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Experimental Neurology 168, 32–46 (2001)doi:10.1006/exnr.2000.7575, available online at http://www.idealibrary.com on

Effect of Lipopolysaccharide on the Morphology and IntegrinImmunoreactivity of Ramified Microglia in the Mouse Brain

and in Cell CultureChristian U. A. Kloss,*,† Marion Bohatschek,* Georg W. Kreutzberg,* and Gennadij Raivich*

*Department of Neuromorphology, Max-Planck-Institute for Neurobiology, Am Klopferspitz 18a, 82152 Martinsried, Germany; and†Department of Neurology, Experimental Stroke Research, Klinikum Großhadern, Marchioninistrasse 15, 81377 Munich, Germany

Received June 2, 2000; accepted September 23, 2000

Microglial cells form the first line of defense in braininfection. They are related to monocytes and macro-phages and can be readily activated by cell wall com-ponents of bacteria such as lipopolysaccharides (LPS).In the present study, we explored the effect of thisendotoxin in mouse on the morphology of microgliaand their immunoreactivity for the integrin family ofcell adhesion molecules in vitro and in vivo. Subcuta-neous injection of LPS led to a dose-dependent activa-tion of aMb2-positive microglia, with a saturating ef-fect at 1 mg LPS in the blood–brain barrier deficientarea postrema, at 10 mg in the directly adjacent tissue,

nd at 100 mg throughout the brainstem and cerebel-lum. Morphologically, this activation was character-ized by the swelling of the microglial cell body, a thick-ening of the proximal processes, and a reduction indistal ramification. Microglial immunoreactivity forthe integrins a4b1, a5b1, a6b1, and aMb2 was stronglyncreased. In vitro, ramified microglia were obtainedsing a coculture on top of a confluent astrocyte mono-

ayer. Two days exposure to LPS resulted in a morpho-ogical activation of the cultured cells with an in-rease of the integrin immunoreactivity for a5 (5.7-

fold), a4 (3.1-fold), b1 (2.3-fold), and aM (1.5-fold), and adecrease in the a6-staining intensity by 39%. Even asublethal dose of LPS (3 mg in vivo and 500 mg/ml invitro, respectively) did not induce the phagocyte-asso-ciated integrin aXb2 (CD11c/CD18, p150,95) and did

ot lead to a morphological transformation of the ram-fied microglia into phagocytes. © 2001 Academic Press

Key Words: coculture; inflammation; mice; neuroglia;sepsis; VLA.

INTRODUCTION

Microglia form an important part in the brain-resi-dent immune system and play a central role in theacute and chronic inflammation. They are implicatedin a wide range of brain pathologies including directand indirect brain injury, ischemia, infectious, autoim-

320014-4886/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

mune, and neurodegenerative disease (20, 44, 45, 53,61). In contrast to the diversity of diseases that lead tomicroglial activation, their reaction repertoire appearsto be stereotype and well conserved during evolution.Thus, different signals lead to a step by step activationthat will culminate in the transformation to brain mac-rophages in the presence of neural debris (63, 73).These phagocytotic microglia can present antigen andrelease a large array of cytotoxic substances, includingreactive oxygen and nitrogen intermediates, protein-ases, and cytokines that will attack intruding organ-isms (4, 25, 66, 71).

Lipopolysaccharide (LPS, endotoxin), a major com-ponent of the cell wall of the gram-negative bacteria, isa potent stimulus for microglial activation. In micro-glial cell cultures, LPS induces a large number of acti-vation markers, including K-channels and the adhe-sion molecules CD4, ICAM-1, and the b1-integrin sub-unit (58, 70, 84). In vivo, LPS-administration closelymimics the clinical picture of septic shock (52) and on amolecular level has been shown to upregulate cyto-kines (interleukin-1a/IL1a, IL1b, IL12p40, MCP1),major histocompatibility complex molecules (MHC1,MHC2), and signaling enzymes such as iNOS andCOX2 in the affected brain microglia (11, 12, 27, 30, 39,47, 57, 59, 79, 82).

Morphologically, LPS also appears to transform mi-croglia into amoeboid, phagocytotic cells both in vitro(29, 75) and in vivo (12, 41, 51). However, this trans-forming action of LPS is regarded with caution. Com-pared to the situation in vivo, microglial ramificationin vitro, in pure microglial cell cultures, is rather poor,suggesting an unstable phenotype. In vivo, the disrup-tion of the blood–brain barrier and direct trauma fol-lowing CNS injection can also lead to the recruitmentof hematogenous monocytes that are difficult to distin-guish from the microglia-derived brain macrophages(3, 36, 83).

In the current study, we therefore explored the ef-fects of LPS on the morphology and the immunohisto-

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33INTEGRINS ON LPS-ACTIVATED MICROGLIA

chemical staining of brain microglia following systemicinjection, avoiding direct physical trauma, and com-pared them with microglial cells cultured on top of aconfluent astrocyte monolayer. The latter procedureleads to a highly stable and well-developed ramifica-tion of the cultured microglial cells that strongly re-semble their in vivo counterparts (38, 42, 72, 76). Asshown by previous studies, microglial activation leadsto the upregulation of large set of cell adhesion mole-cules of the integrin family that are frequently ex-pressed in a stage-specific manner (23, 31, 33, 43).Here, this information was used to stage and comparethe LPS-induced activation of the ramified brain mi-croglia in vivo and in cell culture.

MATERIALS AND METHODS

Cerebral Response to Systemic Lipopolysaccharides

Adult, male C57BL6 mice (Charles River, Sulzfeld,Germany) weighing 20–25 g were subcutaneously in-jected with lipopolysaccharides (LPS) from Eschericiacoli, serotype 055:B5 (Sigma, Deisenhofen, Germany),in doses ranging from 1 mg to 3 mg LPS dissolved in 1ml phosphate-buffered saline (PBS); the control ani-mals only received the vehicle. The animal experi-ments and care protocols conformed to NIH guidelines.Following survival periods of 12, 24, 48 or 96 h, threeanimals per time point were sacrificed, intracardiallyperfused with 200 ml phosphate-buffered saline (PBS;10 mM Na2HPO4, 0.85% NaCl, pH 7.4), followed by 200ml 4% paraformaldehyde in PBS (FA/PBS), removal ofbrainstem, and postfixation in 1% FA/PBS for 2 h. Thetissue was then cryoprotected by overnight immersionin 30% sucrose (30% sucrose, 10 mM Na2HPO4, pH 7.4)on a rotator at 4°C, frozen on dry ice, cut in a cyrostatat 218°C, and 20-mm-thin sections were collected onwarm glass slides, refrozen on dry ice, and stored at280°C for further use.

Bright Field Immunohistochemistry

For immunohistochemistry, sections of the murinebrainstem, including the area postrema, were processedas described by Moller et al. (56). Briefly, they werethawed and rehydrated in bidistilled water, spread ontoglass slides coated with 0.5% gelatine under a dissectingmicroscope, dried, fixed in 4% paraformaldehyde in phos-phate buffer (PB; 100 mM Na2HPO4, pH 7.4) for 5 min,efatted in acetone (50%, 2 min; 100%, 2 min; 50%, 2in), washed twice in PB and then in PB with 0.1%

ovine serum albumin (PB/BSA; Sigma, Deisenhofen,ermany), for some stainings the acetone treatment wasmitted (Table 1). After preincubation with 5% goat se-um (Sigma) in PB, the sections were incubated withifferent rat monoclonal antibodies overnight at 4°C (Ta-le 1). The specificity and the optimal working dilution ofach antibody have previously been determined using an

identical technique in the murine spleen and the axoto-mized facial nucleus as positive controls for the immuno-staining (43). Unbound primary antibody was thenwashed off (PB/BSA, PB, PB, PB/BSA), the sections in-cubated with biotinylated goat anti-rat (or anti-hamster,respectively) secondary antibody (1:100 in PB/BSA; Vec-tor, Wiesbaden, Germany) for 1 h at room temperature(RT), washed again (PB/BSA, PB/BSA, PB, PB), incu-bated for 1 h with ABC-reagent (Vector) in PB at RT,washed (PB, PB, PB, PBS), and finally visualized usingdiaminobenzidine (DAB; 0.5g/l in PBS; Sigma) with0.01% H2O2 for 5 min at RT. The sections were thenwashed again, dehydrated in alcohol and xylene, andmounted in Depex. Digital micrographs of the brainstemwere taken in a Zeiss Axiophot microscope with a 53objective and a Sony 89B CCD (Model CX-77CC) andimported into the OPTIMAS 6.2 imaging system using anImage Technology OFG card (VP-1100-768).

Immuno-Fluorescence Double-Labeling and ConfocalLaser Scanning Microscopy

For the cellular localization of the integrins in thebrainstem, double-labeling experiments were per-formed using IBA1 as microglial marker or GFAP forastrocytes (37, 43), and then combined with monoclo-nal antibodies against the different integrin subunits(Table 1). The sections were initially treated as de-scribed above, but preincubated with 5% donkey serum(Sigma) in PB. For colocalization, the fixed sectionswere simultaneously incubated with the monoclonalintegrin antibody and rabbit-anti-IBA 1 (1:400; gift ofDr. Y. Imai) or rabbit-anti-GFAP (1:6,000; DAKO,Hamburg, Germany) antisera. The sections werewashed, incubated simultaneously with two secondaryantibodies, biotin-conjugated donkey anti-rabbit im-munoglobulin (Ig) and FITC-conjugated goat anti-ratIg or goat anti-hamster Ig (1:100 in PB/BSA; Dianova,Hamburg, Germany), and then washed again and in-cubated with a tertiary FITC-conjugated donkey anti-goat antibody (1:100 in PB/BSA; Sigma) and Cy3-Avi-din (1:1,000 in PB/BSA; Dianova) for 2 h at RT. Afterwashing, the sections were covered with VectaShield(Vector) and stored in the dark at 4°C for further use.For visualizing the immunofluorescence double-label-ing, digital micrographs of the FITC for the integrinstaining, and the Cy3 fluorescence for the respectivecellular marker representing an area of 100 by 100 mm(1024 3 1024 pixels; grayscale 0–255) were taken witha Leica TCS 4-D confocal laser microscope using a1003 objective. The fluorescence was excited using low

rKr laser power (0.25 V) at wavelengths of 488 nm forITC and 568 nm for Cy3 and detected using theP-FITC filter for FITC and the LP590 filter for Cy3,espectively. Nine consecutive, equidistant levels wereecorded and condensed to a single bitmap using theaxIntens algorithm. To measure autofluorescence,

n additional infrared bitmap was recorded using max-

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34 KLOSS ET AL.

imal laser power, an excitation wavelength of 647 nm,and maximal detector voltage with a LP665 filter.

Cell Culture

The ramified microglia were obtained by coculture ona confluent monolayer of astrocytes, as described pre-viously (42, 72), with some minor modifications. Inbrief, for astroglial primary cultures the cortices ofnewborn Wistar rats were homogenized, the suspen-sion was kept in a 75-cm3 uncoated NUNC cultureflask in an incubator (37°C and 5% CO2), and fed every2 days with Dulbecco’s modified Eagle Medium(DMEM) containing 15% Fetal Calf Serum (FCS). After14 days the cells were frequently washed in DMEMand repeatedly vigorously shaken to eliminate oligo-dendrocytes and neurons, trypsinized (0.25% trypsin/PBS), harvested, counted, reseeded into a four-cham-ber Permanox ChamberSlide (NUNC) at a density of85,000 cells/chamber, and fed every 2 days. After 4days the astrocytes formed a confluent monolayer andwere used for coculturing experiments. Purity of ratastrocyte cultures was assessed by staining contami-nating microglia and oligodendrocytes using routineOX42- and GalC-immunocytochemistry (Serotec, Wies-baden, and Sigma, Deisenhofen, respectively). Theseastrocyte cultures showed less than 3% microglia and1.5% of oligodendrocytes.

Murine microglial primary cultures were preparedusing a slight modification of the method described byGiulian and Baker (28). The whole brains of newbornFVB mice were homogenized, the cells were main-tained in 25-cm3 uncoated NUNC culture flask in theincubator, and fed on days 7 and 12 with DMEM con-taining 15% FCS. After 14 days the microglial cellswere harvested by rotary shaking the primary culturefor 2 h at 200 rpm. The cells in the supernatant werecollected, counted, and reseeded on the astroglialmonolayer at a density of 10,000 cells/chamber.

The microglia–astrocyte cocultures were fed every 2days with DMEM/15% FCS. On day 10 LPS (from E.coli, serotype 055:B5; Sigma, Deisenhofen, Germany)was added in concentrations ranging from 5 ng/ml to500 mg/ml (in DMEM/15% FCS), control cultures onlyreceived DMEM/15% FCS. After 2 days of stimulationthe cells were fixed and immediately immunostainedfor the different integrin subunits.

Immunocytochemistry in Cell Cultures andQuantification of the Staining Intensity

For the detection of integrin-immunoreactivity inthe cell cultures, the cocultures were fixed for 30 min inice-cold methanol at 4°C, followed by 4% paraformal-dehyde in phosphate buffer (PB; 100 mM Na2HPO4, pH7.4) for 5 min, after preincubation in 5% goat serum for30 min at RT, the immunocytochemistry for the differ-ent integrin subunits was performed as described

above using the monoclonal antibodies as in the in vivoimmunohistochemistry (Table 1).

For quantification the cultures were scanned at con-stant illumination lamp voltage in a Zeiss Axiophotmicroscope with a 103 objective using a Sony 89B CCD(Model CX-77CC), and then the bitmap of the opticalluminosity values (OLV; grayscale 0–255) was im-ported into the OPTIMAS 6.2 imaging system using anImage Technology OFG card (VP-1100-768). For eachchamber the mean and standard deviation (SD) of theOLV of all pixels in five randomly selected visual fields,containing at least 500 microglial cells, were recordedusing OPTIMAS 6.2. The raw staining intensity (SI) ofthe antibody was determined for each individual cham-ber using the MEAN-SD algorithm as described byMoller et al. (56) and Kloss et al. (43). Then the back-ground staining intensity was measured for eachchamber using the same algorithm and the final stain-ing intensity of the antibody was calculated by thedifference of the raw antibody SI and the backgroundSI. For statistical analysis, the mean of the SI of iden-tically treated cultures was calculated for each anti-body in the LPS-stimulated and control group (eachfour to six cultures) and compared using an unpairedStudent t-test at the 5% level of significance.

Digital Processing of the Micrographs

The images were digitally processed as described indetail elsewhere (43, 65). In brief, for immunofluores-cence illustrations a trimmed/smoothed version (TSV)of the image was obtained by first smoothing the cor-rected image using a 100 3 100 pixel box and thenubtracting the mean optical luminosity value for thehole smoothed file from each individual pixel. Thenal illustration was obtained by adding the TSV tohe corrected image. To obtain corrected images in theright field micrographs, the bitmap was subtractedrom the bitmap of an empty scan and then reinvertedsing OPTIMAS 6.2. A TSV was calculated from theegative image as described above. The final illustra-ion was obtained by subtraction the TSV from theorrected image.

RESULTS

Microglial Morphology in the Normal Brainstem

Microglia in the murine brainstem and in cell culturewere detected by their constitutive expression of theaMb2-integrin (CD11b/CD18, complement receptor 3)with the rat monoclonal antibody 5C6 (43, 62, 67). Inthe normal brainstem (CO in Fig. 1), the microgliashow the typical ramified morphology of resting micro-glia with small cell bodies, multiple, branched, slenderprocesses, and extensive ramification of the distal pro-cesses. In the area postrema they appear mildly acti-vated as judged by the larger cell body, shorter proxi-

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35INTEGRINS ON LPS-ACTIVATED MICROGLIA

FIG. 1. Overview of the microglial morphology in the normal brainstem and 2 days after injection of LPS. Immunohistochemistry for theaMb2-Integrin, a constitutively expressed microglial cell marker. Note the extensive ramification of resting microglia in the unstimulatedcontrol (CO). Only the microglia in the area postrema (AP) have thickened and retracted processes and a more intense aMb2-staining,consistent with mild activation. Systemic injection of 1 mg LPS leads to the activation of microglia in the area postrema, at 10 mg LPS thiseffect is also observed in the adjacent brain regions, and at 100 mg LPS throughout the brainstem at 48 h after the injection. Additionalncrease in the dose of LPS to 1 mg does not alter the microglial morphology. At 3 mg there is a patchy decrease of the microglial stainingn some parts of the brainstem. Staining in area postrema is unchanged. CC denotes the central canal. Scale bar, 250 mm.

36 KLOSS ET AL.

mal processes, reduced ramification of the distal pro-cesses and the increased staining for the aMb2-integrin (63). Here, the blood–brain barrier is absentand serum components have access to the brain paren-chyma (9, 60). Within 100 mm from the area postrema,the microglia gradually changed from the activated tothe resting phenotype.

Effect of Systemic LPS on the Morphology ofBrainstem Microglia

As early as 12 h after a single systemic injection of theE. coli lipopolysaccharide (LPS), a mild increase in mi-croglial activation in the area postrema and the neigh-boring brainstem hemispheres could be observed (datanot shown). This activation became maximal at 48 h anddeclined again 96 h after the stimulation. As shown bythe aMb2-immunoreactivity in Fig. 1, there was a dose-dependent activation of the microglia throughout thebrainstem and in the area postrema 2 days after the

FIG. 2. Morphology of normal (A, B) and LPS-stimulated microglfrom area postrema (A, C). Immunohistochemistry for the aMb2-IntLPS-stimulated microglia show a reduction of ramification in thetransform into round, amoeboid macrophages. Note the difference inmore activated microglia in the area postrema (B) of control animal

LPS-treatment. Systemic injection of 1 mg LPS increasedthe aMb2-staining and led to morphological activation onthe microglial cells in the area postrema but did not affectstaining intensity or microglial morphology in the otherparts of the brainstem. At 10 mg LPS, activation was alsoobserved in the brain region bordering area postrema,and at 100 mg LPS, throughout the brainstem. A furtherincrease in the dose of LPS up to 1 mg did not yield anadditional increase in the staining intensity or furthereffects on microglial morphology. Addition of 3 mg LPSled to a less uniform staining of the microglial staining inthe brainstem, with some regions showing patchy de-crease. Staining in area postrema was not affected. Max-imally activated microglia (0.1–3 mg) had large cell bod-ies, stout proximal processes, a reduction of the ramifieddistal processes, and a marked increase in the aMb2-integrin immunoreactivity. A complete loss of microglialbranches that is characteristic for brain macrophageswas not detected even after a systemic injection of themaximal, sublethal dose of 3 mg LPS (Fig. 2).

C, D) in the area postrema (B, D) and a brainstem region 1 mm awayin, 48 h after injection of 3 mg LPS or saline (normal controls). Thea postrema (D) as well as in the remote brainstem (C) but do note morphology of ramified microglia in remote brainstem (A) and thecale bar, 25 mm.

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37INTEGRINS ON LPS-ACTIVATED MICROGLIA

Microglial Integrin-Immunoreactivity in theBrainstem after LPS-Stimulation

The murine spleen was used as a positive control forthe 11 monoclonal antibodies from rat and hamsterused in this study (Table 1). As described previously(43), all 11 antibodies produced a specific staining pat-tern in the spleen and the clones against the a4-, a5-,a6-, aM-, aX-, b1-, and b2-subunits also showed immu-noreactivity in the injured brain. In the present study,clear immunoreactivity for the aMb2-integrin com-pletely colocalized with the IBA1-positive microglia inthe murine brainstem (Fig. 3). In addition, microgliamoderately stained for the a6- and b1-subunits. A veryweak a4- and a5-labeling could only be identified usingimmunofluorescence double labeling and IBA1 as amicroglial marker.

As shown in Fig. 1, a dose of 100 mg LPS resulted inmaximal morphological activation of the microglia andwas therefore used to study the changes of integrinimmunoreactivity 2 days after the endotoxin stimula-tion. All integrin subunits present in the normal brain(a4, a5, a6, aM, b1, and b2) were upregulated afterLPS-stimulation (Fig. 3). In the gray matter, the im-munostaining colocalized with the IBA1-positive mi-croglia (Fig. 3), but not with GFAP as a marker forastrocytes (data not shown). The aL-, aX-, b3-, b4-, andb7-subunits could not be detected in the normal orLPS-stimulated brain using monoclonal antibodiesthat were able to detect specific immunoreactivity inthe spleen in parallel experiments.

Effect of LPS on the Morphology of Microglia inCulture

In the in vivo experiments, the systemically injectedndotoxin could act directly on neuroglia or be medi-ted by the blood vessels, ependymal cells, choroidlexus macrophages, or neurons (12, 79). In the currenttudy, we used cell culture to address this problem. Aumber of methods have been described to induce mi-roglial ramification in cell culture (14, 28, 29, 50, 75).

TAB

Monoclonal Antibodi

Integrinsubunit CD IgG species Clone

a4 CD49d Rat R1-2a5 CD49e Rat BMA5a6 CD49f Rat GoH3aL CD11a Rat KBAaM CD11b Rat 5C6aX CD11c Hamster N418b1 CD29 Rat MB1.2b2 CD18 Rat YTS 213b3 CD61 Hamster Hmb3b4 CD104 Rat 346-11Ab7 — Rat M293

owever, only the coculture on a confluent monolayerf living astrocytes consistently yields a high numberf strongly ramified microglia and this method could beeproduced by different laboratories (38, 42, 72, 76).his method allows to study a uniform population ofurine microglia seeded at a definite time point on the

op of the astrocyte monolayer, that strongly resemblehe resting microglia in vivo (compare Figs. 1 and 4).

The morphological response to LPS in vitro was veryimilar to that in the brain. In the unstimulated cocul-ures the microglia had a small cell body, multiple longnd slender processes, and some ramification of theistal branches (Fig. 4). A 2-day addition of 50 ng/mlPS to the culture medium was sufficient to induce aild activation of the microglial cells that became max-

mal with 5 mg/ml LPS. At this concentration the mi-croglia had an enlarged cell body, thickened and short-ened proximal processes, reduction of the distal rami-fication, and increase in the aMb2-staining. Theyclosely resembled the activated microglia in the LPS-stimulated brainstem. In parallel to the effect in vivo,addition of further LPS did not lead to a further de-ramification. Interestingly, even the stimulation withas little as 50 ng/ml LPS led to the appearance of asmall population (about 5%) with a large, round, andflat shape (“fried egg” cells), with relatively weakaMb2-staining. A further increase in LPS concentra-tion, up to the maximum of 0.5 mg/ml, did not increasetheir number. Small and round, amoeboid macro-phages were not detected at any time point during thisstudy.

Integrin-Immunoreactivity on Ramified Microgliaafter LPS-Stimulation

The intensity of integrin immunoreactivity in thecontrol and LPS-stimulated cultures of microglia wasdetected using the same panel of monoclonal rat andhamster antibodies that was shown to be capable todetect integrins in the mouse spleen, the axotomizedfacial motor nucleus (43), and the LPS-stimulated

1

Used in This Study

Source PretreatmentOptimaldilution

Pharmingen — 1:1000Chemicon — 1:2000Serotec Acetone 1:3000Serotec — 1:6000Serotec Acetone 1:4000Endogen Acetone 1:100Chemicon Acetone 1:3000Serotec Acetone 1:2000Pharmingen — 1:800Pharmingen Acetone 1:100Pharmingen Acetone 1:1600

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38 KLOSS ET AL.

FIG. 3. LPS increases integrin immunoreactivity on brainstem microglia. Confocal laser scanning immunofluorescence for differentintegrin subunits in green in the control (CO) and LPS-injected mice (LPS). The superimposed microglial profiles in red are shown usingIBA1-immunofluorescence (1IBA). In the normal brain, only the aM- and b2-integrin show strong immunoreactivity. However, there is alsoa weak staining for the a4-, a5-, a6-, and b1-subunits (arrowheads) that colocalizes with the microglia. A 2-day stimulation with 100 mg LPSeads to a massive increase of all the mentioned integrin subunits on the IBA1-positive microglia. The b1-subunit is also present on the

vessels and neurons and the a6-integrin on the blood vessel endothelia. This nonmicroglial immunoreactivity does not change after thenjection of LPS. Scale bar, 50 mm.

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39INTEGRINS ON LPS-ACTIVATED MICROGLIA

FIG. 4. LPS changes the morphology of cultured ramified microglia. Mouse ramified microglia cultured on top of confluent rat astrocytesere visualized with immunoreactivity for the mouse aMb2 integrin using the species-specific monoclonal antibody 5C6. After a coculture

of 7–10 days, these microglia have several long and slim branches and are strongly ramified. A 2-day stimulation with LPS leads to adose-dependent effect on microglial morphology. Most microglia shorten and thicken their processes and increase the aMb2-immunoreac-tivity. A small minority (approximately 5%) adopts a large, round, flat shape with a weak aMb2-staining. The first effects can be observedwith 50 ng/ml LPS, and concentrations higher than 5 mg/ml do not cause a further change. Note the close similarity to the microglialmorphology in vivo (Fig. 1). Scale bar, 250 mm.

40 KLOSS ET AL.

brainstem in the current study. In the unstimulatedcocultures of ramified microglia on a confluent rat as-trocyte monolayer, strong microglial immunoreactivitywas only observed for the aM-integrin subunit, butthere was a clear, though moderate staining for thea5-, a6-, b1-, and b2-subunits. Immunoreactivity forthe a4-integrin was barely visible and staining for theaL-, aX-, b3-, b4-, and b7-subunits could not be de-tected. The cocultured rat astrocytes did not exhibitimmunoreactivity with the monoclonal antibodies usedin this study.

As shown in Fig. 4, 5 mg/ml LPS were sufficient for amaximal morphological activation. This concentrationwas used for subsequent stimulation experiments. Af-ter a 2-day LPS-exposure, the activated microgliashowed a 5.7-fold increase in a5-immunoreactivity, a3.1-fold increase for a4, a 2.3-fold increase for b1 and a1.5-fold increase in the aM-subunit (Figs. 5 and 6).Only the a6-staining significantly decreased by 39%after LPS-stimulation. Interestingly, for all integrinsubunits the immunoreactivity of the 5% of the largeand round “fried egg” cells was much lower than thestaining of the normal reactive microglia in the samecultures (Fig. 5).

DISCUSSION

In the brain, microglial cells are the prime target ofLPS, the major cell wall component of gram-negativebacteria. The current study describes the effects of LPSon the morphology and integrin-immunoreactivity ofmicroglia in the normal mouse CNS and in cell culturesof ramified microglia on top of a confluent astrocytemonolayer. Peripheral injection of LPS led to a strong,dose-dependent morphological activation of the micro-glia, with a maximum effect at day 2. The reversibleactivation was associated with the upregulation of thea4b1, a5b1, a6b1, and aMb2 integrins. This corre-sponds to the activation pattern of the nonphagocytoticmicroglia surrounding a phagocytotic microglial nod-ule in the process of removing neural debris, the socalled bystander activation (63). Despite this strongactivating effect, even sublethal doses of LPS were notable to induce the phagocyte morphology and thephagocytosis-associated integrin aXb2 on the affectedmicroglia. In parallel to the situation in vivo, LPS wasable to activate the ramified cultured microglia both interms of morphology and the integrin profile but couldnot induce the phagocytotic phenotype.

The Microglial Activation by LPS: CNS Entry Routes

LPS is a strong activator of cells of the monocytelineage and plays an important part in the pathogen-esis of septic shock. In addition to peripheral tissues,septic shock also affects neurological function, whichcontributes to the multiorgan failure in this patho-physiological process (52). This effect of LPS on the

FIG. 5. LPS increases integrin immunoreactivity on ramified mi-croglia in coculture. Immunohistochemistry for the mouse a4-, a5-, a6-,b1-, aM-, and b2-integrin subunits. The microglia in the unstimulatedcontrol cultures (CO) are extensively ramified and show a clear immu-noreactivity for the a5-, a6-, b1-, aM-, and b2-subunits. Treatment with5 mg/ml LPS for 2 days leads to a strong increase in immunoreactivityfor a4-, a5-, b1-, and aM-subunits and a decrease of the a6-subunit onmost reactive, ramified microglia with thickened process (open arrow-heads). The small proportion of large, round cells (“fried egg” cells) arestained only slightly (solid arrowheads). Note the absence of staining onthe astrocyte monolayer. Scale bar, 100 mm.

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41INTEGRINS ON LPS-ACTIVATED MICROGLIA

brain has been studied by two different approaches, bya direct injection of the endotoxin into the brain paren-chyma or by a peripheral application. A problem of thedirect parenchymal route is the traumatic disruption ofthe blood–brain barrier and the subsequent early in-flux of blood macrophages (3, 55), which are difficult todistinguish from the resident, activated microglia (45,61, 63).

One way to circumvent this problem is a peripheral,i.e., intravenous or subcutaneous, injection of the en-dotoxin (11–13, 30). Here, a generalized activation ofmicroglia has been observed, but the mechanism bywhich LPS stimulates microglia remains unsolved. Be-cause of its large size, LPS is assumed not to easilycross the blood–brain barrier (BBB) (16). However, aspecific transport through the BBB-competent endo-thelia has been described for transferrin (19) and forlow density lipoproteins (18). Alternatively, LPS couldact on the BBB and facilitate the entry for itself (78) orfor large LPS-binding serum proteins, including albu-min (Bohatschek, unpublished observations) into theCNS. Extracellular pathways circumventing the mam-malian BBB have also been described for other highmolecular weight molecules like the endogenous serumproteins albumin, IgG, complement C9, and IgM andthe exogenous tracer protein horseradish peroxidase(9, 49, 68).

The current study suggests two different routes ofentry for LPS with widely different exchange rates: (a)through the BBB-deficient vessels and by subsequentdiffusion, and (b) by an uptake through a BBB-compe-tent endothelium. The saturating effect of LPS on mi-croglia in area postrema is already observed after asystemic injection of 1 mg, apparently due to a directentry of the endotoxin through the local, BBB-deficientvascular endothelium (9, 66). The subsequent diffusionof LPS into the adjacent parenchyma is probably re-sponsible for the precocious activation of the neighbor-ing microglial cells observed at the dose of 10 mg. Inontrast, there is a uniform activation of microgliahroughout brainstem and cerebellum following the

FIG. 6. Quantification of the effect of LPS on the mouse integrinubunits on cultured ramified microglia. The staining intensity (SI)as determined using the MEAN-SD algorithm (mean 6 SEM, n 5

4–6 separate cultures per bar). Open bars, control side; closed bars,2-day stimulation with 5 mg/ml LPS. *Statistically significant differ-nces between the LPS-stimulated and control cultures (P , 0.05,

2-tailed Student t test).

injection of 100 mg LPS. The absence of an additionaleffect in the area postrema and neighboring regionsclearly suggests the direct entry of LPS through thelocal vascular endothelium throughout the CNS at thisand at higher dosages. However, the 100-fold-higherdose needed for the saturating effect points to a 2orders of magnitude lower exchange rate for LPS inmost of the brain, compared to the BBB-deficient areapostrema.

Regulation and Function of Integrins on LPS-Activated Brain Microglia

Whatever the mechanism, the activation of microgliain the injured CNS proceeds through several, well-defined stages, which show specific changes in the pro-file of expressed cell adhesion molecules (31, 33, 54,64). These changes were documented in particular de-tail for the mouse axotomized facial motor nucleus andcan be used to stage the microglial reaction in this andin other forms of brain pathology (43, 63).

In the normal mouse brain, resting microglia onlyshow moderate immunoreactivity for the aMb2-inte-grin (stage 0). They are rapidly activated by variousforms of injury, enter the state of alert (stage 1) withincreased immunoreactivity for the aMb2-integrin andits ligand ICAM-1, and change morphology with a hy-pertrophy of the cell body and proximal processes anda decrease in distal ramification (40, 56, 69, 81). Overthe following days, these activated microglia graduallybegin to express a5b1 and a6b1 and home on andadhere to damaged cellular structures such as axoto-mized neurons (stage 2: homing and adhesion). Thisstage is also characterized by a reduction in the stain-ing for aMb2 and ICAM-1 and very moderate immu-noreactivity for MHC1 and costimulatory moleculessuch as B7.2 (63). The presence of dead neural cellsleads to further transformation into rounded phago-cytes that remove neural debris and show a very highlevel of immunoreactivity for MHC1, the costimulatoryfactors ICAM-1 and B7.2 and the integrins a5b1, a6b1,and aMb2 (stage 3a: phagocytosis). Of particular sig-nificance is the appearance of the aXb2. In the mousebrain it is only present on the phagocytotic microglia(43). In peripheral tissues, the expression of the mousepromoter for this integrin is restricted to professionalantigen-presenting cells (10). The selective expressionof the aXb2 on phagocytotic microglia and their fre-quent interaction with infiltrating T-cells (66, 81)points in the same direction.

Interestingly, the appearance of phagocytotic micro-glia is accompanied by the activation of the surround-ing microglia, a phenomenon described as bystanderactivation or stage 3b (63). These bystander-activatedmouse microglia are still ramified and are character-ized by the appearance of the a4b1 integrin and by theabsence of aXb2. They are also strongly labeled withantibodies against the a5b1, a6b1, and aMb2 inte-

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42 KLOSS ET AL.

grins, though not as strongly as the adjacent phagocy-totic microglia. Based on this classification, the LPS-stimulated microglia in vivo that express a4b1, a5b1,a6b1, and aMb2, but not aXb2, display a profile thatmatches that of bystander activation. In addition,these LPS-treated microglia also express high levels ofMHC1, B7.2, and ICAM-1 (Bohatschek and Raivich,unpublished observations). This is typical for thisstage. A similar profile of integrin immunoreactivity,with an increase in a4b1, a5b1, and aMb2 and theabsence of aXb2, was also observed in the LPS-treated,ramified microglial cells cultured on top of a confluentastrocyte monolayer.

In the injured CNS the bystander activation is nor-mally induced by diffusible factors emitted by the glialnodules, consisting of neuronal debris, phagocytoticmacrophages, and surrounding astrocytes (56, 66). Inthe axotomized facial motor nucleus, neuronal celldeath is accompanied by a massive increase in proin-flammatory cytokines such as IL1b, TNFa, and IFNg(66). Transgenic deletion of the TNF p55 receptor(TNFR1) strongly reduces this microglial activation (7,17). Interestingly, deletion of the IL1-receptor type 1(IL1R1) or IFNg-receptor type 1 (IFNgR1) had no effect(7), suggesting a selective involvement of TNFa. Onemajor difference to the bystander activation in theaxotomized facial motor nucleus is the absence of CNSphagocytes following the injection of LPS in the cur-rent study. Here, ramified microglia are probably theprimary target of LPS (6). However, not all the effectsof LPS are direct and mediated by a strictly intracel-lular signaling. LPS causes the induction of a largepanel of cytokines, including TNFa, both in vitro and invivo, on microglia (13, 15, 46), as well as on neighboringnonmicroglial cells such as astrocytes (35). These cyto-kines may affect the microglia via an autocrine or aparacrine feedback loop. For example, inhibition of thisloop for TNF with soluble p75TNF receptor stronglycurtails the synthesis of IL12 in pure microglial cellcultures (5). In the context of the current study, thesecytokines, and in particular TNFa, may also be respon-sible for the near identity in the morphology and mo-lecular profile of reactive microglia following LPStreatment and in the neighborhood of cell debris andphagocytotic glial nodules.

The only difference between the microglial responseto LPS in vivo and in vitro was the upregulation of thea6b1 in the brain and its downregulation on culturedramified microglia. Although there are many possibleexplanations, this difference could reflect the highercomplexity of the intact CNS, for example indirect ef-fects of LPS through neighboring neurons, oligoden-drocytes, or vascular endothelia. Whatever the reasonfor these differences in a6b1 expression, the similarityin the morphological response of microglia to LPS invivo and in vitro—the hypertrophy of the cell body andproximal processes, the decrease in territory size, theloss of distal ramification, as well as the normal induc-

tion of the other integrin family members—all suggestthat this integrin is probably not involved in the mi-croglial response to LPS. The same conclusion may alsoapply to most members of the b2-integrin family. Thus,aLb2 and aXb2 were not detected on LPS-stimulatedmicroglia in the current study in vivo or in vitro, al-though they are clearly observed on infiltrating lym-phocytes (aLb2) and phagocytotic microglia (aXb2) inthe injured mouse nervous system (43, 66). Prelimi-nary studies in the aM-deficient mice and the b2-defi-cient mice also show a normal microglial pattern ofmorphological response to injury (8), although thisneeds to be confirmed in LPS-treated animals.

However, there are several lines of evidence thatimplicate the b1 integrins in the maintenance and lossof microglial ramification. Addition of fibronectin, theligand for the a4b1 and a5b1 integrins, to microglialcell cultures induces the ramified morphology (14, 80).Fibronectin is strongly expressed in the vicinity ofbrain abscesses (24) and may mediate the morpholog-ical state of microglia migrating and adhering to theinterface between the healthy and infected tissue. Ad-dition of the RGD tripeptide (arginine-glycine-asparticacid), a competitor for the b1-integrin binding site offibronectin and several other structurally related ex-tracellular matrix molecules, reverses the ramificationin vitro and leads to the appearance of amoeboid mac-rophages (76). Both a4b1 and a5b1 are strongly up-regulated in LPS-treated microglia, in vitro as well asn vivo, and may be responsible for the state of rami-cation, i.e., the change in morphology from a highly tomoderately ramified cell. Antagonistic interactions

etween integrins and their ligands are also possible,s demonstrated by laminin and fibronectin. Laminin,he ligand for a6b1, inhibits microglial ramification;

fibronectin, the ligand for a4b1 and a5b1 has an op-posing function (14, 80). Interestingly, there is a con-spicuous difference in the integrin pattern of thephagocytotic and the adjacent, bystander activated mi-croglia: the phagocytotic cells have very high levels ofa5b1 and lower levels of a4b1, the reverse is true forthe neighboring, nonphagocytotic microglia (43). Thiscould suggest a ramification-stimulating effect for thea4b1 and an inhibiting effect for the a5b1, but this ishypothetical and must be tested in future experiments.However, the simultaneous and apparently coordi-nated expression of a4b1 and a5b1 does point to thefine balance between these receptor–ligand interac-tions, which may be important in the regulation ofmicroglial morphology and function.

LPS and the Stability of the Ramified MicroglialPhenotype

A key finding of the current study was the failure ofsystemically applied LPS to induce phagocytotic mor-phology and the associated cell adhesion molecules onthe affected mouse brain microglia. Microglia clearly

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43INTEGRINS ON LPS-ACTIVATED MICROGLIA

responded to the effect of LPS with cell hypertrophyand the induction of a panel of integrins characteristicof reactive cells. However, they did not transform intoamoeboid macrophages and did not loose their ramifiedappearance. The same stability of the ramified pheno-type was also observed for the vast majority of micro-glia cultured on top of confluent astrocytes. Thus, eventhe maximal doses (3 mg in vivo, 0.5 mg/ml in vitro) didnot reverse the ramification.

At first sight, this may appear to be in contrast withprevious studies in the rat brain. Intracerebral injec-tion of LPS causes the rapid appearance of round cellsin the parenchyma that stain for myelomonocytic an-tigens such as MHC1, MHC2, iNOS, and the aMb2integrin, particularly in the presence of IFNg (35, 41,51). However, the direct trauma following injectionwith inflammation-inducing chemicals and the conse-quent disruption of the blood–brain barrier will alsolead to the recruitment of hematogenous monocytesthat are difficult to distinguish from the microglia-derived brain macrophages (3, 36, 83). The rapid andmassive increase in the density of macrophages thatoutnumber the resident microglia (51), their perivas-cular localization (35), the absence of sizable microglialproliferation (36), and the severe inhibition in leuko-penic animals (3) all point to the blood-derived natureof these rounded and amoeboid cells. Interestingly, asystemic application of LPS can also lead to recruit-ment of monocytes into CNS parenchyma (83). Thiscautions against the assumption of a transformation oflocal microglia into round macrophages in the absenceof direct trauma or neural cell death.

As shown in the present study, the response of cul-tured ramified microglia to LPS is nearly identical tothat of brain microglia in situ, in terms of both mor-phology and expressed cell adhesion molecules. How-ever, there is a remarkable difference to the previouslypublished in vitro work. Thus, addition of LPS to puremicroglial cell cultures appears to transform these cellsinto rounded and amoeboid macrophages (29, 75).Compared to the situation in vivo, microglial ramifica-ion in pure microglial cell cultures is rather moderateven before the addition of LPS, suggesting an unsta-le phenotype. This moderate ramification is also re-ersed by withdrawal of serine and glycine (77), inhi-ition of Cl-channels (21), addition of IFNg (29), or

neutralization of TGF-b1 (34) and induced by severalcolony-stimulating factors such as MCSF and GMCSF(26, 50, 75). Overall, the ramification is considerablystronger and more stable on microglia cultured on topof confluent astrocytes. Thus, none of the proinflamma-tory cytokines tested in this coculture model, such asIL1b, TNFa, or IFNg, led to a reversal of ramification,although they all strongly affected the proliferation ofthis cell type (42). Inhibition of the endogenous MCSF,GMCSF, and the TGFb isoforms 1 to 3 also had noeffect on the ramification (38, 42). Finally, as shown inthe current study, the addition of LPS did lead to a

morphological response in ramified microglia on top ofastrocytes that is typical of reactive microglial cells.However, in the vast majority of the affected cells, itdid not lead to a transformation into small, amoeboidmacrophages, or the complete loss of ramification.

LPS also causes a second reaction in about 5% of thecells, which adopt a large, flat, round “fried egg” mor-phology. This effect was already observed at the lowestdose tested (50 ng/ml), it was not dose-dependent andadditional LPS did not influence number or shape ofthe flat cells. The morphology, motility, and cytoskel-etal organization of these large, round, microglia-de-rived cells was described in detail in previous studiesand includes rearrangement of the actin filaments andmicrotubules, the appearance of two condensed inter-mediate filament rings and a marked downregulationof MHC class II antigen (1). The downregulation of thisantigen presentation molecule and the low level of thephagocytosis-associated integrins argue against theserelatively rare, large and round cells being activatedmacrophages. These “fried egg” cells sometimes appearoverlayed by ramified microglia, suggesting the pres-ence of several cell strata. A similar morphology wasalso observed in rat microglia cocultured with astro-cytes and later overgrown by the confluent astrocytes(42). Recent ultrastructural studies also revealed flat,monocyte-like cells sandwiched between the astrocytesand the culture dish surface (Bohatschek and Raivich,unpublished observations). In the context of the cur-rent study, LPS could cause the migration of a smallpercentage of microglia to leave its normal, superficialposition, migrate through the astrocyte layer, contactthe substrate, and then spread profusely on the dishsurface. Even though this response does occur, it isuncommon, and most microglia cultured in the pres-ence of LPS do maintain their normal position andmorphology.

Overall, the ramified microglia appears to be a re-markably stable cellular phenotype. In the adult brain,transformation into round macrophages is normallyonly associated with neural cell debris in severe formsof brain pathology (44, 63). In the current study, theapplication of LPS caused reactive changes but did nottransform microglia into amoeboid macrophages. In-terestingly, even the initially round monocytes/macro-phages recruited into brain parenchyma are frequentlyobserved to ramify as the inflammation recedes (3, 74,83). From a developmental perspective, this microglialramification is, however, a slow process that normallyevolves during embryogenesis and the postnatal mat-uration (48). In vitro, ramification on top of confluentastrocytes is equally tardy and takes 7–10 days to becompleted (42, 72). Astrocyte-conditioned medium doesenhance ramification, but the effect was observed onlyin some but not in other studies (22, 75), suggesting theneed for a direct astrocyte contact to achieve a stableeffect (76). Such contact between the “mature” astro-cyte surfaces and microglia could also play a key role in

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44 KLOSS ET AL.

the generation of the ramified phenotype in vivo. Pro-toplasmic astrocytes expand over large territories inboth normal and injured brain (2, 63) and carry largeand thin membrane sheets (32) that could provide am-ple contact surface for the neighboring microglia. Atpresent, this mode of action is hypothetic and relies onthe identification of the contact molecules and theirsynthesis by mature astrocytes. However, the overallstability of the ramified microglial phenotype clearlysuggests the importance of such molecules for the in-tegrity of the neural parenchyma in the adult brain.

ACKNOWLEDGMENTS

We thank Helma Tyrlas for her expert technical assistance withthe cell culture. This study is part of the doctoral thesis of ChristianKloss. This work was supported by grant DFG Ra486/3-1 from theDeutsche Forschungsgemeinschaft and BMBF Grants 01KO9401/3and 01KO9703/3 from the Federal Minister for Education and Re-search (G.R.).

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