,1 ,

, , ,

*Vita-Salute San Raffaele University, Center of Excellence in Cell Development, Milan, Italy

�Scientific Institute San Raffaele, Milan, Italy

�CNR Institute of Neuroscience, Milan, Italy

§Bioxell, Milan, Italy

¶Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada

**National Institute of Neuroscience, Milan, Italy

��IIT Network, Research Unit of Molecular Neuroscience, Milan, Italy

The triggering receptor expressed in myeloid (TREM) cells2, referred to with the acronym TREM2, is a single-spanningmembrane receptor of an immunoglobulin/lectin-like family.The members of this family operate through the interactionwith a common coupling protein, DNAX-activation proteinof 12 kDa (DAP12) which upon phosphorylation induces theactivation of various intracellular signalling pathways(tyrosine kinases, phospholipase Cc). Activation of other

Received December 20, 2008; revised manuscript received February 11,2009; accepted April 21, 2009.

Address correspondence and reprint requests to Jacopo Meldolesi,Vita-Salute San Raffaele University, DIBIT, via Olgettina 58, Milan,20132, Italy. E-mail meldolesi.jacopo@hsr.it1The present address of Dr. Stefano is Institute of Research in Bio-medicine, Bellinzona, Switzerland.Abbreviations used: BSA, bovine serum albumin; DAP12, DNAX-

activation protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;hHsp60, human heat shock protein 60; HRP, horseradish peroxidase;Hsp60, heat shock protein 60; PBS, phosphate-buffered saline; PLOSL,polycystic lipomembraneous osteodysplasia with sclerosing leukoence-phalopathy; rHsp60, rat heat shock protein 60; SDS–PAGE, sodiumdodecyl sulfate–polyacrylamide gel electrophoresis; siRNA, short inter-fering RNA; TREM, triggering receptor expressed in myeloid cells.

Abstract

Triggering receptor expressed in myeloid (TREM) cells 2, a

receptor expressed by myeloid cells, osteoclasts and micro-

glia, is known to play a protective role in bones and brain.

Mutations of the receptor (or of its coupling protein, DAP12)

sustain in fact a genetic disease affecting the two organs, the

polycystic lipomembraneous osteodysplasia with sclerosing

leukoencephalopathy (PLOSL or Nasu-Hakola disease). So

far, specific agonist(s) of TREM2 have not been identified and

its (their) transduction mechanisms are largely unknown. Heat

shock protein 60 (Hsp60) is a mitochondrial chaperone that

can also be harboured at the cell surface. By using constructs

including the extracellular domain of TREM2 and the Fc do-

main of IgGs we have identified Hsp60 as the only TREM2-

binding protein exposed at the surface of neuroblastoma N2A

cells and astrocytes, and lacking in U373 astrocytoma.

Treatment with Hsp60 was found to stimulate the best known

TREM2-dependent process, phagocytosis, however, only in

the microglial N9 cells rich in the receptor. Upon TREM2

down-regulation, the Hsp60-induced stimulation of N9

phagocytosis was greatly attenuated. Hsp60 is also released

by many cell types, segregated within exosomes or shedding

vesicles which might then undergo dissolution. However, the

affinity of its binding (Kd = 3.8 lM) might be too low for the

soluble chaperone released from the vesicles to the extra-

cellular space to induce a significant activation of TREM2. It

might in contrast be appropriate for the binding of TREM2 to

Hsp60 exposed at the surface of cells closely interacting with

microglia. The ensuing stimulation of phagocytosis could play

protective effects on the brain.

Keywords: receptor binding, surface-harboured agonist,

western and far-western blotting, phagocytosis, polycystic

lipomembraneous osteodysplasia with sclerosing leukoence-

phalopathy.

J. Neurochem. (2009) 110, 284–294.

JOURNAL OF NEUROCHEMISTRY | 2009 | 110 | 284–294 doi: 10.1111/j.1471-4159.2009.06130.x

284 Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294� 2009 The Authors

receptors of the family, such as the inflammatory amplifierTREM1, results in a potentiation of excitatory cellresponses. In contrast, TREM2 appears to act mostly as aninhibitor.

Knowledge about the physiology of TREM2 is stilllimited. In spite of the intense investigation in the lastdecade, even its endogenous agonist(s) is (are) unknown(Hamerman and Lanier 2006; Klesney-Tait et al. 2006;Turnbull et al. 2006). What is clear at the moment is thatloss-of-function mutations of TREM2 and/or DAP12, inducein man a genetic disease named PLOSL (standing forpolycystic lipomembraneous osteodysplasia with sclerosingleukoencephalopathy) or Nasu-Hakola disease (Palonevaet al. 2002; Klunemann et al. 2005). The structures affectedby PLOSL are not the myeloid organs but the bones and thebrain. Bones develop cysts and fractures because oftheir altered resorption by osteoclasts (Cella et al. 2003;Takegahara et al. 2006; Colonna et al. 2007); the brainexhibits an ample spectrum of lesions including atrophy,extensive leukodystrophy, axonal loss and glyosis, accom-panied by severe neurological and psychiatric symptoms, i.e.convulsions and an early onset, progressive dementia(Paloneva et al. 2000, 2002).

Previous work by us and others identified the microgliaas the brain cells expressing the highest levels of TREM2(Schmid et al. 2002; Sessa et al. 2004). Neurodegenerationtaking place in PLOSL was therefore envisaged as aconsequence of the overactivation of microglial cells. Soonthereafter, work by the group of Neumann demonstratedthat knock-out of the receptor induces a marked decrease ofphagocytosis, a key activity of microglial cells. This effect,accompanied by increased generation of proinflammatorycytokines, ultimately induces the accumulation of apoptoticcell debris and neuronal damage (Takahashi et al. 2005). Inaddition, blockade of the receptor was shown to exacerbatebrain pathology, and intravenous application of TREM2-rich stem cells to facilitate brain repair in experimentalautoimmune encephalomyelitis, the animal model of multi-ple sclerosis (Piccio et al. 2007; Takahashi et al. 2007).Based on these findings, TREM2 is now considered as apromising target for new therapeutic approaches to neuro-degenerative diseases (Piccio et al. 2008; Radhakrishnanet al. 2008).

In the present work an intense effort has been made toidentify endogenous ligand(s) of TREM2. Our results,obtained in a variety of cell lines, provide evidence for thespecific TREM2 binding of the heat shock protein 60(Hsp60, 60 kDa), a cytoplasmic chaperone which, how-ever, is also exposed to the surface and/or released fromvarious types of cells. Moreover, binding of Hsp60 wasfound to induce a marked increase in the phagocyticactivity in the N9 microglial cell line, whereas N2A, aneuroblastoma line where TREM2 is not expressed,remained unaffected.

Materials and methods

MaterialsRat and human recombinant Hsp60, rHsp60 and hHsp60, were

produced in the Gupta’s laboratory; the soluble murine and human

TR2 (mTR2 and hTR2) constructs were produced from the cDNA

fragments encoding the murine and human TREM-2 extracellular

regions cloned in frame with the human IgG1 Fc tail (mTREM2

extracellular domain amplification primers: forward 5¢-ACGGTACCCTCAACACCACGGTGCTG-3¢, reverse 5¢-TAGGATCCATGGAGGAGGTGGGTGGGAA-3¢; human TREM2 extracellular region

amplification primers: forward 5¢-AGTGGTACCGAGCTGTCCGGAGCCC-3¢, reverse 5¢-AAGTCTAGATTCTCCTTCCAAGAGGCT-3¢). These constructs were transiently transfected in CHO-K1

cells with Fugene 6 transfection reagent (Roche Diagnostics,

Monza, Italy) by following manufacturer’s instructions. The fusion

proteins were then purified from supernatants by protein A affinity

chromatography (Hitrap protein A HP; GE Healthcare, Milan, Italy).

The anti-human TREM2 29E3 and 20G2 monoclonals were the

same as used in the study by Sessa et al. (2004); the anti-Hsp60

monoclonal antibody was produced in Gupta’s laboratory; the anti-

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal

antibody was produced by AbD Serotec-MorphoSys (Oxford, UK);

fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG

antibodies were obtained from Southern Biotechnology (Birming-

ham, AL, USA); goat anti-mouse horseradish peroxidase (HRP)-

conjugated antibodies were obtained from Bio-Rad Laboratories

(Hercules, CA, USA); human IgGs and the 3,3¢,5,5¢, tetramethyl-

benzidine liquid substrate system for ELISA was obtained from

Sigma (St Louis, MO, USA); trypsin was obtained from Biowhit-

taker (Walkersville, MD, USA); the streptavidin–HRP conjugate

was obtained from Zymed (South San Francisco, CA, USA);

immobilized protein G, EZ-Link-Sulfo-NHS-LC-Biotin, streptavi-

din-agarose resins and EZ-Link-activated peroxidase antibody

labelling kit was obtained from Pierce Biotech (Rockford, IL,

USA); The ToxiLight, non-destructive cytotoxicity BioAssay Kit

was obtained from Lonza (Rockland, ME, USA). The other reagents

were purchased from Sigma.

Cell culturesPrimary rat cortex astrocytes (P2, 9 days in vitro), a kind gift of D.

Zacchetti, were cultured as in De Pietri Tonelli et al. (2004). Murine

neuroblastoma N2A cells were grown at 37�C under a 5% CO2

atmosphere in Dulbecco’s modified Eagle’s medium supplemented

with 10% fetal calf serum clone III (Hyclone, Logan, UT, USA),

20 mM HEPES–KOH, pH 7.4, 2 mM L-glutamine, 100 U/mL

penicillin and 100 mg/mL streptomycin (Biowhittaker, Verviers,

Belgium); human osteoblast SaOS-2 and human neuroblastoma SH-

SY5Y in Dulbecco’s modified Eagle’s medium supplemented with

10% fetal bovine serum (Cambrex Corp., Rockland, ME, USA) and

the antibiotic mixture; human U373 astrocytoma cells, in the same

medium supplemented in addition with 1 mM Na pyruvate; N9

murine microglial cells (Righi et al. 1989) in Iscove’s modified

Dulbecco’s medium (Invitrogen Life Technologies) were supple-

mented with the two antibiotics and with 2 mM L-glutamine;

BWZ.36 cells, transfected or not with TREM2 and DAP12 (Piccio

et al. 2007) were grown in RPMI-1640 medium containing 10%

fetal calf serum and the antibiotic mixture.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294

Hsp60, an agonist of the TREM2 receptor | 285

Confocal immunofluorescenceN2A, U373, BWZ.36 and primary astrocytes were plated on poly-

(L-lysine)-coated coverslips, transferred on ice and fixed for 10 min

in 4% paraformaldehyde dissolved in phosphate-buffered saline

(PBS), pH 7.4, then quenched with 0.1 M glycine and processed as

such for surface immunolabeling without detergent permeabiliza-

tion. For surface TREM2 detection, the TREM2/IgG (TR2)

construct proteins dissolved in binding buffer (25 mM Tris–HCl,

pH 7.4, 120 mM NaCl, 1.2 mM MgSO4, 2.5 mM KCl, 0.1% bovine

serum albumin (BSA) as in Alokail (2005) were used as primary

antibodies. Incubation was performed overnight at 4�C, then

coverslips were washed twice with PBS and incubated for 2 h at

22–24�C with FITC-conjugated anti-human IgGs. For surface

Hsp60 detection, incubation with the anti-Hsp60 monoclonal was

performed for 2 h at room temperature. After washing in PBS, the

cells were incubated in sequence for 1 h at 22–24�C with the anti-

mouse, tetraethylrhodamine isothiocyanate-conjugated secondary

antibody and 1% in PBS/0.1% BSA. Immunolabelled cells were

studied in a Bio-Rad MRC 1024 confocal microscope.

Cell surface biotinylationCells grown in Corning flasks were washed twice with PBS and then

incubated in 2 mM biotin for 15 min at 4�C. After a 5-min treatment

with BSA (final concentration 1%) at 4�C, they were washed twice

with PBS, harvested, centrifuged at 1000 g for 5 min at 4�C and

lysed in extraction buffer (for whole-cell extract preparation) or

homogenized (for differential centrifugation).

Differential and gradient centrifugation of cell homogenatesCells suspended in a mixture of 0.32 M sucrose, 5 mM HEPES,

pH 7.4, and a cocktail of protease inhibitors, were gently

homogenized in a cell cracker and then centrifuged at 1000 gfor 5 min. After resuspension of the low-speed pellet in 1.6 M

sucrose/HEPES, a partially purified plasma membrane fraction was

separated by floatation in a density gradient as described by

Cocucci et al. (2007). The floating band was collected, its sucrose

was diluted to 0.32 M, and the membranes were sedimented by

centrifugation at 100 000 g for 1 h, then suspended either in

binding buffer (for immunoprecipitation) or in extraction buffer

[for sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS–PAGE)].

SDS–PAGE, western blotting and peptide sequencingWhole-cell lysates, prepared by 15-min incubation on ice in

extraction buffer (10 mM Tris–HCl, pH 7.5, 0.5% Triton X-100,

120 mM NaCl, 25 mM KCl, 50 mM NaF, 0.2 mM phenylmethyl-

sulfonyl fluoride, 1 mM Na3VO4 and 5 mM Na4P2O7) were

centrifuged at 10 000 g at 4�C for 10 min. The supernatants, boiled

for 5 min at 95�C in Laemmli buffer, were loaded on 12%

polyacrylamide gels. In the case of 2D SDS–PAGE, the fraction was

purified with the 2D Clean-Up Kit (Amersham Biosciences, Little

Chalfont, UK), dissolved in the rehydration buffer (8 M urea, 2%

CHAPS, 40 mM dithiotreitol, 0.5% carrier ampholyte mixture, pH

3–10, and 0.02% bromophenol blue) and then used. The 2D SDS–

PAGE and peptide sequencing by mass spectrometry were per-

formed as described by Lorusso et al. (2006). Immunolabelled

western blot bands were revealed by the chemiluminescent reaction,

using the Western Blotting Detection Reagent (Amersham Bio-

sciences), as described in Borgonovo et al. (2002). Surface-

biotinylated cells were solubilized in binding buffer containing

1% Nonidet P40 (for immunoprecipitation) or extraction buffer (for

pull-down) and centrifuged. The supernatants were incubated for 3–

12 h at 4�C in the presence of immobilized protein G-agarose beads

coated with either mTR2 or streptavidin. After washing in binding

buffer or PBS, respectively, the beads were heated for 5 min at 95�Cin Laemmli buffer and then centrifuged. The supernatants were

analysed by SDS–PAGE. Bands were silver stained or decorated by

antibodies or by the streptavidin–HRP conjugate. Quantification of

western and biotin-/streptavidin-decorated bands was performed in a

Personal Densitometer SI of Molecular Dynamics (Sunnyvale, CA,

USA).

Far-western blottingAliquots of recombinant murine and human Hsp60 and TR2, run in

triplicate on 12% or 2D SDS–PAGE, were transferred onto

nitrocellulose membranes which were incubated overnight at 4�Cwith the following proteins and antibodies, all resuspended in Tris-

buffered saline/BSA 0.1%: mTR2 and hTR2 revealed either by anti-

IgG antibodies or by rHsp60 and hHsp60, respectively, were

subsequently labelled by anti-Hsp60 antibodies; rHsp60 and hHsp60

by anti-Hsp60 antibodies or by mTR2 and hTR2, respectively, were

subsequently labelled by anti-IgG antibodies. Likewise, a-synuc-lein, b-actin and human IgGs were revealed with their specific

antibodies or with either TR2 or Hsp60 subsequently labelled by the

anti-IgG or anti-Hsp60 antibodies, respectively. Finally, 2D blots of

surface-biotinylated N2A were incubated overnight at 4�C with the

mTR2. The washed filters were incubated for 1 h with the

corresponding secondary antibodies and processed for conventional

western blotting.

Cytotoxicity assayTriplicate cell samples in 96-well plates, incubated for up to 3 h at

37 or 42�C, were centrifuged at 800 g for 10 min, and 50 lL of the

supernatants were transferred to luminescence-compatible 96-well

plates. After addition of 100 lL of reconstituted AK Detection

Reagent from the Toxilight BioAssay Kit, followed by a 5-min

incubation protected from light, the luminescence was measured in a

BertholdTech CENTRO Reader (Berthold Technologies, Bad

Wildbad, Germany). Whole-cell lysates were employed as positive

and normalizing controls.

Hsp60/TR2 binding assayThe wells of 96-well plates were coated with mTR2 or hTR2

(1 lg/mL) in PBS and incubated overnight at 4�C. After an

additional 2 h saturation with 5% BSA in PBS at 4�C, they

received, for 3 h at 4�C, HRP-conjugated rHsp60 or hHsp60

(prepared with the EZ-Link Activated Peroxidase Antibody

Labelling Kit) in 25 mM Tris–HCl pH 7.4, 120 mM NaCl,

1.2 mM MgSO4, 2.5 mM KCl, 0.1% BSA, either in increasing

amounts or, alternatively, in fixed amount (1 lg/mL), mixed,

however, with increasing concentrations of unlabelled rHsp60 or

hHsp60. Control plates were processed without TR2. After

washing five times with PBS, 100 lL/well of the peroxidase

substrate, 3,3¢,5,5¢, tetramethylbenzidine, were added and the

colorimetric reaction was measured at 650 nm in a Microplate

Reader Model 680 (Bio-Rad Laboratories).

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286 | L. Stefano et al.

Discharge of Hsp60 to the mediumConfluent cell monolayers were incubated at either 37 or 42�C for

various periods of time after which the media were centrifuged at

1000 g to discard cells and debris. Supernatants were incubated for

3 h at 4�C with anti-Hsp60-coated, immobilized protein G beads.

After washing three times with PBS, the beads were heated at 95�Cfor 5 min in Laemmli buffer and analysed for Hsp60 by western

blotting. In some experiments the supernatants of the low-speed

centrifugation were processed before application to the beads (i) by

high-speed centrifugation (100 000 g for 1 h at 4�C). The final

supernatants and the solubilized pellets were then analysed

separately (ii) by centrifugation as in (i), followed by resuspension

of the pellet and gradient floatation as described above under

differential centrifugation. Alternatively, aliquots of the resuspended

high-speed pellets were incubated with trypsin (10 mg/mL, 3 min)

followed by an excess of BSA; with Triton X-100 (0.5%, 3 min) or

with Triton X-100 followed by trypsin and BSA. At the end, all

samples were diluted with Laemmli buffer, heated and western

blotted with the anti-Hsp60 antibody.

PhagocytosisN9 and N2A cells (�100 000/sample) were plated on poly-(L-

lysine)-coated coverslips, incubated overnight in their culture

medium and then processed according to the pHrodo Escherchiacoli bioparticle procedure of Invitrogen (Carslbad, CA, USA). After

two washes in the medium they were incubated at 37�C in the

OPTIMEM medium containing 100 lL/mL of pHrodo E. colibioparticles (1 lm in diameter) with or without Hsp60 (1 lg/mL) for

1–6 h. One hour before the end of the experiment the medium was

added with 1 lM Cell Tracker Green, then the cells were washed

gently for three times with cold medium, fixed with cytoskelfix

(Cytoskeleton, Denver, CO, USA) for 4 min, washed, mounted and

examined in the Bio-Rad confocal microscope. The cells appeared

stained green, the bioparticles in acidic environment, orange. The

same treatment was applied to N9 cells pre-transfected twice with a

TREM2 short interfering RNA (siRNA) (Piccio et al. 2007) and

exposed 48 h after the first transfection to pHrodo E. coli bioparticleswith and without Hsp60. These cells were not exposed to the Cell

Tracker Green staining. The cells positive for the siRNA were

distinguished from the negative ones by TREM2 immunocytochem-

istry (see Cocucci et al. 2008). For each time point, at least 40 cells

were analysed for orange fluorescence quantitation by the Image J

program (National Institute of Health, Washington DC, USA).

Results

The chaperone Hsp60 is a TREM2-binding proteinMany of the initial experiments were carried out by the use ofthe mTR2 and hTR2 constructs, composed by the extracel-lular domain of the mouse or human TREM2 fused to the Fcdomain of the human IgG1 (Fig. 1a). As these hybridproteins are directly recognized by anti-hIgG antibodies(Fig. 1b), they can be used to investigate the expression anddistribution of TREM2 ligand(s) by far-western blotting andimmunofluorescence.

To reveal the surface-bound hybrid TREM2/IgG protein,three cell types were first fixed with paraformaldehyde, then

exposed to the mTR2 without previous membrane permea-bilization and finally labelled with a fluorescent anti-hIgGantibody. Figure 1d–f show that both the murine neuroblas-toma N2A cells and the primary astrocytes prepared from therat brain cortex, but not the human U373 astrocytoma cells,exhibited a specific immunolabeling documenting theirsurface binding of the TR2 constructs.

The TR2s were used also to pull-down their bindingproteins exposed to the cell surface. Partially purified plasmamembrane fractions were isolated by gradient floatation ofthe low-speed pellets isolated from surface-biotinylated cells.After solubilization with 1% Nonidet P40 in the bindingbuffer these fractions were mixed with the mTR2 and thenwith beads coated with G protein. The proteins pulled-downby the beads were then separated by SDS–PAGE. As can beseen in Fig. 1c, various bands, including a major one ofapproximately 60 kDa, appeared in the silver-stained gels ofboth N2A and U373 cells. However, when the blots preparedfrom those gels were exposed to streptavidin conjugated toHRP to reveal the biotinylated proteins, i.e. the proteins that inintact cells were exposed to the surface, only two labelledbands (�60 and 56 kDa) appeared in N2A and none in U373cells (Fig. 1c). In both streptavidin-positive bands of N2Acells, peptide sequencing revealed the presence of the HSPD1gene product, the wild-type form of Hsp60, a chaperoneprotein concentrated in the mitochondria which, however, isknown to be exposed also to the surface of various cell types(Soltys and Gupta 2000; Gupta et al. 2008). When the plasmamembrane fractions of surface-biotinylated N2A cells wereprocessed by 2D SDS-PAGE, only a few of the numeroussilver-stained spots appeared decorated also by streptavidin–HRP (compare panels h to g in Fig. 1). Three of these spots,closely aligned at 60 kDa, were specifically labelled by themTR2 (Fig. 1i). Polypeptide sequencing revealed the threespots to be all composed by Hsp60.

Further evidence of the Hsp60 binding to TREM2 wasobtained by the far-western blotting experiments summarizedin Fig. 2a–c. The anti-Hsp60 antibody was able to decoratenot only the bands of the rHsp60 and hHsp60 (Fig. 2a) butalso those of the mTR2 and hTR2 previously incubated withthe two Hsp60s, respectively (Fig. 2b). Likewise, TR2(revealed by anti-hIgG1 antibodies) was able to bind thebands of both rHsp60 and hHsp60 (Fig. 2c).

Figure 2d–k summarizes a number of control resultsconfirming the specificity of the Hsp60/TR2 interaction:labelling of the two subunits of the hIgG1 by the anti-hIgGantibody (Fig. 2d) and not by Hsp60 followed by the anti-Hsp60 antibody (Fig. 2g) and lack of immunolabeling of twocontrol proteins, b-actin (40 kDa) and a-synuclein (18 kDa)(Fig. 2e and f), by the Hsp60/anti-Hsp60 antibody (Fig. 2g)and by the mouse or hTR2/anti-hIgG1 antibody (Fig. 2h).Finally, the anti-Hsp60 antibody, when applied to non-permeabilized cells, was found to immunodecorate the

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294

Hsp60, an agonist of the TREM2 receptor | 287

surface of N2A cells and of primary rat astrocytes (rightimages of Fig. 2i and j). In contrast, U373 cells remainedunlabelled (Fig. 2k). The results of Fig. 2i–k confirm thesurface localization of Hsp60 in N2A and astrocytes but notin U373, already revealed by Fig. 1d–f. A further control wasmade using another type of cell line lacking surface Hsp60,

the lymphocyte BWZ.36, analysed as such and aftertransfection of TREM2 (Piccio et al. 2007). These cellswere exposed first to the chaperone followed or not, uponwashing, by the anti-Hsp60. Figure 2l shows that in the non-transfected cells, exposure to Hsp60 alone or followed by theantibody failed to reveal any surface fluorescence signal. In

(a)

(d) (e) (f)

(b)

(g) (h) (i) (j)

(c)

Fig. 1 The TREM2 binding protein harboured at the surface of N2A

cells and primary astrocytes is Hsp60. Panel a compares the structure

of TREM2, extracellular (yellow, OUT) transmembrane (orange, K)

and intracellular (magenta, IN) domains, with the human (h) and

mouse (m) TR2 constructs. The constructs were generated from the

cDNA fragments encoding the TREM-2 extracellular regions (EC,

yellow; L19-S171, 152 amino acids and T13-I168, 156 amino acids,

respectively) amplified by PCR and cloned in a vector containing the

exons for hinge, CH2, and CH3 regions of human IgG1 (236 amino

acids) (Fc-hlgG, green). To increase the expression of the construct,

the endogenous TREM-2 leader peptide was substituted with that of

CD33 (red); moreover, a glycine spacer (blue) was inserted to sepa-

rate the TREM-2 extracellular region from the fc-hIgG. Panel b shows

the SDS–PAGE bands of the mouse and human TR2 (mTR2 and

hTR2) forms employed in this work, immunolabelled by anti-hIgG

conjugated to HRP (1 : 3000 dilution, 2 h, 22–24�C). Panels d–f show

that both murine neuroblastoma N2A cells and cultured rat primary

astrocytes bind TR2 on their surface, as revealed by the increased

surface immunofluorescence of the right with respect to the left

(controls, no TR2) images. In contrast, in the case of human astro-

cytoma U373 cells the TR2s failed to induce any appreciable increase

in the background surface immunofluorescence. Panel c (left) shows

that several proteins are pulled down by TR2 from partially purified

plasma membrane fractions isolated from surface-biotinylated N2A

and U373 cells. However, only two bands of N2A cells (60 and

56 kDa), and none of U373, bound streptavidin–HRP, documenting

their exposure to the cell surface (Panel c, right). Panel g shows the

2D SDS–PAGE, silver-stained protein pattern of a partially purified

plasma membrane fraction isolated from surface-biotinylated N2A

cells; Panel h shows that only a few spots of that pattern bound

streptavidin–HRP and were therefore exposed to the cell surface.

Panel i shows a 2D western blot from the same experiment showing

that, when subtracted of the artefactual spots (revealed by the anti-

hIgG antibody administered without previous TR2 treatment, Panel j),

only three spots, aligned at an apparent molecular weight of 60 kDa,

were positive for TR2 binding. Peptide sequencing revealed all these

spots to contain only Hsp60 (not shown). The bar in d, valid also for e

and f, corresponds to 25 lm.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294� 2009 The Authors

288 | L. Stefano et al.

contrast, the cells transfected with TREM2 and exposed insequence to Hsp60 and then to the antibody, exhibited astrong immunofluorescence, confirming the binding of theprotein to the receptor (Fig. 2m).

The affinity of the Hsp60 binding to the TREM2 receptorwas investigated by incubating multiwell plates coated attheir bottom with mTR2 with increasing concentrations ofthe rHsp60–HRP conjugate protein (Fig. 3a) or mixtures of afixed amount of the conjugate with increasing concentrationsof unlabelled rHsp60. The Kd calculated from the Scatchardanalysis of the data was 3.8 lM (Fig. 3b). Analogous resultswere obtained using hHsp60 and hTR2 (not shown).

The surface exposure of Hsp60 had been reportedpreviously in various types of cells (Soltys and Gupta

1996, 2000; Gupta et al. 2008). To obtain quantitativeinformation about N2A and U373, pull-down experimentswere carried out using extracts prepared from surface-biotinylated cells solubilized with 0.5% Triton X-100 in theextraction buffer and then exposed to streptavidin-coatedagarose beads. A band immunodecorated by the anti-Hsp60antibody was observed in the extract but not in the fractionpulled-down from U373 cells (Fig. 4d). In contrast a clearlyappreciable band was present in the fraction pulled-downfrom N2A extracts (Fig. 4b). In order to calculate the fractionof Hsp60 exposed to the surface of N2A cells, we firstcalculated the percentage of the total labelled protein pulled-down from our surface-biotinylated cells. This value (22%)was then used to correct the score obtained by simply

(a)

(d) (e) (f) (g) (h)

(b) (c)

(i)

(j)

(k)

(l)

(m)

Fig. 2 The binding of Hsp60 to TREM2 is specific. The figure illus-

trates the results of a series of experiments demonstrating the spec-

ificity of the results shown in Fig. 1i. The western blot of Panel a shows

that the anti-Hsp60 antibody (1 : 1000 dilution, 2 h, 22–24�C) used in

this work binds both the rHsp60 and hHsp60 recombinant proteins; the

blot of Panel b shows that mTR2 and hTR2 (10 lg/lane) bind re-

combinant rHsp60 and hHsp60 (15 lg/mL in TBS/0.1% BSA, over-

night at 4�C), respectively; the blot of Panel c show conversely that

rHsp60 and hHsp60 (10 lg/lane) bind mTR2 and hTR2 (15 lg/mL in

TBS/0.1% BSA, overnight at 4�C), respectively. The human IgGs

(10 lg/lane) used to reveal TR2 are recognized by the anti-human IgG

antibody (Ab) (Panel d) but not by Hsp60 (Panel g). Moreover, two

purified control proteins, b-actin and a-synuclein (10 lg/lane), are

immunodecorated by their specific antibodies (rabbit anti-actin,

1 : 2000 and mouse anti-a-synuclein, 1 : 1000, 2 h, Panels e and f)

but neither by rHsp60 (and hHsp60, not shown) nor by human (and

mouse, not shown) TR2 (Panels g and h). The surface of both N2A

and rat primary astrocytes is immunodecorated by the anti-Hsp60

(1 : 100 dilution, 2 h, 22–24�C), and not by the anti-mouse tetraeth-

ylrhodamine isothiocyanate-conjugated IgG antibodies (1 : 200, 1 h,

22–24�C) (right and left images of Panels i and j), whereas the surface

of U373 cells is immunodecorated by neither of them (Panel k). Panel l

shows that the lymphocyte BWZ.36 cells exhibit no surface immu-

nolabeling when exposed to the anti-Hsp60 (1 : 100 dilution, 2 h, 22–

24�C), (left) even when pre-exposed to the antigen (1 lg/mL, 1 h, 22–

24�C) and washed (right). The same cells transfected with TREM2

(Panel m) were negative when exposed only to the anti-Hsp60 anti-

body (left), but became immunolabelled when the antibody was pre-

ceded by exposure to Hsp60 (1 lg/mL, 1 h, 22–24�C) followed by

washing (right). The bar in i, valid also for j–m, corresponds to 25 lm.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294

Hsp60, an agonist of the TREM2 receptor | 289

dividing the pulled-down Hsp60 for the total Hsp60 of thecell extract, as revealed by western blotting (Fig. 4b). Thecorrected score, i.e. �5% of the total cellular Hsp60, is in thesame range of the values calculated by Soltys and Gupta(1996) in CHO and human lymphoblast cells, based on acompletely different approach (quantitation of immunogoldlabelling).

Hsp60 was also recovered in the incubation mediumRecovery of Hsp60 to the extracellular medium wasinvestigated not only in the neuroblastoma N2A andastrocytoma U373 cells but also in a few more lines: thehuman neuroblastoma SH-SY5Y, the human osteoblastSaOS-2 and the mouse microglial N9 cell lines. Figure 5a

shows that although quantitatively variable from experimentto experiment and in single experiments among the varioustimes of incubation, the media of all the investigated celllines, centrifuged at low-speed to remove the cell debris,contained significant amounts of Hsp60. These amountsincreased when the temperature of cell incubation wasincreased from 37 to 42�C. Recovery in the media did notappear to result from cytotoxicity. In fact, during incubationthe cell scores of a cytotoxicity test remained very low(Fig. 5b). Furthermore, in all cells except N2A the release ofHsp60 was not accompanied by the release of typicalcytosolic proteins such as GAPDH (Fig. 5a).

Previous recoveries of chaperones in the medium bathingmacrophages were attributed to transmembrane transport(Johnson and Fleshner 2006). Alternatively, the chaperoneswere reported to be accumulated and released withinvesicles, either exosomes discharged upon exocytosis ofthe multivesicular bodies (Clayton et al. 2005; Lancaster andFebbraio 2005), or shedding vesicles which are released inhigh number from the surface of the microglia (Bianco et al.2005). In order to distinguish between soluble and particulatemechanisms, the media recovered from N2A, U373 andSaOS-2 cells incubated for 30 min at either 37 or 42�C werecentrifuged at low speed (1000 g, 5 min) and the superna-tants were further centrifuged 100 000 g for 1 h to sedimentparticulate structures up to small vesicles. Figure 5c showsthat most Hsp60 from the media of both N2A and U373 cellswas recovered in the pellet of the high-speed centrifugation.To establish whether the sedimented Hsp60 was free orassociated with membranes, we resuspended the high-speedpellets obtained from both the N2A and U373 media inconcentrated (1.6 M) sucrose which was covered in sequenceby two cushions, one of 1.5 M, the other of isotonic sucrose.Upon centrifugation (100 000 g, 1 h), Hsp60 was recoveredin the band floating over the 1.5 M sucrose and not in thepellet, a result typical of membrane-associated proteins(Fig. 5d and not shown). The distribution of Hsp60 withrespect to the membrane, associated with the external surfaceor segregated within the lumen, was checked in resuspendedpellets by either solubilization with Triton X-100, treatmentwith trypsin or combination of the two. Figure 5e shows thattrypsin, when applied to the resuspended pellets, had hardlyany effect showing that Hsp60 was protected from itsdigestion. In contrast, when trypsin was applied to theresuspended pellets upon membrane solubilization by TritonX-100, it did induce the complete disappearance of Hsp60.We concluded that the recovery of Hsp60 in the N2A andU373 media was because of the segregation of the chaperonewithin vesicles released by the cells.

Hsp60 strengthens the phagocytic activity of theTREM2-expressing microglial cellsA question that remained to be answered was whetherbinding of Hsp60 to TREM2 is functionally significant, i.e.

(a) (b)

Fig. 3 Binding of Hsp60 to TR2. Panel a shows the concentration

dependence of the rHsp60 specific binding to wells coated with mTR2

(0.1 lg/well). Data shown are averages of three experiments ±SD.

Panel b shows the Scatchard plot of the results in a. The calculated Kd

of the binding was 3.8 lM. Analogous results were obtained by using

hHsp60 and hTR2 (not shown).

(a) (b) (c) (d)

Fig. 4 Streptavidin pull-down of Hsp60 from N2A and not from U373

surface-biotinylated cells. Panel a shows the biotinylated proteins of

the N2A cell extract (WCE) and the proteins pulled-down from the total

cell extract by streptavidin-agarose beads (PD); Panel b shows that

the Hsp60 of N2A cells, decorated by its specific antibody, is present

not only in the extract but also in the pulled-down fraction. Panels c

and d show the same data concerning U373 cells which exhibit Hsp60

in the extract (Panel c) but not in the pull-down fraction (Panel d).

Following quantitation of the data corrected for the recovery, the

Hsp60 exposed to the surface of N2A was calculated to be �5% of the

total Hsp60 of the cells (not shown).

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294� 2009 The Authors

290 | L. Stefano et al.

able to induce cellular responses. To investigate the problemwe focused on phagocytosis, the response known to beinhibited in the TREM2 knock-out microglia, with ensuingincrease of neurodegeneration (Takahashi et al. 2005, 2007).As the pro-phagocytic role of TREM2 has been recentlyconfirmed in myeloid cells (N’Diaye et al., 2009), it may bea general effect of the receptor activation. Figure 6 shows thephagocytic accumulation of fluorescent pHrodo-conjugatedE. coli bioparticles in the cytoplasm of two cell types, themicroglial N9, which expresses TREM2, and the neuroblas-toma N2A, which lacks it, incubated in the presence or not ofHsp60. As can be seen in the N9 cells incubated withoutHsp60 and in N2A cells incubated both with and without thechaperone, the fluorescence increased only during the firsthour and remained stable thereafter. In contrast, in the N9cells exposed to the chaperone the fluorescence increasedalmost linearily. Its values, significantly higher than those ofthe other incubated cells already at 2 h (+2.5-fold), reachedlevels almost 10-fold higher after 6 h (Fig. 6a–c). Theexperiments were repeated with N9 cells down-regulated forTREM2 by two applications of the specific siRNA (Piccioet al. 2007). In the cells expressing low levels of TREM2 thephagocytic accumulation of pHrodo-conjugated E. colibioparticles in response to Hsp60 was significantly lowerthan in non down-regulated cells exposed to the chaperone.

We concluded that the Hsp60 binding induced an activationof TREM2, with ensuing stimulation of at least one criticalprocess governed by the receptor.

Discussion

The physiological importance of TREM2, a receptor that, inspite of its general structure, appears to play mostly aninhibitory function, is now widely documented in the variouscell systems and organs where it is expressed, i.e. myeloidcells, bones and brain. In addition, the defect of either thereceptor or its coupling protein, DAP12, is known to inducefunctional and developmental alterations of both osteoclastsand microglia (Klesney-Tait et al. 2006; Takegahara et al.2006; Colonna et al. 2007; Neumann and Takahashi 2007;Piccio et al. 2007), ultimately responsible for the develop-ment of the PLOSL disease. The health of bones and brainappears therefore to depend substantially on TREM2 signal-ling. So far, however, little is known about TREM2agonist(s) (Hamerman and Lanier 2006; Klesney-Tait et al.2006). In a paper devoted to the problem, an ample spectrumof structures and molecules from bacteria to yeast andastrocytoma cells, from lipopolysaccharide to dextran sulfateand many other anionic ligands were reported to bindTREM2. However, the binding process was not characterized

(a) (b) (c)

(e)

(d)

Fig. 5 Release of Hsp60 from various cell lines incubated at 37 and

42�C. Panel a (left images) shows that not only monolayers of N2A

cells, which exhibit Hsp60 at their surface (Figs. 1 and 2), but also

those of U373 and of microglial N9, osteoblast SaOS-2 and neuro-

blastoma SH-SY5Y cells, that exhibit no surface Hsp60, release the

chaperone to the medium. Such a release, which in general appears

higher at 42�C than at 37�C, could not be due only to necrotic cells

because no appreciable recovery of cytosolic proteins, such as

GAPDH (right images panel a) was observed in the media bathing the

cells, with the exception of N2A. Panel b shows the results of the

cytotoxicity Toxilight Bioassay. Notice that the cells of all five lines,

incubated at either 37 or 42�C, exhibited only very small values. The

values of the various cell extracts (WCE) are shown to the right of

Panel b. Upon high-speed centrifugation (100 000 g, 1 h) of the

medium obtained by 1 h incubation of N2A, U373 (c) and SaOS-2 (not

shown) monolayers, Hsp60 was recovered in the pellets (P) and not in

the supernatants (S). In the pellets the chaperone was segregated

within membrane-bound particles. In fact (a) when the high-speed

centrifugation pellets obtained from the medium of N2A (Panel d) and

U373 (not shown) cells were resuspended in concentrated sucrose

and centrifuged in a gradient, Hsp60 was recovered in the floating

bands and not in the pellets; and (b) (Panel e) the Hsp60 recovered in

the medium of N2A and U373 cells was unaffected by trypsin (10 mg/

mL, 3 min) unless the incubation with the protease was preceded by a

membrane solubilization with Triton X-100 (0.5%, 3 min; Tx-Tryp).

n.t. = untreated sample.

� 2009 The AuthorsJournal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294

Hsp60, an agonist of the TREM2 receptor | 291

in detail (Daws et al. 2003). Moreover, binding of bacteriaand dextran, and interaction with astrocytoma cells werereported to induce activation of the receptor based, however,on reporter assays that required overnight incubation of thecells (Daws et al. 2003; Piccio et al. 2007). Finally, elegantexperiments carried out with cells expressing TREM2/DAP12, either spontaneously or by transfection, demon-strated the interaction of the receptor with one (or more)ligands exposed at the surface of other cells, and also theensuing phagocytic response. However, the nature of theligand(s) was not established (Hamerman et al. 2006; Hsiehet al. 2009).

In our study, the only protein exposed by the neuroblas-toma N2A cells found to bind TREM2 was Hsp60, achaperone usually indicated as mitochondrial, which inaddition is known to be also harboured at the surface ofvarious cell types (Soltys and Gupta 2000; Gupta et al.2008) and to be discharged to the extracellular space(Calderwood et al. 2007; Habich and Burkart 2007). InN2A cells and in primary astrocyte cultures, the harbouringof Hsp60 at the external face of the plasma membrane wasdocumented by the surface decoration not only with an anti-Hsp60 antibody but also with TR2, a chimeric proteincomposed by the extracellular domain of TREM2 fused tothe Fc domain of human IgGs. The specificity of theTREM2 to Hsp60 binding was confirmed by the experimentwith mouse thymoma BWZ.36 cells which are negative forsurface Hsp60 but became positive upon transfection ofTREM2 followed by incubation with the chaperone protein.Moreover, Hsp60 was the only surface protein, identified bybiotinylation, that was pulled-down in detectable amountsfrom N2A plasma membrane extracts by TR2-coated beads.The surface decoration with both TREM2 and anti-Hsp60was observed also in primary rat astrocytes but not in thehuman astrocytoma U373 (which also exhibited no pull-down of Hsp60) and mouse microglial N9 cell lines.Therefore the surface harbouring of Hsp60 does not appearto be a general property of cells but a property of only someof them.

The interaction of TREM2 with Hsp60 was not limited tobinding but induced in the microglial N9 cells, which are richin the receptor (Sessa et al. 2004), the best characterizedresponse to its activation (Takahashi et al. 2005; N’Diayeet al., 2009), a rapid and large stimulation of phagocytosis.This response was decreased in N9 cells upon down-regulation of TREM2 expression and was absent in neuro-blastoma N2A cells which have no TREM2. Our findingsmay be of importance also because of the harbouring ofHsp60 at the surface of astrocytes, which are known tointeract continuously with the microglia in both physiolog-ical and pathological conditions (see among others, Bezziet al. 2001; Suzumura et al. 2006), and possibly also ofneurons. Moreover, a paper (Hsieh et al. 2009), appearedduring the revision of the present paper, demonstrated that anunidentified ligand of TREM2 exposed at the surface ofN2A, of neurons and of other types of cells, increased 5–10fold upon apoptosis. These results may be consistent with ourfindings because a marked surface accumulation of theintracellular Hsp60 has been reported during apoptosis invarious cells types (see, for example, Sapozhnikov et al.1999; Lin et al. 2007). Therefore, it may be hypothesizedthat under normal conditions, the surface binding of Hsp60 toTREM2, bridging the space between neuronal/astrocyticapoptotic debris and microglia, participates in the regulationof the phagocytosis by the latter cells and therefore in theprotection of the brain.

(a)

(b) (c) (d)

Fig. 6 Stimulation of phagocytosis by Hsp60 in N9 but not in N2A

cells. Panel a shows the time-course of fluorescence increase in the

cytoplasm of the two cell types, documenting the accumulation of

pHrodo-conjugated Escherichia coli bioparticles (1 lm in diameter) in

an intracellular acidic environment corresponding to phagosomes.

Each value shown is the average of at lest 40 cells ±SD. Notice that in

the two untreated cells and in N2A even after exposure to Hsp60

(1 lg/mL, room temperature), the fluorescence is low after 1 h and

does not increase thereafter. In contrast, in N9 cells treated with

Hsp60 the fluorescence increases progressively, reaching significantly

higher values already after 2 and 3 h, and an almost 10-fold increase

after 6 h incubation. In the N9 cells where, before treatment with

Hsp60, TREM2 had been down-regulated by transfection of a specific

small interfering RNA, the increase of the phagocytosis induced by the

chaperone was much lower (�1/3 at 6 h). Panels b–d illustrate the

results at 6 h in N9 cells, untreated (b), treated with Hsp60 (c) and

exposed to the same treatment after down-regulation of TREM2 (d).

Notice that in the untreated cells the orange puncta of E. coli biopar-

ticles were small and a few, in the cells treated with Hsp60 they were

many more, and in the cells down-regulated for TREM2 (as docu-

mented by their low immunolabeling for the receptor, green) before

Hsp60 treatment, they were intermediate. The green staining in b and

c is due to Cell Tracker Green Staining. The bar in b is 10 lm.

Journal Compilation � 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 284–294� 2009 The Authors

292 | L. Stefano et al.

This conclusion may appear surprising because Hsp60,together with the other chaperone, Hsp72, is known to act as apro-inflammatory agent (Calderwood et al. 2007) that caninduce degeneration also in the brain (Lehnardt et al. 2008).Growing evidence has shown, however, that the action ofHsp60 is a multifaceted process, dependent also on itsinteraction with one or more receptors distinct from those ofHsp72 (Habich et al. 2002; Habich andBurkart 2007). Its role,therefore, is not limited to pro-inflammation during infectionthat appears to be largely mediated by the binding of thebacterial lipid, lipopolysaccharide (Habich and Burkart 2007),but includes also anti-inflammatory actions (Zanin-Zhorovet al. 2005; Hamerman et al. 2006; Yin Ji et al. 2007).

At variance with the results of Hsp60 surface decoration,those of Hsp60 discharge to the medium were positive withall five cell lines investigated, N2A and U373 together withSH-SY5Y, SaOS-2 and N9, which is rich in TREM2 (Sessaet al. 2004). The process was not because of necrotic releaseof the cytoplasm. Rather, Hsp60 was discharged whilesegregated within the lumen of membrane-bound vesicles, asdemonstrated by experiments of differential centrifugation,gradient floatation and trypsin digestion of the cell culturesupernatants. At least two types of discharged vesicles exist,the exosomes, released upon exocytosis of multivesicularbodies, and the vesicles shed directly from the surface ofcells (see Lakkaraju and Rodriguez-Boulan 2008; Cocucciet al. 2009). Both of them are known to accumulate specificcytosolic proteins, therefore either one or both could beresponsible for the discharge of Hsp60. Interestingly, life ofdischarged vesicles can be short. Vesicles, therefore, can bethe source of a free pool of extracellular Hsp60, which isbelieved to play important roles especially in immuneresponses and inflammation (see Calderwood et al. 2007).However, in view of the relatively low affinity of its binding(apparent Kd, 3.8 lM), the importance of the free Hsp60 poolat TREM2 is probably lower than that of the pool harbouredat the plasma membrane surface.

In conclusion, our evidence identifies Hsp60, a chaperoneoften believed to operate only in mitochondria, which,however, is harboured onto the surface of many types ofcells, including astrocytes and neuroblastoma N2A cells, asthe agonist that binds the TREM2 of microglial cells andactivates at least one of its intracellular responses, phagocy-tosis. Our results suggest that, in physiological conditions,the Hsp60/TREM2 system has a role in the coordinatefunctioning of the various brain cell types. However, they donot exclude the possibility that TREM2 binds also exoge-nous, and possibly also endogenous ligands, such as thoseproposed by Daws et al. (2003), and that it is activatedindirectly by some form of cross-talk taking place in thecontext of multiprotein complexes, as previously proposedby Klesney-Tait et al. (2006) and Takegahara et al. (2006).In other cell types, on the other hand, Hsp60 has been shownto bind and activate receptors distinct from TREM2. This

could happen also in the microglia, especially when thesecells undergo profound changes in their signalling phenotypeupon activation. So far, many properties of the receptor,including the signalling steps linking initial events forstimulation of phagocytosis, are unknown. The data we havepresented could provide information and tools to be used infurther studies focussed on the role of TREM2 in physiologyand on the pathogenesis of the PLOSL disease.

Acknowledgments

We are grateful to our colleagues of the San Raffaele Institute, D.

Zacchetti and A. Bachi, for the primary astrocytes and the peptide

sequencing of Hsp60, respectively. This study was supported by

grants from Telethon Italy (GGP030234) and from the Italian

Ministry of Research (PRIN 2006059395).

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