ELSEVIER Neuroscience Letters 188 (1995) 70-74

H[HOSCIgiC[ [EIT[IIS

Reciprocal control of inflammatory cytokines, IL-1 and IL-6, and fl-amyloid production in cultures

Roberto Del Bo, Nadia Angeretti, Elisa Lucca, Maria Grazia De Simoni, Gianluigi Forloni*

Istituto di Ricerche Farmacologiche 'Mario Negri', via Eritrea 62, 20157 Milano, Italy

Received 1 December 1994; revised version received 25 January 1995; accepted 6 February 1995

Abstract

To investigate the role of IL-6 in the pathogenesis of Alzheimer's disease (AD) its effect on amyloid precursor protein (APP) mRNA expression was evaluated. The levels of APP mRNA were determined by Northern blot analysis in primary cultured rat cortical neurons and glial cells exposed to IL-6 (50-200 ng/ml). The cytokine increased neuronal APP mRNA expression about 100% at the highest dose after 6 h of exposure. APP mRNA expression was unaffected in astroglial cells exposed to IL-6. Since IL-lfl also increased neuronal APP mRNA, the combination of IL-lfl and IL-6 was tested. The effects were partially additive. The ability of fl-amyloid fragment 25-35 to induce IL-1 or IL-6 mRNA was also investigated in astroglial cells. IL-1/3 mRNA was strongly induced by/325-35 (25-100ktM) while the expression of IL-6 mRNA remaining unchanged. The results suggest roles for both IL-1 and IL-6 in the neuronal mechanisms related to fl-amyloid protein deposition in AD.

Keywords: Alzheimer's disease; fl-Amyloid; Astrocytes; Gene expression; Cytokines

Alzheimer's disease (AD) is neuropathologically char- acterized by senile plaques, neurofibrillary tangles and cerebrovascular amyloidosis (for review see Ref. [29])./3- Amyloid (flA), aggregating in /3-pleated sheets, is the major component of the senile plaques (SP) and is depos- ited in cortical and meningeal blood vessels. This 39-43 residue polypeptide derives from the proteolysis of a larger transmembrane glycoprotein precursor with multi- ple isoforms generated by differential splicing of a gene mapped on the human chromosome 21 and highly con- served across the species. The deposits of/3A are associ- ated with degenerative changes of neuronal cells and neu- ronal death. Numerous findings suggest a causal role of /3A in the pathogenesis of AD. Yankner et al. [31] de- scribed a neurotoxic effect of synthetic flA and its frag- ments in vitro and linkage studies have shown an associa- tion between/3A precursor protein (APP) gene mutations and some cases of familial early-onset AD [ 14].

Chronic inflammatory activity sustained by altered immune responses may well be involved in the patho- genesis of AD. Many immune system proteins, including inflammatory cytokines, acute phase proteins and com-

* Corresponding author, Tel.: +39 2 39014462; Fax: +39 2 3546277.

plement factors, have been identified in the AD brain, where they are associated with senile plaque tangles and dystrophic neurites [23]. The accumulation of cytokines IL-1 [16] and IL-6 [3,8] in AD does not appear to be merely a consequence of the degenerative processes, but may play a role in the cascade of events inducing neu- ronal death. The presence of IL-1 in AD might possibly be associated with the accumulation of/3A. We have re- cently demonstrated that APP mRNA is induced by IL-1 in neuronal but not astroglial cells [11], while IL-1 excre- tion in glial cells is activated by a synthetic homologue of /3A 1-42 [2]. Furthermore, Buxbaum et al. [6] have shown the ability of IL-1 to regulate the processing and secretion of APP in PC12.

Many IL-1 actions are mediated by IL-6 which plays a major role in the acute phase protein synthesis. In the CNS, IL-1 induces the synthesis of IL-6 in astrocytes and microglia [18]. Since no information is available on whether IL-6 direct controls APP production in primary tissue cultures, we investigated how IL-6 induces APP mRNA in neuronal and astroglial cells and the conse- quence when cortical neurons where exposed simultane- ously to both cytokines. Finally we analyzed the capacity of a biologically active fragment of/3A,/325-35 [9], to

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R. Del Bo et al. I Neuroscience Letters 188 (1995) 70-74 71

induce the expression of IL-1 and IL-6 mRNA in astro- glial cells.

Brains were removed from fetal rats at embryonic day 17. Cortical cells were dissociated in serum-free medium containing 0.1% trypsin (DIFCO) and 25 ~g/ml deoxyri- bonuclease for 5 min .'it room temperature, and plated in Primaria (Falcon) 35-mm dishes, pre-coated with poly-D- lysine (50/ag/ml; SIGMA), 106 cells/dish, in basal Ea- gle's medium (BME-Hanks' salt, GIBCO) supplemented with 10% fetal calf serum (FCS, GIBCO) and glutamine (2 raM). Cultures were kept at 37°C in a humidified CO2 atmosphere. After 5-7 days in vitro, non-neuronal cell division was halted by exposure to 10 -5 M cytosine arabi- noside. IL-1 (human IL-1/3) and IL-6 (25-200ng/ml) were added once after 9 days of culture.

Glial cell cultures were taken from newborn rat pups. Using a sterile technique, each pup was decapitated, and the brain was removed and placed in c Petri dish. The tissue was separated from meninges and dissociated by trituration with a Pasteur pipette. Glial cells were grovcn to confluence (3 weeks) in Primaria (Falcon) dishes, in Dulbecco's modified minimal essential medium (DMEM; GIBCO) supplemented with 10% FCS and 2 mM glu- tamine, and cultured at 37°C in a water-saturated atmos- phere of 95% air/5% (;02. The massive presence of astro- cytes (>90%)in our preparation was investigated by im- munocytochemistry [1t0]. All results reported were ob- tained from cells cultured for 2 weeks.

Total cellular RNA was isolated according to the acid guanidinium/phenol/chloroform (AGPC) method de- scribed by Chomczynski and Sacchi 1987 [7]. Using a denaturing solution containing 4 M guanidinium thiocy- anate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 0.1 M 2-mercaptoethanol, the lysate was extracted twice with 0.1 M sodium acetate (pH 4)/phenol/49:l chloro- form-isoamyl alcohol mixture (1:10:2), and nucleic acids were precipitated with equal volumes of isopropyl alco- hol. Northern blot analysis was done as described by Maniatis et al. [21]. Total RNA was separated on 1.2% agarose-formaldehyde gels, transferred to Nylon 66 filters (Gene Screen Plus, Du Pont). On the basis of spectro- phometric analysis an equal amount of total RNA was loaded on each line (15-20/~g).

The total APP mRNA probe was a 1.0-kb E c o R I frag- ment of a mouse cDNA clone representing the/3-amyloid and the proximal 3'-untranslated portion of the APP mRNA [4]. APP695 probe was a 40-base oligodeoxynu- cleotide (GGCTGCCGTCGTGGGAACTCGGACTACC- TCCTCC) made on a Beckman 200 A DNA synthesizer. The/3-actin probe was a 0.8 kb fragment from a human cDNA clone corresponding to the sequence published by Hanuklogu et al. [17]. IL-6 mRNA probe corresponding to a 0.65 kb fragment from mouse cDNA [27]; IL-1/3 mRNA probe corresponding to a 1.3 kb fragment from human cDNA [15], GFAP mRNA probe corresponding to a 2.5 kb fragment from human cDNA [19]. cDNA probes

were labeled using a randomly primed DNA labeling kit from Amersham and [32p]dCTP, the oligonucleotide was labeled by 3'-end labeling deoxynucleotidyltransferase (Bethesda Research Laboratories) [32p]dATP. All labeled probes were purified through a Sephadex G-50 column (Pharmacia). Membranes were prehybridized overnight at 42°C in a solution containing 50% formamide, 1% SDS, 1 M sodium chloride and 10% dextran sulphate and 100 ~g/ml of denatured salmon sperm DNA, then hybrid- ized with the appropriate 32p-labeled cDNA probe (0.8- 1 x 106 cpm/ml) at 42°C for 16-18 h.

Blots were washed first in 2 x SSC and 0.05% SDS at room temperature and then in 2 x SSC and 0.1% SDS (1% for oligonucleotide) at 65°C. The blots were exposed to X-ray film at -80°C with intensifying screens for the time necessary for the signal to be in a linear range for quantification. The exposure time was the same in all experiments for each probe. Densitometric analysis of autoradiograms was done with an IBAS 2 image analyzer (Zeiss) integrating the optical density with the area of the hybridized bands [22]. The signal associated with the presence of/3-actin mRNA was used as an internal stan- dard to normalize APP, IL-1/3 and IL-6 expression.

IL-6 mRNA was also determined by PCR amplifica- tion: 1/tg of total RNA was reverse transcribed into the complementary DNA strand (cDNA) using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Perkin-Elmer) according to the manufacturer's protocol. The cDNA amount corresponding to 50 ng total RNA was used for PCR amplification. The primers and cycles used in the PCR reaction were selected according to Ga- dient and Otten [13],/3-actin expression serving as con- trol.

Northern blot analysis of RNA extracted from cortical neurons exposed to IL-6 (50-200 ng/ml) at the optimal time of 6h, established in preliminary experiments, showed a dose-dependent increase of total APP mRNA expression (Fig. 1A). The maximal effect, more than 100% compared to the control, was detected at 200 ng/ml; the increase at 100 ng/ml was similar but the variability was higher in this group. An identical pattern of induction was observed when only the expression of APP695 tran- script was considered, using a specific oligoprobe (data not shown). These data indicate a direct control of IL-6 on APP expression, suggesting a potential role in amyloi- dogenesis. The inducibility of APP gene expression by IL-6 was previously suggested [28] in view of consensus sequence found in the promoter region of most acute phase proteins induced by IL-6. According to this hy- pothesis, Altstiel and Sperber [1] have shown an increase of synthesis of specific isoforms of APP contain a domain homologous to Kunitz serine protease inhibitors in PC 12 cells exposed to IL-6. The levels of APP mRNA in cul- tured astroglial cells were comparable to the expression in neurons [ 11] but in astrocytes, the APP mRNA expression was not altered by exposure to IL-6 (Fig. 1B). Thus, both

72 R. Del Bo et al. / Neuroscience Letters 188 (1995) 70-74

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Fig. 1. Effect of IL-6 on APP mRNA expression. Densitometric quanti- fication of Northern blot analysis of total RNA extracted from cortical neuron cells exposed to IL-6 for 6 h (A) or astroglial cells exposed to IL-6 (200 ng/ml) (B) and hybridized with probes recognizing/5-actin and total APP mRNA./5-Actin mRNA was used as internal standard. The data are the means _+ SE of 7-12 determinations, **P < 0.01 versus control group (Dunnett's test).

IL-6 and IL-1 [11] activate APP mRNA in neurons but

not in glial cells. To identify a common mechanism of action we tested

the effect of simultaneous exposure of cortical cells to both cytokines. IL-1 (100ng/ml) increases the APP mRNA levels by about 50%, while IL-6 at the maximal dose (200 ng/ml) raised then about 80%. When the cells

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Fig. 2. APP mRNA in cortical neurons exposed to IL-6 and IL-1. Total RNA was extracted from cortical neurons after 6 and 14 h of exposure to IL-6 (200 ng/ml) and IL-I (100 ng/ml), respectively. The data are the means _ SE of ¢-6 determinations, **P < 0.01 versus control group (Dunnett's test).

were exposed to both cytokines the effect was approxi- mately the sum of the single effects (130%). Northern blot analysis suggested a distinct mechanism of neuronal APP mRNA induction by IL-1 or IL-6. Thus, IL-1 and IL-6 might contribute independently to the APP produc- tion and promote flA deposition in AD. Since IL-1 in- duced IL-6 expression in astrocytes and microglia in AD, the immune reaction may be triggered by IL-1, our data indicate that the production of IL-6 further amplifies the

consequence on APP expression. Moreover, recent find- ing showed that in AD temporal cortex IL-6 was elevated, but there was no significant accumulation of IL-1 in this

area [30]. The role of IL-1 and indirectly of IL-6 in the cellular

mechanisms related to flA deposition is further supported by the results depicted in Fig. 3, where astroglial cells

were exposed to the biologically active fragment fl25-35. The expression of IL-lfl, IL-6 and glial fibrillary acidic

A

actin

IL l

Astroglial cells

I ~ 25-35 I~ 25-35 50 gM 25 gM controls

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8004

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I ZOO-

IL-1B mRNA [ ] GFAP mRNA

Control 2 Sp M S OM M 1001/M

g 25-35

Fig. 3. Effect of/525-35 on IL-I/5 and GFAP mRNA expression in astroglial cells. (A) Example of Northern blot of total RNA extracted from astroglial cells treated with/525-35 and hybridized with/5-actin, IL-I/5 and GFAP probes. (B) Dose-response effect of/525-35 on IL-1/5 and GFAP mRNA expression. IL-6 mRNA was also measured but no signal was detectable in any group. Total RNA was extracted from astroglial cells after 14 h of exposure to /525-35. The data are the means + SE of 8-12 determinations, **P < 0.01 versus control group (Dunnett' s test).

R. Del Bo et al. / Neuroscience Letters 188 (1995) 70-74 73

protein (GFAP) mRNA was evaluated after 24 h exposure to fl25-35 (25, 50, 100~M). The expression of IL-1 mRNA was progressively increased from 2-fold at 25/tM to 5-fold at 50/~M and 9-fold at 100/tM fl25-35 (Fig. 3) but no induction of GFAP mRNA was observed. Densi- tometric quantification corrected for the constitutively expressed fl-actin mRNA showed a robust and specific activation of IL-1 mRNA by fl25-35. No signal was de- tectable for IL-6 mRNA either in control or in f125-35- treated cells on Northern blot analysis. The PCR amplifi- cation technique demonstrated the presence of IL-6 mRNA in astroglial cells but the signal was not affected by exposure to fl25-35 (data not shown).

Astrocytes and microglia are components of SP and our data indicate that in AD, gliosis provoked by an initial neurodegenerative process may activate a cyclic mecha- nism mediated by IL-1 directly or through IL-6 which increases the neuronal production of APP while flA in- duces the expression of IL-1. Araujo and Cotman [2] ob- served an increase of IL-lfl secretion from cultures of astrocytes and microglial after the exposure to a synthetic homologue of the entire flA protein (fll-42). They also reported that f l l -42 stimulated the proliferation of mi- croglia, whereas we found fl25-35 had negligible effect on astrocytes proliferation [10].

The relationship between flA and immunological re- sponse in AD is further supported by Rogers et al. [24] who reported the capacity of flA to activate complement. They suggested that/~A may be necessary but not suffi- cient in AD pathogenesis and full complement activation is a crucial step in the passage between simple flA depos- its and flA associated with neuritic damage. Several im- portant markers of immune function, including cytokines, complement factors and complement receptors, have now been demonstrated in AD brain tissue, co-localized with AD pathological strucI:ures.

The concept that the accumulation of immune system factors is not a simple consequence of the degenerative processes, but may play a role in the cascade of events inducing neuronal death in AD stimulated clinical inves- tigations to verify the efficacy of anti-inflammatory drugs in AD patients [26]. Numerous epidemiological observa- tions [5,12], includinG; our retrospective study [20], have found a negative association between anti-inflammatory drug use and AD. A pilot study with the classical non- steroidal anti-inflammatory drug indomethacin, did find a reduction of cognitive decline in treated AD patients [25]. However, more specific pharmacological approaches and larger clinical trials are necessary to prove the pathoge- netic role of inflammatory markers in AD and their clini- cal relevance.

In conclusion, our ~esults indicate that IL-6 can induce APP mRNA expression in neurons but not in glial cells and this action seems independent of IL-1. In turn, IL-lfl mRNA, but not IL-6 mRNA, is induced by flA fragment 25-35 in astroglial cells. Both inflammatory cytokines

appear involved in flA production, suggesting a causal role of the immunological response in AD pathogenesis.

This work was supported by Consiglio Nazionale delle Ricerche, Rome, Italy, c. n. 93.0174.CT04 and Convenzi- one Psicofarmacologia.

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