Brain Research 985 (2003) 89–97www.elsevier.com/ locate/brainres

Research report

C ompromised reactive microgliosis in MPTP-lesioned IL-6 KO mice*Hernan Cardenas, Laurel M. Bolin

The Parkinson’ s Institute, 1170 Morse Avenue, Sunnyvale, CA 94089,USA

Accepted 9 June 2003

Abstract

Reactive gliosis, the cellular manifestation of neuroinflammation, is a pathological hallmark of neurodegenerative diseases includingParkinson’s disease. The persistent gliosis observed in the Parkinson’s disease substantia nigra (SN) and in humans and animals exposedto the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) may represent a chronic inflammatory response thatcontributes to pathology. We have previously shown that in the absence of interleukin-6 (IL-6) dopaminergic neurons are more vulnerableto MPTP. Since IL-6 is both an autocrine and paracrine proliferation factor for CNS glia, we investigated reactive gliosis inMPTP-lesioned IL-6 (2 /2) mice. While astrogliosis was similar in injured IL-6 (1 /1) and IL-6 (2 /2) SN pars compacta (pc),microgliosis was severely compromised in IL-6 (2 /2) mice. In the absence of IL-6, an acute reactive microgliosis was transient with acomplete absence of reactive microglia at day 7 post-lesion. Extensive reactive microgliosis was observed in the SNpc of MPTP-lesionedIL-6 (1 /1) mice. Because glial derived inducible nitric oxide synthase (iNOS) has been implicated in dopaminergic cell death, weexamined glial iNOS expression in the IL-6 genotypes to determine if it correlated with the greater vulnerability and reduced microgliosisobserved in the MPTP-lesioned IL-6 (2 /2) nigrostriatal system. Both reactive microglia and astrocytes expressed iNOS in the lesionedSNpc. In the IL-6 (2 /2) mice, microglial iNOS expression diminished as reactive microgliosis declined. The data suggest IL-6 regulationof microglia activation, while iNOS expression appears to be secondary to cell activation. 2003 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative disease: Parkinson’s

Keywords: IL-6; Astrogliosis; Microgliosis; MPTP; Nigrostriatal degeneration; iNOS

1 . Introduction While reactive astrocytes and microglia have been iden-tified in MPTP-lesioned and Parkinson’s disease SN, their

Although the neuronal deficit in Parkinson’s disease has possible contribution to pathogenesis has not been de-long been known, the process that leads to dopaminergic termined.cell death is still unknown. The loss of dopaminergic MPTP-lesioning of both mice and non-human primatesneurons in parkinsonian substantia nigra (SN) is accom- has been widely used to model the selective dopaminergicpanied by reactive gliosis[3,16,20]. While this cellular cell injury, which occurs in Parkinson’s disease. In theactivation is a normal response in injured CNS tissue, its mouse model of MPTP-induced nigrostriatal degeneration,persistence in Parkinson’s disease has led to speculation reactive gliosis has been characterized with both reactiveregarding immunotoxicity[31]. Examination of the SN of astrogliosis and microgliosis observed in the striatum andhumans, who self-administered the neurotoxicant 1- SN[17,22,24,25,34,37].This reactive gliosis is the cellularmethyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 3–16 manifestation of the complex cascade of neuroinflamma-years prior to death, supports the possibility of a chronic tion. Multiple cytokines, growth factors and chemokinesneuroinflammation long after the exogenous injury[26]. that drive reactive gliosis, could contribute to an immuno-

toxic or an immunoprotective milieu. Interleukin-6 (IL-6),a major regulatory cytokine in the primary immune*Corresponding author. Tel.:11-408-542-5611; fax:11-408-734-response, has pro- and anti-inflammatory properties as well8522.

E-mail address: lbolin@thepi.org(L.M. Bolin). as neurotrophic activity[5,7,30,40].We have previously

0006-8993/03/$ – see front matter 2003 Elsevier B.V. All rights reserved.doi:10.1016/S0006-8993(03)03172-X

90 H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97

shown that the nigrostriatal system of IL-6 (2 /2) mice is 2 .3. Immunohistochemistrymore vulnerable to MPTP neurotoxicity, with far greaterstriatal dopamine depletion and dopaminergic neuron loss The SN was serially sectioned (12mm) in its entiretythan in control mice[6]. We now report that reactive and frozen sections were mounted on gelatin coatedmicrogliosis is severely compromised in MPTP-lesioned microscope slides. Each slide contained three sections for aSN pars compacta (SNpc) of IL-6 (2 /2) mice. The total of 10 slides, which were stored under desiccation atexpression of iNOS protein is concomitant with both 280 8C. Frozen sections were fixed in ice cold 4%astrocyte and microglia activation and is thus diminished paraformaldehyde immediately upon removal from freezerin lesioned SNpc of IL-6 (2 /2) mice but not in IL-6 followed by two 3 min washes in Hank’s balanced salt(1 /1) control mice. These data have implications for the solution (HBSS) (Life Technologies, Grand Island, NY,immunoprotective role of IL-6 and microglia as well as the USA) plus 0.01% saponin (Sigma, St. Louis, MO)contribution of reactive nitric oxide (NO) to nigrostriatal (HBSS1S) and 1% goat serum (Life Technologies). Afterinjury. An understanding of cell-specific mechanisms of wash, sections were incubated in blocking buffer [(1 mlgliosis and the contributions of specific immunoeffector HBSS: 320ml 1 M NaN : 10 ml H O (all from Sigma)]3 2 2

cells to the inflammatory milieu in the degenerating SN for 30 min, followed by a second 10-min block in HBSS1

could lead to anti-inflammatory therapeutics to inhibit S plus 10% goat serum. Alternate slides were incubated inimmunotoxicity in Parkinson’s disease. anti-mouse Mac-1 and the remaining slides were incubated

in anti-mouse GFAP diluted in buffer [HBSS1S11% goatserum] for 48 h at 48C. Immunoreactivity was detected by

2 . Materials and methods the peroxidase-antiperoxidase (PAP) method. Briefly, sec-tions were washed and incubated for 30 min with rat IgG

2 .1. Animal tissue (1:200) to detect Mac-1 or mouse IgG (1:600) to detectGFAP. After rinsing, slides were incubated with rat PAP

Male mice of two different IL-6 genotypes [i.e. wildtype (1:300) or mouse PAP (1:800) (all from Sternberger(1 /1) and knock-out (2 /2)] on a C57Bl /6 genetic Monoclonals, Lutherville, MD, USA) for 30 min at roombackground were bred at the Parkinson’s Institute as temperature. Slides were washed and colorimetric signaldescribed[6]. Briefly, founder IL-6 (2 /2) males on a was developed using metal enhanced diaminobenzidineC57Bl /6 background were bred with C57Bl /6 wild type according to manufacturers instructions (DAB, Pierce,females and progeny were backcrossed to establish IL-6 Rockford, IL, USA). After dehydration through a series of(2 /2) and IL-6 (1 /1) colonies of genetic homogeneity graded alcohols [70% ETOH, 95% ETOH, 100% ETOH,]on a C57Bl /6 background. At 10–12 weeks of age, both followed by xylenes, slides were coverslipped in DePexgenetic groups i.e. IL-6 (1 /1) and IL-6 (2 /2) mice, mounting medium (Electron Microscopy Sciences,received a single subcutaneous (s.c.) injection of either Washington, PA, USA). Sections were viewed and photo-physiologic saline (n59) or MPTP [30 mg/kg] (n515). graphed with a Nikon Eclipse E 400 microscope fitted withAnimals were sacrificed by cervical dislocation 1, 2 and 7 a Nikon FDX-35 mm camera (Melville, NY, USA).days post-injection. The midbrain was blocked, snapfrozen in isopentane and stored at280 8C. 2 .4. Immunofluorescence

2 .2. Antibodies Tissue for dual-labeled fluorescence was fixed andblocked as described above. Each primary antibody (anti-

Microglia were detected using three antibodies. A rat GFAP and anti-Mac-1) was simultaneously incubated withanti-mouse Mac-1 antibody that is specific for theaM anti-iNOS (Transduction Laboratory) diluted in buffersubunit of the b2 integrin receptor (1:1000; Roche, (HBSS1S) for 2 h at room temperature, followed by threeIndianapolis, IN, USA); a rat anti-mouse CD11b, which is 5-min washes in buffer. Sections were sequentially incu-specific for thebM subunit of theb2 integrin receptor bated for 30 min with donkey anti-mouse Cy3 conjugated(1:100; Serotec, Raleigh, NC, USA)[35,36] and CD45 IgG (1:2000) for GFAP and donkey anti-rat Cy3 conju-(1:50; BD Pharmingen, San Diego, CA, USA), the gated IgG (1:2000) for Mac-1 detection (all fromleukocyte common antigen, which recognizes all monocyte Chemicon). A donkey anti-rabbit fluorescein isothio-lineage cells [39]. Astrocytes were detected using an cyanate-conjugated IgG (1:100; Jackson Immunoresearch,antibody that recognizes glial fibrillary acidic protein West Grove, PA) was used for iNOS detection. Sections(mouse monoclonal anti-mouse GFAP, 1:800; Sternberger were washed with buffer three times for 15 min betweenMonoclonals, Lutherville, MD, USA) [12]. To detect sequential incubations of secondary antibodies. SectionsiNOS, two different polyclonal antibodies were used were coverslipped with fluorescent mounting medium(rabbit anti-mouse iNOS; 1:100; Chemicon, Temecula, (Dako, Carpinteria, CA, USA) and images were visualizedCA, USA) and (rabbit anti-mouse iNOS, 1:100; Transduc- and captured with a laser scanning confocal microscopetion Laboratory, San Diego, CA, USA). Zeiss LSM 5 PASCAL (Thornwood, NY, USA).

H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97 91

2 .5. Quantitation reactive microglia were observed in the SNpc of both IL-6genotypes at 1 day (Fig. 1B,F). At 7 days, there was robust

Sections of SN from each genotype were examined for microgliosis in the SNpc (Fig. 1C) with greatly enlargedspecific glial quantification at 7 days post-lesion; MPTP cell bodies and numerous processes (Fig. 1D, arrow). This[n55/genotype], saline [n53/genotype]. Cells in the persistent MPTP induced microgliosis was observed in theSNpc were counted in every third section from each SNpc and the ventral tegmental area of IL-6 (1 /1) mice.experimental group. GFAP positive astrocytes with clearly However, reactive microglia were completely absent at 7visible cell bodies and a minimum of two processes were days post-lesion in the IL-6 (2 /2) SNpc (Fig. 1G). Thiscounted in at least five sections per animal. In the same impaired microgliosis was not due to a lack of microglia innumber of sections per animal, Mac-1 positive microglia IL-6 (2 /2) mice, as resident articulated microglia werewith enlarged cell bodies and thick processes were counted present in the SN reticulata (Fig. 1G,H,arrowhead). These.

Cells were counted at a magnification of 203. Statistical ‘resting’ microglia were also observed in both genotypesanalysis was performed by Student’st-test and one-way under all conditions. To exclude the possibility thatANOVA. detection of reactive microglia was limited due to a down-

regulation of Mac-1 expression, SN sections were incu-bated with antibodies to CD45 and CD11b at 2 days post

3 . Results MPTP-lesioning. Both of these markers detected similarreactive microgliosis in MPTP-lesioned SNpc of IL-6

3 .1. Microgliosis in MPTP-lesioned SN of IL-6 (1 /1) genotypes. As observed with the Mac-1 antibody, reactiveand IL-6 (2 /2) mice microgliosis was more pronounced in IL-6 (1 /1) SNpc as

determined by CD45 or CD11b antibodies (Fig. 2A,B),To investigate MPTP-induced reactive gliosis, immuno- compared to the IL-6 (2 /2) SNpc (Fig. 2C,D).

histochemistry was performed on sections of both IL-6genotypes at 1, 2 and 7 days post-injection. Reactive 3 .2. Astrogliosis in MPTP-lesioned SN of IL-6 (1 /1)microgliosis was evaluated at these time points by examin- and IL-6 (2 /2) miceing the expression of Mac-1. As seen inFig. 1A,E,a singles.c. injection of saline did not elicit microglia activation 7 Reactive astrogliosis was evaluated at 7 days becausedays post-treatment or at any other time points (data not others have found that astrocyte activation is at its peak 1shown). In MPTP-lesioned mice, moderate numbers of week after MPTP intoxication[33,36]. Reactive astrocytes

Fig. 1. Microgliosis in IL-6 (1 /1) and IL-6 (2 /2) SN. No reactive microglia were observed in SNpc of saline treated IL-6 (1 /1) (A) or IL-6 (2 /2)1(E) mice. In MPTP-lesioned IL-6 (1 /1) (B) and IL-6 (2 /2) (F) SNpc, comparable Mac-1 reactive microglia were observed at 1 day. At 7 days post

MPTP-lesioning, there was vigorous microgliosis in the IL-6 (1 /1) SNpc (C). This reactive microgliosis was manifest by enlarged cell bodies with shortprocesses (D, arrow). In the IL-6 (2 /2) mice (G), reactive microglia were not seen, although resident microglia were detected in the SN reticulata (G,arrowhead). These ramified microglia have small cell bodies with articulated processes (H, arrowhead). Scale bars represent 120mm (A,B,E,F), 300mm(C,G) and 25mm (D,H).

92 H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97

Fig. 2. Microgliosis in SNpc IL-6 (1 /1) and IL-6 (2 /2) mice 2 days post MPTP-lesioning. In IL-6 (1 /1) SNpc CD11b (A) and CD45 (B)immunopositive microglia were detected in the SNpc. In IL-6 (2 /2) SNpc there were fewer CD11b (C) and CD45 (D) immunopositive microglia. Scalebar represents 100mm.

that expressed glial fibrillary acidic protein (GFAP) were (1 /1) mice, there was a significant increase (P,0.0001)counted in the SNpc of saline and MPTP injected mice of reactive astrogliosis in the SNpc of MPTP-lesioned(Table 1). Cell counts were based on two criteria; the mice compared to saline injected controls (Table 1). Thisrobust expression of GFAP protein and the morphology of was true for the IL-6 (2 /2) mice as well, with athe astrocytes, which have a stellate appearance with a significant increase (P,0.01) in SNpc astrogliosis inminimum of two processes greater than twice the diameter response to MPTP. In both genotypes, MPTP-lesioningof the soma. While only SNpc reactive astrocytes were resulted in similar reactive astrocyte numbers, with acounted, astrogliosis was observed throughout the SN. In 50–55% increase compared to saline injected controls.response to a single s.c. injection of saline, there were a Although there were significant differences within each

1moderate number, approximately 30 GFAP cells per genotype between saline and MPTP injected mice, theresection, of reactivate astrocytes in the SNpc of IL-6 (1 /1) were no differences in reactive astrogliosis between theand IL-6 (2 /2) mice. However, MPTP-lesioning in- two genotypes at 7 days.creased the number of GFAP positive cells. In the IL-6

3 .3.T able 1 iNOSinductioninSNofMPTP-lesionedIL-6(1 /1)Reactive gliosis in the SNpc and IL-6 (2 /2) mice

Astrocytes Microglia

To investigate iNOS expression, which has been impli-IL-6 (1 /1) Saline 3566 0IL-6 (1 /1) MPTP 7763*** 3367 cated in dopaminergic cell death, SN sections were dualIL-6 (2 /2) Saline 3266 0 labeled with iNOS and glial-specific antibodies. IL-6 (1 /1)IL-6 (2 /2) MPTP 64611* 0 and IL-6 (2 /2) sections from MPTP-lesioned SNpc at 1Reactive glial cells expressing either GFAP (astrocytes) or Mac-1 (Fig. 3A,B) and 7 days (Fig. 3C,D) were incubated in(microglia) were counted 7 days after a single injection of saline or 30 antibodies to Mac-1 or iNOS. At 1 day in both the IL-6mg/kg MPTP. Values represent the mean6S.E.M. of three saline treated

(1 /1) SNpc (Fig. 3A) and the IL-6 (2 /2) SNpc (Fig.mice and four MPTP treated mice.3B), iNOS was colocalized with engorged microglia in*** P,0.0001, MPTP vs. saline IL-6 (1 /1); * P,0.01, MPTP vs.

saline IL-6 (2 /2); one-way ANOVA. MPTP-lesioned but not in saline injected mice (data not

H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97 93

1 1Fig. 3. Colocalization of iNOS in Mac-1 microglia in MPTP-lesioned SNpc. At 1 day post-lesion, Mac-1 (red) reactive microglia (A,B) of both IL-6genotypes expressed iNOS (green). At 7 days iNOS was colocalized in activated microglia in IL-6 (1 /1) SNpc (C) and was occasionally observed in

1‘resting’, articulated Mac-1 microglia located in the IL-6 (2 /2) SN reticulata (D). Scale bar represents 25mm.

shown). Reactive iNOS immunopositive microglia were Reactive astrocytes also expressed iNOS in MPTP-also observed 2 days after lesion (data not shown). At 7 lesioned SNpc. At 1 day (Fig. 4) and 2 days post-lesion

1days post MPTP-lesioning, similar colocalization was seen iNOS was colocalized in SNpc GFAP astrocytes in thein the IL-6 (1 /1) SNpc (Fig. 3C), however in the IL-6 IL-6 (1 /1) (Fig. 4A) and IL-6 (2 /2) mice (Fig. 4B).(2 /2) SNpc no reactive microglia were observed. Only This was also true at 7 days after neurotoxicant lesioning‘resting’, articulated microglia (Fig. 3D), located in the SN in both IL-6 (1 /1) (Fig. 4C) and IL-6 (2 /2) (Fig. 4D)reticulata, appeared to express iNOS in IL-6 (2 /2) SN at mice. The fluorescent images are representative of the data,7 days post-lesion. Again, iNOS was not observed in SNpc indicating that not all reactive astrocytes in the SNpc wereof saline injected mice of either genotype (data not shown). iNOS immunopositive.

94 H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97

1Fig. 4. Colocalization of iNOS in GFAP astrocytes in MPTP-lesioned SNpc. In both IL-6 genotypes, at 1 (A,B) and 7 days (C,D), astrocytes were duallabeled with anti-GFAP (red) and anti-iNOS (green) antibodies. Scale bar represents 25mm.

4 . Discussion Here we report that SNpc astrogliosis was similar inMPTP-lesioned IL-6 (1 /1) and IL-6 (2 /2) mice, while

Neuroinflammation is a primary response to CNS injury microgliosis is severely compromised in IL-6 (2 /2) mice.and may secondarily participate in pathology. The deleteri- The lack of a robust, persistent reactive microgliosis isous effects of neuroinflammation, particularly reactive concomitant with a greater loss of dopaminergic neurons ingliosis, have been demonstrated in Alzheimer’s disease the lesioned SNpc of IL-6 (2 /2) mice [6].[29,32,34].While it is not yet known if reactive gliosis Reactive gliosis was examined in MPTP-lesioned IL-6contributes to dopaminergic neuron death in Parkinson’s (1 /1) and IL-6 (2 /2) SNpc at 1, 2 and 7 days post-disease, there is mounting support for that hypothesis from injection (Figs. 1 and 2). The extent of reactive astrogliosisinvestigations of the MPTP model of nigrostriatal damage. and microgliosis in the injured SNpc was determined at 7

H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97 95

days post-lesion. This time point was chosen because in induced dopaminergic neuron death. In the MPTP-lesionedthis model, maximal nigrostriatal damage has occurred as IL-6 (2 /2) mice, microgliosis is compromised and itsdetermined by striatal dopamine depletion[19] and loss of diminution is not neuroprotective.

1TH neurons in the SNpc[9]. Additionally, peak reactive To determine if production of ROS may be a factor ingliosis has been observed at 7 days as determined by the increased vulnerability of the IL-6 (2 /2) nigrostriatalGFAP [37] and Mac-1[22] immunohistochemistry. system, we examined iNOS expression in the MPTP-

Immunochemical analysis revealed a similar acute acti- lesioned SNpc. Co-localization immunohistochemistry re-1vation of Mac-1 , enlarged microglia in IL-6 (1 /1) and vealed microglial and astrocytic expression of iNOS in

IL-6 (2 /2) SNpc at 1 and 2 days post-lesion that was not MPTP-lesioned SNpc of both IL-6 genotypes. Reactiveobserved in saline-injected animals. Dopaminergic cell microglial iNOS expression persisted in enlarged cells ofinjury occurs within this time frame, as it has been shown IL-6 (1 /1) SNpc but was barely detected in ‘resting’,

1that MPP is no longer detectable in the mouse midbrain ramified microglia in the SN reticulata of IL-6 (2 /2) miceafter 16 h post-injection[9]. The engorged microglia are (Fig. 3). Thus, IL-6 (2 /2) microglial iNOS expressionprobably phagocytic, engulfing dopaminergic neuron cell diminished in the absence of reactive microglia. Reactivedebris. Microgliosis did not persist through 7 days in the astrocytes expressed iNOS through 7 days post-lesion inIL-6 (2 /2) SNpc, in contrast to the robust reactive both genotypes (Fig. 4). If microglial-derived reactivemicrogliosis observed in IL-6 (1 /1) SNpc. Thus, in the nitrogen species contribute to dopaminergic neuron deathabsence of IL-6, microglia mount a limited response to in this model, the early acute induction of iNOS could beneurotoxicant driven injury in the SNpc. This was not the responsible and is compatible with previous reports

1case for reactive astrogliosis. Comparable levels of GFAP [11,28]. Our data at 7 days post-MPTP suggests a persis-astrocytes were seen in saline-injected mice of both IL-6 tent astrocytic iNOS expression, which may inhibit re-genotypes. An equally persistent reactive astrogliosis was covery of damaged neurons. This possibility has beenobserved in MPTP-lesioned IL-6 (1 /1) and IL-6 (2 /2) suggested by observation of an increased density of iNOSSNpc. The 50–55% increase in MPTP driven SNpc immunoreactive glial cells in parkinsonian SNpc as com-astrogliosis in both genotypes (Table 1) suggests that these pared to control tissue[21,23]. Further exploration of theimmunoeffector cells react to neurotoxicant damage in the astrocytic neuroinflammatory response observed in bothpresence or absence of endogenous IL-6. Since IL-6 has IL-6 genotypes may elucidate mechanisms involved in thebeen shown to regulate astrogliosis by autocrine and chronic neuroinflammation observed in Parkinson’s diseaseparacrine mechanisms[27,42], other factors must induce SN. The cytokine knock-out mice, particularly IL-6 (2 /2)the observed astrogliosis in the MPTP-lesioned IL-6 (2 /2) mice, have illuminated the redundancy of regulatorymice. Cytokines capable of driving astrogliosis include function in the inflammatory response. Our data showingIL-1b and tumor necrosis factor alpha (TNFa) both of vigorous reactive astrogliosis in both MPTP-lesioned IL-6which have been shown to be up regulated in the absence genotypes illustrates that redundancy. Conversely, a sus-of IL-6 [1,14,15]. These cytokines also drive astrocyte tained MPTP triggered reactive microgliosis appears torelease of granulocyte monocyte-colony stimulating factor require the presence of IL-6 and/or other cytokines that(GM-CSF), which in turn stimulates microgliosis[2,43]. IL-6 regulates. The increased vulnerability of the nigros-Additionally, IL-6 has been shown to directly trigger triatal system in the IL-6 (2 /2) mice [6] suggests thatmicroglia proliferation [4,41]. Thus, diminished microg- IL-6 and possibly microgliosis, offer neuroprotection toliosis in MPTP-lesioned IL-6 (2 /2) SNpc suggests a injured dopaminergic neurons. It is probable that astrocyticselective inhibition of glial activation and a role for IL-6 in expression of IL-6 in control animals not only regulatesthe sustained neuroinflammatory response to neurotoxic- microglial proliferation, it may also stimulate expression ofant. immunoprotective cytokines. In the degenerating SNpc of

Compromised microgliosis in the SNpc of MPTP- IL-6 (1 /1) mice, synergism between reactive astrocyteslesioned IL-6 (2 /2) mice is interesting because microglial and microglia may buffer their immunotoxic activities.reactive oxygen species (ROS) production has been impli- Such cross-talk has been suggested to modulate astrocytecated in dopaminergic cell death in the MPTP model. Mice and microglia activities in the 6-hydroxydopamine modelwith a targeted deletion of iNOS[10,28] were less of nigrostriatal degeneration[38]. Additionally, astrocyte–vulnerable to MPTP than control mice. In control mice, microglia interactions that enhance astrocytic IL-6 neuro-iNOS was colocalized in reactive microglia[28]. Inhibition protection have been demonstrated in vitro in response toof iNOS expression by minocycline was, also, neuro- nontoxic methylmercury treatment[13]. In the IL-6 (2 /2)protective in the MPTP model[11,44] as well as in a SNpc, neuroinflammation is dysregulated with a resultant6-hydroxydopamine injury[18]. Recently, inhibition of disruption of reactive gliosis and immunoeffector cell–cellmicrogliosis through administration of pioglitazone, a interactions. Increased vulnerability of the nigrostriatalperoxisome proliferator-activated receptor-g agonist, was system to neurotoxicant may be a result of that disruption.found to be neuroprotective in the MPTP model[8]. In Dysregulation of the inflammatory response may alsothese investigations reactive microgliosis enhances MPTP- contribute to nigrostriatal vulnerability in the aging SNpc

96 H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97

[15] E . Fattori, M. Cappelletti, P. Costa, C. Sellitto, L. Cantoni, M.in Parkinson’s disease and the IL-6 deficient mice provideCarelli, R. Faggioni, G. Fantuzzi, P. Ghezzi, V. Poli, Defectivean excellent model for exploring that possibility.inflammatory response in interleukin 6-deficient mice, J. Exp. Med.180 (1994) 1243–1250.

[16] L .S. Forno, L.E. DeLanney, I. Irwin, D. Di Monte, J.W. Langston,Astrocytes and Parkinson’s disease, Prog. Brain Res. 94 (1992)A cknowledgements429–436.

[17] J .W. Francis, J. Von Visger, G.J. Markelonis, T.H. Oh, NeuroglialWe thank Iwona Strycharska-Orczyk and Erik Nelson responses to the dopaminergic neurotoxicant 1-methyl-4-phenyl-

for excellent technical assistance. This work was supported 1,2,3,6-tetrahydropyridine in mouse striatum, Neurotoxicol. Teratol.17 (1995) 7–12.by a fellowship from The Klatt Family Research Endow-

[18] Y . He, S. Appel, W. Le, Minocycline inhibits microglial activationment to HC and NIH grant NS40065-02 to LMB.and protects nigral cells after 6-hydroxydopamine injection intomouse striatum, Brain Res. 909 (2001) 187–193.

[19] R .E. Heikkila, F.S. Cabbat, L. Manzino, R.C. Duvoisin, Effects of1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine on neostriatal dopa-R eferencesmine in mice, Neuropharmacology 23 (1984) 711–713.

[20] E .C. Hirsch, S. Hunot, P. Damier, B. Faucheux, Glial cells and[1] A . Ait-Ikhlef, D. Hantaz-Ambroise, C. Jacque, L. Belkadi, F. Rieger, inflammation in Parkinson’s disease: a role in neurodegeneration?,

Astrocyte proliferation induced by wobbler astrocyte conditioned Ann. Neurol. 44 (1998) S115–120.medium is blocked by tumor necrosis factor-alpha (TNF-alpha) and [21] S . Hunot, F. Boissiere, B. Faucheux, B. Brugg, A. Mouatt-Prigent,interleukin-1beta (IL-1beta) neutralizing antibodies in vitro, Cell. Y. Agid, E.C. Hirsch, Nitric oxide synthase and neuronal vul-Mol. Biol. (Noisy-le-grand) 45 (1999) 393–400. nerability in Parkinson’s disease, Neuroscience 72 (1996) 355–363.

[2] F . Aloisi, A. Care, G. Borsellino, P. Gallo, S. Rosa, A. Bassani, A. [22] J .N. Kay, M. Blum, Differential response of ventral midbrain andCabibbo, U. Testa, G. Levi, C. Peschle, Production of striatal progenitor cells to lesions of the nigrostriatal dopaminergichemolymphopoietic cytokines (IL-6, IL-8, colony-stimulating fac- projection, Dev. Neurosci. 22 (2000) 56–67.tors) by normal human astrocytes in response to IL-1 beta and tumor [23] C . Knott, G. Stern, G.P. Wilkin, Inflammatory regulators in Parkin-necrosis factor-alpha, J. Immunol. 149 (1992) 2358–2366. son’s disease: iNOS, lipocortin-1, and cyclooxygenases-1 and -2,

[3] R .B. Banati, S.E. Daniel, S.B. Blunt, Glial pathology but absence of Mol. Cell. Neurosci. 16 (2000) 724–739.apoptotic nigral neurons in long-standing Parkinson’s disease, Mov. [24] M . Kohutnicka, E. Lewandowska, I. Kurkowska-Jastrzebska, A.Disord. 13 (1998) 221–227. Czlonkowski, A. Czlonkowska, Microglial and astrocytic in-

[4] J . Bauer, F. Herrmann, Interleukin-6 in clinical medicine, Ann. volvement in a murine model of Parkinson’s disease induced byHematol. 62 (1991) 203–210. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), Immuno-

[5] H . Baumann, J. Gauldie, The acute phase response, Immunol. Today pharmacology 39 (1998) 167–180.15 (1994) 74–80. [25] I . Kurkowska-Jastrzebska, A. Wronska, M. Kohutnicka, A. Czlon-

[6] L .M. Bolin, I. Strycharska-Orczyk, R. Murray, J.W. Langston, D. Di kowski, A. Czlonkowska, The inflammatory reaction following 1-Monte, Increased vulnerability of dopaminergic neurons in MPTP- methyl-4-phenyl-1,2,3, 6-tetrahydropyridine intoxication in mouse,lesioned interleukin-6 deficient mice, J. Neurochem. 83 (2002) Exp. Neurol. 156 (1999) 50–61.167–175. [26] J .W. Langston, Epidemiology versus genetics in Parkinson’s disease:

[7] L .M. Bolin, A.N. Verity, J.E. Silver, E.M. Shooter, J.S. Abrams, progress in resolving an age-old debate, Ann. Neurol. 44 (1998)Interleukin-6 production by Schwann cells and induction in sciatic S45–52.nerve injury, J. Neurochem. 64 (1995) 850–858. [27] S .W. Levison, F.J. Jiang, O.K. Stoltzfus, M.H. Ducceschi, IL-6-type

[8] T . Breidert, J. Callebert, M.T. Heneka, G. Landreth, J.M. Launay, cytokines enhance epidermal growth factor-stimulated astrocyteE.C. Hirsch, Protective action of the peroxisome proliferator-acti- proliferation, Glia 32 (2000) 328–337.vated receptor-gamma agonist pioglitazone in a mouse model of [28] G .T. Liberatore, V. Jackson-Lewis, S. Vukosavic, A.S. Mandir, M.Parkinson’s disease, J. Neurochem. 82 (2002) 615–624. Vila, W.G. McAuliffe, V.L. Dawson, T.M. Dawson, S. Przedborski,

[9] P . Chan, J.W. Langston, D.A. Di Monte, MK-801 temporarily Inducible nitric oxide synthase stimulates dopaminergic neurode-prevents MPTP-induced acute dopamine depletion and MPP1 generation in the MPTP model of Parkinson disease, Nature Med. 5elimination in the mouse striatum, J. Pharmacol. Exp. Ther. 267 (1999) 1403–1409.(1993) 1515–1520. [29] H .J. Luth, G. Munch, T. Arendt, Aberrant expression of NOS

[10] T . Dehmer, J. Lindenau, S. Haid, J. Dichgans, J.B. Schulz, De- isoforms in Alzheimer’s disease is structurally related to nitro-ficiency of inducible nitric oxide synthase protects against MPTP tyrosine formation, Brain Res. 953 (2002) 135–143.toxicity in vivo, J. Neurochem. 74 (2000) 2213–2216. [30] Y . Maeda, M. Matsumoto, O. Hori, K. Kuwabara, S. Ogawa, S.D.

[11] Y . Du, Z. Ma, S. Lin, R.C. Dodel, F. Gao, K.R. Bales, L.C. Yan, T. Ohtsuki, T. Kinoshita, T. Kamada, D.M. Stern, Hypoxia/Triarhou, E. Chernet, K.W. Perry, D.L. Nelson, S. Luecke, L.A. reoxygenation-mediated induction of astrocyte interleukin 6: aPhebus, F.P. Bymaster, S.M. Paul, Minocycline prevents nigrostriat- paracrine mechanism potentially enhancing neuron survival, J. Exp.al dopaminergic neurodegeneration in the MPTP model of Parkin- Med. 180 (1994) 2297–2308.son’s disease, Proc. Natl. Acad. Sci. USA 98 (2001) 14669–14674. [31] P .L. McGeer, E.G. McGeer, Glial cell reactions in neurodegenera-

[12] L .F. Eng, Glial fibrillary acidic protein (GFAP): the major protein of tive diseases: pathophysiology and therapeutic interventions, Al-glial intermediate filaments in differentiated astrocytes, J. Neuroim- zheimer Dis. Assoc. Disord. 12 (1998) S1–6.munol. 8 (1985) 203–214. [32] P .L. McGeer, E.G. McGeer, K. Yasojima, Alzheimer disease and

[13] C . Eskes, P. Honegger, L. Juillerat-Jeanneret, F. Monnet-Tschudi, neuroinflammation, J. Neural Transm. Suppl. 59 (2000) 53–57.Microglial reaction induced by noncytotoxic methylmercury treat- [33] R .E. Mrak, W.S. Griffin, Interleukin-1, neuroinflammation, andment leads to neuroprotection via interactions with astrocytes and Alzheimer’s disease, Neurobiol. Aging 22 (2001) 903–908.IL-6 release, Glia 37 (2002) 43–52. [34] J .P. O’Callaghan, D.B. Miller, J.F. Reinhard Jr., Characterization of

[14] G . Fantuzzi, C.A. Dinarello, The inflammatory response in the origins of astrocyte response to injury using the dopaminergicinterleukin-1 beta-deficient mice: comparison with other cytokine- neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Brainrelated knock-out mice, J. Leukoc. Biol. 59 (1996) 489–493. Res. 521 (1990) 73–80.

H. Cardenas, L.M. Bolin / Brain Research 985 (2003) 89–97 97

[35] V .H. Perry, D.A. Hume, S. Gordon, Immunohistochemical localiza- Interleukin-6 (IL-6) as an anti-inflammatory cytokine: induction oftion of macrophages and microglia in the adult and developing circulating IL-1 receptor antagonist and soluble tumor necrosismouse brain, Neuroscience 15 (1985) 313–326. factor receptor p55, Blood 83 (1994) 113–118.

[36] G . Raivich, G.W. Kreutzberg, Pathophysiology of glial growth factor [41] J . Tilgner, B. Volk, C. Kaltschmidt, Continuous interleukin-6 appli-receptors, Glia 11 (1994) 129–146. cation in vivo via macroencapsulation of interleukin-6-expressing

[37] J .F. Reinhard Jr., D.B. Miller, J.P. O’Callaghan, The neurotoxicant COS-7 cells induces massive gliosis, Glia 35 (2001) 234–245.MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) increases [42] N .J. Van Wagoner, J.W. Oh, P. Repovic, E.N. Benveniste,glial fibrillary acidic protein and decreases dopamine levels of the Interleukin-6 (IL-6) production by astrocytes: autocrine regulationmouse striatum: evidence for glial response to injury, Neurosci. Lett. by IL-6 and the soluble IL-6 receptor, J. Neurosci. 19 (1999)95 (1988) 246–251. 5236–5244.

[38] R .W. Rodrigues, V.C. Gomide, G. Chadi, Astroglial and microglial [43] L .Vitkovic, J.J. Chatham, A. da Cunha, Distinct expressions of threereaction after a partial nigrostriatal degeneration induced by the cytokines by IL-1-stimulated astrocytes in vitro and in AIDS brain,striatal injection of different doses of 6-hydroxydopamine, Int. J. Brain Behav. Immun. 9 (1995) 378–388.Neurosci. 109 (2001) 91–126. [44] D .C. Wu, V. Jackson-Lewis, M. Vila, K. Tieu, P. Teismann, C.

[39] J .D. Sedgwick, S. Schwender, H. Imrich, R. Dorries, G.W. Butcher, Vadseth, D.K. Choi, H. Ischiropoulos, S. Przedborski, Blockade ofV. ter Meulen, Isolation and direct characterization of resident microglial activation is neuroprotective in the 1-methyl-4-phenyl-microglial cells from the normal and inflamed central nervous 1,2,3,6-tetrahydropyridine mouse model of Parkinson disease, J.system, Proc. Natl. Acad. Sci. USA 88 (1991) 7438–7442. Neurosci. 22 (2002) 1763–1771.

[40] H . Tilg, E. Trehu, M.B. Atkins, C.A. Dinarello, J.W. Mier,