interleukin-2 receptors in cerebrospinal fluid from individuals with various neurological disorders. Ann. Neural. 28, 168-173.

754

D. M. Araujo

and P, A. Lapchak

46. Lapchak P. A. (1992) A role for interleukin-2 in the regulation of striatal dopaminergic function, NeuroReport 3, 1655168. 47. Lapchak P. A. and Araujo D. M. (1993) Interleukin-2 regulates monoamine and opioid peptide release from the hypothalamus. Neuroffeporf 4, 303-306. 48. Lapehak P. A., Araujo D. M. and Hefti F. (1993) Systemic interleukin-1~ decreases brain-derived neurotrophic factor messenger RNA expression in the rat hippo~mpal formation. .~euros~~~~ce 53, 2X-301. 49. Lapchak P. A.. Araujo D. M., Quirion R. and Beaudet A. (1991) Immunoautoradiographic localization of interleukin 2-like immunoreactivity and interleukin 2 receptors (Tat antigen-like immunoreactivity) in the rat brain. Neuroscience 44, 173-184. 50. Licinio J., Wong M.-L. and Gold P W. (1992) Neutrophil-activating peptide-l/interleukin-8 mRNA is localized in rat hypothalamus and hippocampus. NeuroReport 3, 753-756. 51. Ling E. A., Dahlstrom A., Polinsky R. J., Nee L. E. and McRae A. (1992) Studies of activated microglial ceils and macrophages using Afzheimer’s disease cerebrospinal fluid in adult rats with ex~~mentaily induced lesions. ~euros&~ence 51, 815-825. 52. Luber-Narod J. and Rogers J. (1988) Immune system associated antigens expressed by cells of the human central nervous system. Neurosci. Lett. 94, 17-22. 53. McGeer P. L., Akiyama H., Itagaki S. and McGeer E. G. (1989) Immune system response in Alzheimer’s disease. Ccai. J. neural. Sci. 16, 516-527. 54. McGeer P. L., Akiyama H., ltagaki S, and McGeer E. G. (1989) Activation of the classical complement pathway . . in brain tissue of Alzheimer patients. Neurosci. Let!. 107, 341-346.’ 55. McGeer P. L., Itagaki S.. Boves B. E. and McGeer E. G. (19881 Reactive micro& are uositive for HLA-DR in the substantia nigra oT Parkinson’s and Aizheimer’s disease brains.’ Nearoiogy 38, I%-1291. 56. McGeer P. L., Itagaki S., Tago H. and McGeer E. G. (1987) Expression of HLA-DR and interleukin-2 receptors on reactive microglia in senile dementia of the Alzheimer type. 1. Neuroimmun. 16, 122-129. 57. McGeer P. L. and Rogers J. (1992) Anti-inflammatory agents as a therapeutic approach to Alzheimer’s disease. Neurology 42, 447-449. 58. McRae A., Ling E. A., Polinsky R.. Gottfries C. C. and Dahlstrom A. (1991) Antibodies in the cerebrospinal fluid of some Alzheimer’s disease patients recognize amoeboid microglial cells in the developing central nervous system. neuroscience 41, 7399752. 59. Merrill J. E. (1990) Interieukin-2 effects in the central nervous system. Ann iv.X Acud. Sci. 594, 188-199. 60. Nieto-Sampedro M. and Chandy K. G. (1987) Interleukin-Z-like activity in injured rat brain. Neurockem. Res. 12, 7233127. 61. Perlmutter L., Barron E. and Chui C. H. (1990) Morphologic association between microglia and senile _. plaque amyloid in Alzheimer’s disease. Neurosci. Lett. 119, 32-36. _ 62. Pike C. J.. Walencewicz A. J., Glabe C. G. and Cotman C. W. (1991) In uilro agina of O-amvloid protein causes _ peptide _ aggregation and neurotoxicity. Brain Res. 563, 31 l--314. - _ . 63. Probst A., Brunnschweiler H., Lautenschfager C. and Uhich J. (1988) A special type of senile plaque, possibly an initial stage. Acta neuropufhol. 74, 133- 14 1. 64. Rogers J., Cooper N. R., Webster S., Schultz J., McGeer P. L., Styren S. D., Civin W. II., Brachova L., Bradt B.. Ward P. and Lieberbura I. (1992) Complement activation by. a-amyloid in Alzheimer’s disease. Proc. natn. Acad. Sci. . U.S.A. 89, 10016-1020~ 65. Rogers J. and Luber-Narod J. (1988) Immune actions in the nervous system: a brief review with special emphasis on Alzheimer’s disease. Drug Devl Res. 15, 227-235. 66. Rogers J.. Luber-Narod J., Styren S. D. and Civin W. H. (1988) Expression of immune system-associated antigens by cells of the human central nervous system: relationship to the pathology of Alzheimer’s disease. ~~rob~o~. Aging 9, 339-349. 67. Rothwell N. 3. and R&on J. K. (1993) Involvement of cytokines in acute neurodegeneration in the CNS. Neurosii. Biobehaa. Rev. 17, 217-227. 68. Schobitz B.. Voorhuis D. A. M. and de Kloet E. R. (1992) Localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain. Neurosci. Left. 136, 1891192. 69. Shimojo M., Imai Y., Nakajima K., Mizushima S., Uemura A. and Kohsaka S. (1993) Interleukin-2 enhances the vjability of primary cultured rat neocortical neurons. Neurusci. Lett. 151, 170-173. 70. Spranger M., Lindholm D., Bandtlow C., Heumann R., Gnahn H., Naher-Noe M. and Thoenen H. (1990) ReguIation of nerve growth factor (NGF) synthesis in the rat central nervous system: comparison between the effects of interleukin-1 and various growth factors in astrocyte cultures and in &o. Eur. J. Neurosci. 2, 69-76. 71. Strauss S., Bauer J., Ganter U., Jonas U., Berger M. and Volk B. (1992) Detection of interleukin-6 and a-2 macroglobulin immunoreactivity in cortex and hippocampus of Alzheimer’s disease patients. Lab. Invest. 66, 223-230. 72. Taupin V., Toulmond S., Serrano A., Benavides J. and Zavala F. (1993) Increase in IL-6, IL-1 and TNF levels in rat brain following traumatic lesion. 1. Neuroimmun. 42, 177-186. 73. Van Duijn C., Hofman A. and Nagelkerken L. (1990) Serum levels of interleukin-6 are not elevated in patients with Alzheimer’s disease. ~eurosc~. Lerr. RI& 350-354. interleukin-2 receptor. A. Rev. Biochem. 58, 875-911. 74. Waldmann T. A. (1989) The multi-subunit 15. Walker D. G. and McGeer P. L. (1992) Complement gene expression in human brain: comparison between normal and Alzheimer disease cases. Molec. Brain Res. 14, 109-116. 76. Wekerle H., Linington C., Lassman H. and Meyermann R. (1986) Cellular immune reactivity within the CNS. Trends Neurosci. 9, 271-277. L., Oster-Granite M. L. and Neve R. L. (1989) Neurotoxicity 77. Yankner B. A., Dawes L. R., Fisher S., Villa-Komaroff of a fragment of the amyloid precursor associated with Alzheimer’s disease. Science 245, 417-420. and neurotoxic effects of amyloid fi protein: 78. Yankner B. A., Duffy L. K. and Kirschner D. A. (1990) Neurotrophic reversal by tachykinin neuropeptides. Science 250, 279-282. 19. Yasukawa IL., Hirano T., Watanabe Y., Muratani K., Matsuda T., Nakai S. and Kishimoto T. (1987) Structure and expression of human B ceil stimulatory factor-2 (BSF-2/IL-6) gene. Eur. molec. Biof. Org. J. 6, 2939-2945. (Accepted 15 January 1994)