Vol. 179, No. 2, 1991 September 16, 1991

BIOCHEMICAL

BACTERIAL

TOXINS

Hensler,

INDUCE HEAT SHOCK PROTEINS

HUMAN NEUTROPHILS

IN

T.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 872-879

M. KSller,

J.E.

Alouf'

arrd W. Kiinig

Lehrstuhl fiir Med. Mikrobiologie und Immunologie Arbeitsgruppe Ihfektabwehrmechanismen Ruhr-UniversitHt Rochum, UniversittitsstraOe 150, D-4630 Rochum 1, F.R.G. 'Unit&

des Toxines Ract&iennes, Institut Pasteur, Paris &dex 15, France

75724 Received

July

25,

1991

SUMMARY We studied the influence of different bacterial toxins (alveolysin; toxic shock syndrome toxin 1, TSST-1 and erythrogenic toxin A, ETA) on the expression of heat shock proteins (hsps) in isolated human polywrphonuclear granulocytes (PMNs). As was shown by wstern blotting (anti-hsp72) ETA and TSST-1 were potent inducers of hsps at low toxin concentrations (10 rig/ml). Alveolysin led to the expression of hsps at hemolytic concentrations (1 HU; 700 rig/ml) whereas at subhemolytic concentrations (7 rig/ml) no heat shock response was observed. The induction of heat shock proteins WBS also accompanied by increased mRNA levels for hsp70 as was determined by 0 1991Academic Press,Inc. PCR-analysis.

Heat to

shock

the

expression

organisms duced

treatment

so far

during

-esponse

of

(3)

stress

examined

infection

against

,escribed

or other

the

physiological or

(1).

heat

and provides

The heat

known about

ability

bacterial

toxins

to

of

bacterial

induce

a stress

shock

for

response

regard

role

In contrast,

(1).

pathogenicity response

is

lead

in all also

in-

the

immune

GroEL hsp has been well

a pathogenic

mimicry

conditions (hsps)

In this

antigen

evidence

based on molecular

proteins

(2).

bacterial

immune diseases the

shock

and inflammation comwn

stress

factors

in autoless

is

such as

in human polymrpho-

Abbreviations: ETA, erythrogenic toxin A; HETE, hydroxyeicosatetraenoic acid; hsp, heat shock protein; HU, hemolytic unit; LUH, lactate dehydrogenase; PBS, phosphate buffered saline; PMNI polymorphonuclear granulocyte; PMSF, phenyl methyl sulfonyl fluoride; TEST-1, toxic shock syndrome toxin 1. 0006-291X/91

Copyright All rights

$1.50

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

872

*1

Vol.

nuclear acute

granulocytes

(PM'&)

inflammatory

toxins

induced

from

the

signed cytolysin (TSST-1; shock,

of

context

evaluate

the

(alveolysin

from

ETA) with scarlet

expression

effects

well

of

defined

associated

fever;

7) but

still

of

stress

response

the

MATERIALS

the

bacterial

(leukotrienes,

demonstrated

Therefore,

alvei;

of

shown that

a cholesterol

Bacillus

component

mediators

we recently (6).

RESEARCH COMMUNICATIONS

cellular

previously

lipid

hsps in human leukocytes to

major

We have

generation

In this

AND BIOPHYSICAL

as the

response.

PMNs (4,5).

induced

the

BIOCHEMICAL

179, No. 2, 1991

this

7) and of disease

that

study

binding

12-HETE

was de-

bacterial

bacterial

syndromes

unknown pathogenic

HETEs)

toxins (toxic

mechanisms

on

in human PM?&..

AND METHODS

Ficoll 400 ~8s obtained from Pharmacia, Uppsala, Sweden; w/v) was from knoll, Ludwigshafen; sodium metrizoate w/v) rrlas from Nycomed, Oslo, Norway. The bacterial xins (alveolysin, TSST-1 and ETA) rJere purified as was previously ribed (8,9,10). ~ZsI]-rProtein A (2.6-3.7 MBq/pg) was purchased NEN-DuPont, Dreieich, FRG). Cellulose nitrate (0.45 urn pm-e th) was from Schleicher 8 Schiill, Dassel, FRG. Monoclonal es against hsp72 were obtained from Amersham-Buchler, The phosphate-buffered saline (PBS) contained 120 r&i NaCl/ PO& 3 mt4 KH~PO~ (pH 7.4). tion and incubation of cells. Human polymorphonuclear neutroBPMNs) were isolated from heparinized (15 U/ml) peripheral blood of healthy donors using a Ficoll-metrizoate gradient and subsequent dextran sedimentation as was described elsewhere (11). The erythrocytcs were lysed by exposing the cell suspension to hypotonic conditions. The purified cell fraction contained mOre than 95% viable PMNs. The PM& were diluted to a final concentration of 2 x lo7 cells/ml in PBS. Isolated cells were incubated with the bacterial toxins or iwith PBS as control in the presence of CaZ+t4gZ+ (1 III& 0.5 r&l. Incubation proceeded at 37OC under conditions described in the elqphrirents. The toxins were used at subcytolytic concentrations for p)(k, as was confirmed by the analysis of LDH-release and by trypanblue exclusion test. Hemolysin activity was determined as previously described (5). One hewlytic unit of alveolysin was equivalent to 700 ngper ml of PBS. TSST-1 and ETA were used in a subhemolytic range for red blood cells. . After cell rupture by sonication equal protein concentrations (lo-20 ug) of the cell lysates supplemented with protease inhibitors (1mM EDTA, 1mM leupeptin, 1@4 PE6F) were suspended with an equal volume of Laemmli's sample buffer (12; up to a final velure of 50 ~1) containing dithiothreitol (0.1 M) and resolved on a 10x SDS-polyacrylamide gel in a vertical slab unit (Mighty Small II, Atlanta, Heidelberg, FRG). Proteins were blotted on cellulose nitrate for the detection of hsp70-group proteins. The bound rmnoclonal antibodies were detected using a rabbit-anti-mouse-I% antibody and Oligo d(TIla (2.5 uM) following the method described by K. Mullis (13). 10 ~1 of cDNA solution was amplified in reactions (50 ~~11 containing 1 NM of 3-actin primer as control