Immunoreactive S100 proteins of blood immunocytes and brain cells

Journal of

Neuroimmunology

Immunoreactive S 100 proteins of blood immunocytes and brain cells Vijendra K. Singh a.b.r,Jui-Fen Cheng b

Brain SLOO,as acidic proteinwith Ca”-binding and neumuophicproprties, may be involvedin the genesisof neumdegmcmtin diseases. BasedM stine sf cmmmmantieensbetweenUu immuneandnervousswlen~ we cafonned a commrativeanalvsisof SIW

1. Introduction Moore (1965) first identified a soluble protein of the brain and called it SIOO protein becauseof ils solubility in saturated ammonium sulfate solution at neutral PH. Recemly, a family of SloO proteins with &‘-binding property have been described (Kimma et al., 1984; Donato, 1991). They exist a~ three dimeric isoforms, namely SlOOa, SlWao and SlOOb. The &forms SlOOao and SVXlb are homcdimers consisting of (~a- and &¶-subunits, respeclively. whereas isofmm SIODa is a hetemdimer of o(- and psubunits. The molecular mxssesof dimem and indidividual subunits are 21 kDa and 10.5 kDa, respectively. In brain, SlOO,9 is the predominantform presentin astrocytes (Kimurn et al., 1984; Donato. 1991). Although the physiological function of tie most extensively studied &form SlWp is not known, it has been implicated in the palhophsyiologyof Alaheimer’s disease(AD) and Down’s syndrome CDS) Kiriff~n et al.. 1989; Marsh& 1990). In addition to brain, S IOQ has also been localized in non-neural cells including peripheral blad lymphocytes (Cccchia

et al.. 1981; Haimoro et al., 1987; Takahashi et al., 1987: Fermri et al., 1988; Sin&, 1994). but the antigen has not been studied. In this p$-zr, we describe a comparative analvsis of SICU in human blood immunacytes and brain

2. Materials and metbads 2.1. Separation of blmd

immunocytes

Blood donors were healthy adults aged 25-40 years. After informed consentfrom each donor. approximately25 ml of blood was drawn by venipuncwe into syringes containing preservative-free he&win (20 U/ml). Hep_ tinized blood was separatedby density-gradient cent&fueation usine Ficoll-Hwaaue [Histouwue fmm Sicma, St. Liuis, MO)-as described &viousl; C.&h. 198% After cenuifugation, the mononuch?afcells also known as iamunwytes (lymphocytes and monocytes) were collected. They were washed three times with RPM1 1640 growth medium and adjustedto a cell count of 10 X I@ cells/ml. In some experiments, immunwytes were separated info pure lymphocytes and monwytes essenbally according to

our pevious repcn Wigh, 1989). Immunocytes were assayed ti-eshfor SlOil by EIA or kept frozen in phosphateboffer?d saline at - 7WC until analysis by immune blonir;.

’

Small pieces of cerebral cortex of human brain and moose brain were previously stored frozen. Tissue homogenates (10% k/v) w&e prepared in phosphatebuffered saline (PBS: pH 7.4) sod sooicated for I min before “se. Human as-oma U373 cell line was parchased from the American Tvoe Culture Collection (ATCC), Rwkville, MD. CtdNrk ilesks ~otttaining u373 cells were maintained inside the homitied chamber (5% CO,) of an incubator at 37Y!. The cells were cultivated in RPh 1640 growth medium cootainiog 10% (v/v) feNl bwioe semm. Everv 4-5 days. the monolaven were sobcoINred by trypsioization &.5% trypsin for 3 mitt) 8s described elsewhere (Sit& and Van AIstvme. 1978). The cells were pelleted by c&rifugation st & rpm for IO min. washed three times with RPMl 1640 and used in experiNents described.

As describedelsewhere (Singh. 1994). freshly isolated immunocytes (1 X IO6 cells in pellet form) in test tubes were mixed with 23 I.CIof I:100 diluted rebbit-aotibovineSIOD. After a 60.min incubation at room temperaNre, the tubeswere ceowifuged~1 ID00 rpm for 10min. The pellets were washed three times wilh RPMI 1640, each time by breakiog the pellet sod centrifogatioo. The plleu were then incubatedwith 2.5 @I of 1:.5OUdiluted goat-aotirsbbitIgG-alkaline phosphatase(Sigma, St. Louis. MO) at rwn Ntttperarun for 60 min. The pellets were collected by centrifogatioo, warhed three times as above. and mixed with MO aI of a 1 mg/ml solution of p-oitrophenyl phosphatedissolved in sodium carbonate buffer (SO mM Na,CO, and 1 mM MgCI,, pH 9.6). To stop the enzyme reaction. IO ~1 of I N NaOH were added. After IO mitt of ceotrifogation a5 above, the soperoatant(I00 pi/well) was viwtted into duplicate wells of a 96.~11 microtiter plate: Additionally, & optical density wss read at 405 ton osinr: B Micro&& Reader Model 4% (Bio-Rad. Rich-

m&l. CA).

This immunoassay was performed according to wr recent report (Singh et al., 1992). The method involved protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) under denaturing conditions, proein transfer from gels to nitrocellulose membraoes,and immuo&tection by enzyme-linked antibody. Tissue or cell homogenates,after mixing in I: I ratio

with sample buffer (0.0625 M Tris HCI, pH 6.8, 5% (V/V, 2-mcrcapt~ethanol, 35% (w/v) SDS, 10% (v/v) glycerol, and 0.1% (w/v) Bromophenol blue), were densNred by immersing the Nbe in boiling water for 3 min. It was allowed to cool nt room temperature.Using a Mini-Gel unit (Hoefer Scientific Iostntmenfs, San Francisco, CA), the proteins were septuatedin 14% (w/v) acrylamide gels ran st 60 V for 4 h. To visualize the protein pattern, the eels were stained witi Coomassie brilient blue. For blotting, the protein transfer was carried cut for IS-20 h 81 room temperatureby sandwich diffusion in transfer buffer 00 mM Tris HCI, pH 8.3, 20% (v/v) metiwool and 0.2 M glycine). The blots w!! blocked with I% bovine serum albumin in TBST buffer (10 mM Tris.HCI, I50 mM NaCl. 0.05% (v/v) Twern-20, pH 8.0) for 60 min at room temperaNte. The blots were washed three times, 10 min each time with TBST. They were incubatedwith a I:lW diluted [email protected] st room temperaolre for 30 mio. After three washings of 10 mitt each. the blots were incubated with o I:Moo diluted go;u-antirabbit-I@-alkaline phosphatase@ma SL Louis. MO) at trmpershlre for 30 min. After three washings with TBST, the blob were developed within IO min in substratesolution prepared by mixing Resgents A and B in the boffer of AP-Substrate Kit (B&Rod. Richmood, CA). Tissue protein coxeotmtioo was determined by Protein Assay Kit (Bi*Bad, Richmond. CA). F’restainedprotein markers of 6-2fJO kDa range (Novex, San Diego, CA) were used as calibrators of immunobIots. Authentic SlCUl was a 99% pore protein of bovme brain Welbiocbem. La Jolla, CA). Polyclonal rabbit-antibody to SlW, directed against pore SIOO of bovine brain. wits perchasedfrom Sigma (code #2644; St Louis, MO).

e.e-

I

3. Results Antibcdies to SIC0 showed a positive reaction with both U373 cells and blood immunocyfes detected by EL4 (Fig. I). Tbe U373 cells were used as a natural source of SIMI. With both immuoDcv!es and U373 cells. there was an incre.w in absorbance with increasing n&&r of cells reaching a maximum at around 1 X lo6 cells and declining thereafter. llx U373 cells exmesxd a much hither level of Sl00 as compared to immunocytes. lmmunocyt& from six blood donors were mixed with sample buffer containing reducing agent and analyzed und& denaturing conditi&s of pro& elecuophore&Fig. 2A shows a protein pattern of these samples resolved by Cwmassie brilliant blue staining. A sharp protein band conespowkd, but sli@ly larger. to the band of monomeric form of authentic SILO (10.5 kDa). Fig. 2B shows a typical immunoblot of pure SIOU fro& bovine brain

t

2

3

4

5

+

(authentic sample) and blocd immunacytes. Pure SIOO showed a major band of monomeric form (IO.5 kDa) and a minor band of dimeric form (21 IDa). The immunOeytes from three blood donors also showed a positive reaction. There were at least two bands: a major band of 12-14 kDa in lymphccyte pellets and a minor band of approximately 100 kDa in both lymphocyte pellets and supematams. The blots of monwytes purified from immunocytes by adhermce to piystyrene phtic SU&XC did not show ?JIY reaction wbb rabbit-antiSlO (data not included here). When compaed to immunoeyres. the U373 cells contained SIOO mainly in a polymeric form (NJ-IOLI kDa) (Fig. 3). Likewise. Ule immunoblots of human and mwse brain (Fig. 4) showed two lo three bands of mainly polymeric forms(M)- 100 kDa> and a minor band of monomeric form

(105

kDa).

&&a

Ahhowh

the low molecular

in intensity.it

of brain-derived

reproducibly

mre

derived SIC0 (12-14 with monoclonal moncclond

(ID.5

kDa) and immunocvte-

antibody

SSLI

(data not shown).

is specific

proteins

for migration

(MRP)

designated

U373

differences between SILO of blood lymphocytes.

astrocytoma

and brain

inhibitory

toma cells. Srcondly, predominantly

and

a

monomer

slightly

larger

than

.

.

unlike Iym~hcates.

a monomer

was consistently

et al.. 1984: Donate. Since the original

report of brain SILO (Moore,

at least ten proteins

have teen

Their phydological

cept that they all bid phosphorylation

to Ca’+

Gmtra

described

significance

and may regulate protein

et

al.,

1984;

et al.. 1992). Additionally. Caz’

fluxes

survival

(Barge‘

(Marsh&

19921, and glial

D&wo.‘

1991;

the iroform SIOOP

and Van

19w:

proliferation

SILO proteins are distibuted

1965).

in the SloO

is not known ex-

T&&sh~et Since multiple

1994).

immunocyte-derived

1991).

from differing

forms of rhis protein

SloO/3

from one

and SlWa

enriched in brain and muscle, respectively (Kinura

without

are et al..

protein.

(i) our EIA

de-

antigen because we used

any permeability

~eatment;

assay detected SILO predomi-

nantly in the lymphocyte pellets. 7he differences between and brain-derived

compositions,

isofomu.

merefore,

terization

of various

understandine generative

of multiple

we suggest that a derailed chara~.isoforms

the clinical

diseases

may result

or glycasyl&n, is critically

important

and padmphysm~ical

cance, if any: of increased expres&n Down’s

SILO

for instance protein-protein

interaction or phosphorylation

al., 1987; Ferrari et al.,

for example

lymphocytes

and (ii) our immunoblottinp

et al..

et ii..

are known to exist, their levels vary significantly tissue to another,

to be a membrane-assoctated relied on two iindinge

tected a cell surface-associated

Bhattacharwa

tissues including blood lymphocytes (Cocchia et al.. 1981; 1988: Singh,

mainly

This conclusion viable

lo

(Kimura

1991). but some are membrane-bound

et al.. 1989). In this respect, the lymphocyte SIOO

appeared

1992).

(Selinfreund

Haimoto et al.,i987:

(Donato

Eldik,

in both neural and non-neural

the U373

localized.

brain, the majority of SlCXl proteins are cytowlic

4. Discussion

neuronal

astmcy-

immunacytes was

and brain cells mainly contained a polymeric

S I00 akhougb

Bairnbridge

the SIC0 of bled

was also seen. Thirdly,

astnxytorna

stimulates

and mouse

monomeric Gain protein although a minor polymeric comwnent

(Hessian et al.. 1993).

family.

cells (human

blocd lymphocytes expressed a consider-

ably lower level of SIGU as compared to U373

This

MRP-8

inuiguing

brain). At first

kDa). Immunocyte blots did not r&t

antibody

factor-related MRP-I4

SIM)

mass band was

corresponded to bands

such

of !?I&

as Alzheimer’s

in

simdfi-

in we&edisease

and

syndrome.

1984: Takahashi et al.. 1987). Becauw of its abundance in mam&ian

brain and availability

of pure protein. SloOp

is by far the most extensively studied form (Donate, Kimura

et al..

1984).

both monomeric SIC0

and polymeric

in lympimcytes.

This

forms of immunoreactive

antigen,

reaction to monoclonaJ atmbcdy MRP-8

and MRP-14

1991;

due to Jack of

5.5LI.

This research was swwrted a

is not related to

of neutrophils and monocytes. These

two proteins are Ca’+-binding~proteins

that b&g

to the

SILO family (Hessian et rd.. 1993). We are now ConductinC further characterization of lymphocyte-derived SIM) in: eluding distinguishmenl

of ~11and p isofomxs using mono-

clonal antibadies. Increased

brain expression

gested a patiophysiological AD or DS (Griffin

of SIOO in patients

sug-

association between SILO and

et al., 1989; Marshak,

1990). Interest-

ingly, the increaSe of SILO has also been demonstrated in peripheral blood lymphocytes of AD (Singh, 1994) and DS patients &to

et al.. 1990). In DS. trisomy of chromosome

21 is the main cause of neurological ing mental retardation. amyloid

protein

abnommlities

Chromosome

(Sisodia

includ-

21 also encodes p

and Price,

1995~ and SlCOp

protein (Allore et al.. 1988). both of which are upregulated and causally linked to AD (Griffin

et al.. 198% Marshak,

1990; Siscdia and price, 1995). In AD, the pathogenetic role of pamyloid protein has been extensively studied but the role of SlCiI remains unknown. Based

on our

Acknowledgemenls

In our present study. we detected

comparative

studies,

we found

sorae

Biotechnology

Center

id

student (J.F.C.)

in Molecular

bv a erant to V.K.S.

a &ion-waiver

Biology Program, Utah State

University,

Logan. Utah. V.K.S.

(University

of Michigan)

antibody

5.5.Ll

(Macrophage

thanks Dr. A. Blackwood

for a small sample of monoclonal

originally

Laboratory,

from

to graduate

provided by Dr. Nancy ICRF,

London,

UK).

Hogg

Oanato.R.. Gramba.nco. 1.. Ais& MT. andCcccarclli.P. 0989) IdrolitiMoore. B.W. (1965) A ralubk molein charaerisiic of ti netwus rysttm. Biochem.Biopbyr. Re;. Ccmmun. 19.739-744. Sclinfreund.R.H., Bqer, S.W.. PIedger. WJ. and Van El% L.J. (1991) NeumvopbicpfotelnSlCQb stimulatesglial cell pmlifemUn. Pmt. Nad. Acad.Sci. USA 88. 3554-3558. Griffm, W.S.T., Slanley.L.C.. Lhg. C.. while, L. Mac!_eod.V.. PenoL W.. Wlir. CL. and Am% C. (19S9) Brain interleukin-Iand SloO immunorextivityUC elsvati h Down syndromeand Alzheimer diwau. From.Natl. Acad. ki. USA 86.7611-7615. Haimao. H., Hox&. S. ;md Kate K. (19671 Differeaial diswibudonof innnunaunivc Slma and SIC+@ pmeins in normaln0nnewou~ humanLissycs.Lab. Invest 57.489-498. Hessian.P.A.. Edgswtmh.J. md Hog& N. (IW3) MRP-8 and MRP-14. two abundantCf’-bindbig prMEinsof neutrophilsand monwyw. J. kuka. Bial. 53. 197-204. Kao. K.. Suzuki. F., Kurok. N.. Ok&a. K.. Ogawvara. N., Nagoya. hi. and Yz.mm&a. T. (19R)) EnhanccmcntofSIo(LbDnrkin in blocd

ldr expression.J. Nc&ok&nol~ 23.257-262 Siwh. Y.K. (1994) Sludies of neumimmullemarkersin

braineflablishcdin cmlinuovscuRwe. Bnin Bcs. 155, 418-421. Singh.V.K.. Yang. Y.Y. [email protected].(1992) lmmunobl~de!ectionof antibodies10 @in basic pmtem in Akkimcr’s d&se pat vs. Neumsi. Leu. 147.25-28. Siscdia. S.S. and Rice, D.L. (19%) Role of the Pamyloid proteinin .AWtcimer’sdiwasc FASEB J. 9. 366-370. Taiahorhi. K.. Yoshino. T.. Hayashi. K.. Sonate, H. and OhtsuM. Y. (1987) S- 100 bxa wsirivo humanT lymphocytes: theirchnracarirdcr and behavior under normal nnd p!hologic condirirrnr RlowJ 70. 214-220.