 |
INTRODUCTION |
The 70-kDa heat shock proteins
(Hsp70s)1 are highly
conserved proteins expressed both constitutively (Hsc70) and under
stressful conditions (Hsp70) in all prokaryotes and eukaryotes (1, 2). Members of the Hsp70 protein family play essential roles as molecular chaperones in the cytosol, mitochondria, and endoplasmic reticulum. Hsp70s are required for nascent or misfolded protein folding (3, 4),
protein translocation into endoplasmic reticulum and mitochondria (5,
6), and uncoating of clathrin-coated vesicles (7-9). These molecular
chaperone functions of Hsp70s require the enzymatic hydrolysis of ATP
and cycles of bound nucleotide exchange.
Recently, Hsp70s were found to be present in circulation, and their
levels were increased in a number of pathological conditions (10-12).
Studies also show that Hsp70 is a potent activator of the innate immune
system (13, 14). Using recombinant human Hsp70 (rhHsp70), it has been
demonstrated that Hsp70 induces the production of pro-inflammatory
cytokines such as tumor necrosis factor-
(TNF
) and interleukin-6
via the CD14 and Toll-like receptor (TLR)-mediated signal transduction
pathway (12, 15-17). Thus, it has been suggested that Hsp70, through
its cytokine function, serves as a danger signal and that Hsp70 could
be the endogenous ligand for the TLRs, both TLR-2 and TLR-4 (16,
17).
The CD14 and TLR complexes are pattern recognition receptors involved
in the innate immunity for the pathogen recognition and host defense
(18, 19). CD14, the endotoxin (lipopolysaccharide (LPS)) receptor, is a
glycophosphatidylinositol-anchored membrane protein lacking
transmembrane and intracellular domains (20, 21). TLRs are Type I
transmembrane proteins with an extracellular domain containing a
leucine-rich repeat and a cytoplasmic domain analogous to that of the
interleukin-1 receptor family (18, 22). Together with CD14, TLR4
initiates signaling cascades in response to LPS, an abundant glycolipid
of the outer membrane of the Gram-negative bacterial cell wall, whereas
TLR2 initiates the signal cascades in response to bacterial
lipoproteins, Gram-positive bacteria, mycoplasma, yeast, and
spirochetes (18, 22-24).
Because rhHsp70 is produced by Escherichia coli expressing
human Hsp70 cDNA, the final preparation may be contaminated with bacterial products such as LPS and lipoproteins. Contamination of
rhHsp70 with LPS and/or lipoproteins could be responsible for the
reported cytokine functions of Hsp70. In the current study, we
demonstrated that the ability of commercially available rhHsp70 preparations to induce TNF
production by murine macrophages is solely due to the contaminating LPS.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Recombinant human Hsp70 proteins were purchased
from StressGen Biotechnologies Corp. (Victoria, British Columbia,
Canada). Recombinant Hsp70 was cloned from a human embryonic cDNA
library and expressed in E. coli. Two preparations were
available, catalog No. ESP-555 (previously ESP-755) and NSP-555
(previously SPP-755). None of the two rhHsp70 preparations contained
the constitutively expressed Hsp70 (Hsc70). Both rhHsp70 preparations
were assayed for their abilities to bind and hydrolyze ATP. The ESP-555
preparation was the low endotoxin preparation containing <50 EU
(endotoxin units)/mg of rhHsp70 as determined using the Limulus
Amebocyte lysate (LAL) assay, which was recommended for use in assays
requiring low endotoxin. The NSP-555 preparation was not tested for
endotoxin levels and was used in protein-binding assays. For the
purpose of this report, the low endotoxin preparation, ESP-555, was
designated rhHsp70-1, whereas the NSP-555 preparation was designated
rhHsp70-2.
Protein-free LPS (from JM83 E. coli K-12, rough strain) was
kindly provided by Dr. John E. Somerville of Bristol-Myers Squibb Co.
Before use, LPS was dissolved in sterile, pyrogen-free water, sonicated
on ice for 30 s with a sonic dismembrator (Fisher), and diluted
with phosphate-buffered saline (Invitrogen). Polymyxin B sulfate
(catalog No. P4932, cell culture-tested), phosphocreatine kinase,
creatine phosphate, and ATP were purchased from Sigma. Polymyxin
B-agarose (Detoxi Gel, catalog No. 20339) was obtained from Pierce.
Enhanced chemiluminescence (ECL) Western blotting detection solutions
were purchased from Amersham Biosciences.
Cell Culture--
RAW 264.7 murine macrophages (from American
Tissue Culture Collection, Manassas, VA) were cultured in Dulbecco's
modified Eagle's medium (Invitrogen) supplemented with 10% fetal
bovine serum (Invitrogen). Subcultures of macrophages were prepared
every 2 days by scraping cells into fresh medium.
Determination of TNF
Release by Murine
Macrophages--
Murine macrophages were seeded in 24-well plates at
2.5 × 105 cells/well the day before the experiment.
After washing 3 times with the medium, the cells were treated with or
without rhHsp70 (0.1-5 µg/ml) and/or LPS (0.1-100 ng/ml) in 250 µl of medium containing 10% fetal bovine serum for 4 h at
37 °C. At the end of the treatment, media were collected and
clarified by centrifugation at 10,000 rpm for 5 min in a
microcentrifuge (Hermle-Labortechnik, Wehingm, Germany). TNF
content of the media was then determined by a quantitative sandwich
enzyme-linked immunosorbent assay using the Quantikine M mouse TNF
immunoassay kit (catalog No. MTA00, R & D Systems, Minneapolis, MN)
according to the manufacturer's recommendation. All experiments were
done in duplicate.
In some experiments, rhHsp70 and LPS were heated for 60 min in a
boiling water bath before being added to the cells. Likewise, in some
experiments macrophages were preincubated with or without polymyxin B
sulfate (10 µg/ml) for 30 min at 37 °C before the addition of
rhHsp70 or LPS to inactivate LPS.
Measurements of Endotoxin Activity--
The endotoxin activities
of rhHsp70 and LPS preparations were determined using the LAL assay kit
(catalog No.50-648U, BioWhittaker, Walkersville, MD) according to the
manufacturer's recommendation.
Quantification of rhHsp70 Protein by Gel Electrophoresis and
Western Blotting--
The rhHsp70 preparations and fractions from
polymyxin B-agarose columns (see "Removal of Endotoxin from
rhHsp 70-2 Using Polymyxin B-agarose") were analyzed by
SDS-PAGE using 7.5% polyacrylamide gels followed by staining with
Coomassie Blue or by Western blotting using an antibody to recombinant
human Hsp70 (catalog No. SPA-812, StressGen) followed by ECL detecting
system as described previously (25). Densitometric quantification of
rhHsp70 of the Coomassie Blue-stained SDS gels or immunoblots were
performed using the FluorChem 8000 digital imaging system (Alpha
Innotech Corp., San Leandro, CA).
Assay for the Activity of Removing Clathrin from Clathrin-coated
Vesicles (Uncoating Reaction)--
This was performed as described
previously (8). Briefly, the uncoating reaction mixture contained 35 µg/ml (0.5 µM) rhHsp70, bovine clathrin-coated vesicles
containing 0.5 µM clathrin triskelions (kindly provided
by Drs. Lois Greene and Evan Eisenberg of the NHLBI, National
Institutes of Health, Bethesda, MD), 1 mM Mg-ATP, and an
ATP-regenerating system consisting of 30 units/ml phosphocreatine kinase and 15 mM creatine phosphate. The mixtures were
incubated at 25 °C for 10 min, and vesicles in the reaction mixture
were removed by centrifugation at 1 × 105 rpm for 6 min in a TL-100 tabletop centrifuge (Beckman Instruments). Clathrin
released from the coated vesicles in the supernatant was analyzed by
SDS-PAGE, Coomassie Blue staining, and densitometric quantification.
The results, clathrin-uncoating activities of rhHsp70, were expressed
as optical density ratios of clathrin/rhHsp70.
Removal of Endotoxin from rhHsp70-2 Using Polymyxin
B-agarose--
Endotoxin in rhHsp70-2 was removed using polymyxin
B-agarose (Detoxi Gel, Pierce) according to manufacturer's
recommendation. Briefly, aliquots of 0.5 ml of polymyxin B-agarose were
poured into Poly-Prep disposable columns (Bio-Rad) and equilibrated in phosphate-buffered saline. Columns were washed with 5 volumes of 1%
sodium deoxycholate followed by 10 volumes of phosphate-buffered saline. 250 µl of rhHsp70-2 at 100 µg/ml was loaded onto each 0.5-ml Detoxi column and incubated at room temperature for 60 min. The
column was then eluted with phosphate-buffered saline in 250-µl
fractions. The Detoxi column fractions were analyzed by SDS gels,
stained with Coomassie Blue, and quantified as described above.
Statistical Analysis--
Results were expressed as the
mean ± S.D. Levels of significance were determined using a
two-tailed Student's t test (26), and a confidence level of
greater than 95% (p < 0.05) was used to established
statistical significance.
 |
RESULTS |
Induction of TNF
Release by rhHsp70 and LPS--
The reported
induction of proinflammatory cytokine release by rhHsp70 was similar to
the effect of LPS, i.e. mediated through the CD14 and TLR-4
receptor complex signal transduction pathway (12, 15-17). Because
induction of manganese superoxide dismutase (MnSOD) by LPS is also
dependent on CD14 and TLR-4 (27), we wanted to know whether rhHsp70
could also induce MnSOD. To avoid the potential confounding effect of
LPS, we first studied the highly purified, low endotoxin preparation of
rhHsp70, i.e. rhHsp70-1. We observed to our surprise that
rhHsp70-1 not only did not induce manganese superoxide dismutase but
also failed to induce TNF
production by RAW264.7 murine macrophages
(data not shown). Failure to induce TNF
release from macrophages by
rhHsp70-1 was in marked contrast to the reported effect of rhHsp70.
For this reason, we decided to study the effects of both rhHsp70-1 and
the less purified rhHsp70-2 preparation to determine whether rhHsp70
could in fact induce TNF
production by macrophages.
As shown in Fig. 1A,
rhHsp70-1 at 1 µg/ml, as compared with control, did not cause an
increase in TNF
release by murine macrophages. In contrast,
rhHsp70-2 at the same concentration induced a marked increase in
TNF
release to a similar extent as induced by 100 ng/ml LPS. Dose
response studies revealed that rhHsp70-1 at concentrations up to 5 µg/ml failed to induce TNF
release. However, rhHsp70-2 at a
concentration as low as 0.1 µg/ml induced a marked release of TNF
(Fig. 1B). Likewise, LPS at a concentration as low as 0.1 ng/ml induced a marked release of TNF
from murine macrophages (Fig.
1C).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 1.
Induction of TNF
release from RAW264.7 murine macrophages by LPS and rhHsp70
preparations. Macrophages were treated with either LPS or rhHsp70
preparations at the indicated concentrations for 4 h. TNF
concentration in conditioned media was then determined. A,
TNF -inducing activities of rhHsp70 preparations and LPS.
B, dose response of rhHsp70-induced TNF release by murine
macrophages. C, dose response of LPS-induced TNF release
by murine macrophages. Values in A and C
represent the means ± S.D. of 3-6 experiments. *, p < 0.05 (versus control).
|
|
Endotoxin Contents and Functional Properties of rhHsp70-1 and
rhHsp70-2--
There were two possible interpretations for the above
observations. First, rhHsp70 had no effect on the TNF
release by
macrophages; the observed effect of rhHSP70-2 was due to some
contaminant(s) present in the rhHsp70-2 preparation. Second, rhHsp70
did have TNF
-inducing effect; failure of rhHsp70-1 to induce TNF
release by macrophages was due to the presence of functionally inactive rhHsp70 in the rhHsp70-1 preparation. To distinguish these two possibilities, we determined the functional characteristics as well as
endotoxin contents of the rhHsp70-1 and rhHsp70-2 preparations.
As shown in Fig. 2, A and
B, SDS-PAGE and immunoblot analyses of rhHsp70-1 and
rhHsp70-2 revealed that both preparations were identical in rhHsp70
protein content, molecular weight, and ability to interact with an
anti-rhHsp70 antibody. In addition, both preparations had similar
molecular chaperone activities as determined by the uncoating of
clathrin-coated vesicles (Fig. 2C). In contrast, rhHsp70-2
contained a markedly higher content of endotoxin than rhHsp70-1 as
determined using the LAL assay. As shown in Fig. 2D, the
endotoxin activity of rhHsp70-1 was 4.1 ± 0.2 EU/mg, that of
rhHsp70-2 was 577.0 ± 74.2 EU/mg, and that of E. coli
LPS was (2.9 ± 0.7) × 106 EU/mg
(n = 3). Thus, the endotoxin content in the rhHsp70-2
preparation was 140 times higher than that in the rhHsp70-1
preparation. The calculated equivalent LPS concentration in the
rhHsp70-1 was 1.4 pg/µg, whereas it was 0.2 ng/µg in the
rhHsp70-2. As shown in Fig. 1C, the concentration of LPS
present in 1 µg/ml rhHsp70-2, i.e. 0.2 ng/ml, was
sufficient to cause the observed TNF
release from macrophages by
rhHsp70-2.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Enzymatic and endotoxin activities of two
rhHsp70 preparations. A, 2 µg of rhHsp70-1
(lane 1) or rhHsp70-2 (lane 2) were analyzed on
SDS gels and stained with Coomassie Blue. B, 0.5 µg of
rhHsp70-1 (lane 1) or rhHsp70-2 (lane 2) was
analyzed by Western blotting using an antibody to rhHsp70.
C, both rhHsp70 preparations were tested for their
activities in removing clathrin from coated vesicles. The uncoating
reaction was carried out with clathrin-coated vesicles containing 0.5 µM clathrin triskelion, 0.5 µM either
rhHsp70-1 or rhHsp70-2, 1 mM ATP-Mg2+, and an
ATP-regenerating system. After removing the coated vesicles by
centrifugation, the amount of clathrin released and rhHsp70 in the
supernatant were quantified by SDS-PAGE and densitometric scanning. The
relative uncoating activities of the two rhHsp70 preparations were
plotted as optical density (OD) ratios of clathrin/rhHsp70.
D, the endotoxin activity of LPS or rhHsp70 preparations was
determined by LAL assay. Values in bar graphs shown in
C and D represent the means ± S.D. of three
experiments.
|
|
Role of LPS in the Induction of TNF
Release by
rhHsp70-2--
To determine whether the contaminating LPS in the
rhHsp70-2 preparation was responsible for its TNF
-inducing
activity, we first used polymyxin B-agarose (Detoxi gel) to remove LPS
from rhHsp70-2. The concentration of rhHsp70 in the fractions
collected from polymyxin B-agarose columns was determined using
SDS-PAGE and densitometric quantification. The same amounts of rhHsp70 present in the polymyxin B column fractions as in the rhHsp70-2 preparation before passing through the column (Fig.
3A) were then used to
determine the endotoxin activity as well as the TNF
-inducing activity.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of removing endotoxin with polymyxin
B-agarose on the endotoxin activity and
TNF -inducing activity of rhHsp70-2. The
endotoxin in rhHsp70-2 was removed by passing through a polymyxin
B-agarose column. Two fractions were collected from the column, PB-1
and PB-2. rhHsp70 concentrations in polymyxin B column fractions were
determined by Coomassie Blue-stained SDS gels and densitometric
scanning. A, 1 µg of rhHsp70-2 before polymyxin B column,
PB-1 and PB-1, containing 1 µg of rhHsp70-2 were analyzed by
SDS-PAGE. B, the endotoxin activities of rhHsp70-2 or
polymyxin B (PB) column fractions were determined by LAL
assay. C, the TNF -inducing activity of 0.5 µg/ml
rhHsp70-2 or polymyxin B column fractions containing 0.5 µg/ml
rhHsp70 was determined. the values in the bar graphs shown
in B and C represent the means ± S.D. of
three experiments. *, p < 0.01 (versus
rhHsp70-2).
|
|
As shown in Fig. 3B, the polymyxin B column removed more
than 95% of the endotoxin activity from rhHsp70-2. The endotoxin activity of rhHsp70-2 before the polymyxin B column was 577.0 ± 74.2 EU/mg, whereas it was 26.0 ± 14.2 EU/mg and 22.0 ± 2.8 EU/mg in the two fractions of the polymyxin B column. Likewise, polymyxin B column removed most of the TNF
-inducing activity of
rhHsp70-2 (Fig. 3C). Thus, polymyxin B column removed LPS
from the rhHsp70-2 preparation along with its TNF
-inducing activity.
In several similar studies (12, 15, 17), polymyxin B has been directly
added into the incubation medium to determine whether the observed
effect is due to LPS. Therefore, we also tested the effect of polymyxin
B in the incubation medium. In these experiments, macrophages were
preincubated with polymyxin B at 10 µg/ml before rhHsp70-2 or LPS
was added to the incubation media. To make the comparison relevant, we
used an amount of LPS (0.2 ng/ml) with an endotoxin activity equivalent
to that found in 1 µg/ml rhHsp70-2. As shown in Fig.
4, polymyxin B markedly inhibited the
TNF
-inducing activities of both LPS and rhHsp70-2.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of preincubation with polymyxin B on
TNF -inducing activities of LPS and
rhHsp70-2. Macrophages were preincubated with (solid
bars) or without (open bars) 10 µg/ml polymyxin B
(PB) for 30 min at 37 °C before LPS or rhHsp70-2 was
added at the indicated concentrations. Macrophages were incubated for
an additional 4 h before the media were collected and analyzed for
TNF concentration. Values represent the means ± S.D. of three
experiments. *, p < 0.01 (versus no
polymyxin B).
|
|
The above findings strongly suggest that the observed TNF
-inducing
effect of rhHsp70-2 was due to the contaminating LPS. If this were the
case, one would expect that the addition of LPS at a concentration
found in rhHsp70-2 to the rhHsp70-1 should result in the same
TNF
-inducing activity as the rhHsp70-2. As shown in Fig.
5, rhHsp70-1 at 1 µg/ml alone had no
TNF
-inducing activity. However, the addition of LPS to the
rhHsp70-1 at a final concentration of 0.2 ng/ml resulted in a similar
TNF
-inducing activity as that of 0.2 ng/ml LPS or 1 µg/ml
rhHsp70-2.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 5.
The TNF -inducing
activity of LPS added to rhHsp70-1. Macrophages were treated with
1 µg/ml rhHsp70-1, 0.2 ng/ml LPS, 1 µg/ml rhHsp70-2, or 1 µg/ml
rhHsp70-1 + 0.2 ng/ml LPS for 4 h. TNF concentrations in the
culture media were then determined. Values represent the means ± S.D. of three experiments.
|
|
Effect of Heat Inactivation on TNF
-inducing Activities of LPS
and rhHsp70-2--
One of the characteristics of LPS is its relative
heat resistance. Previous studies show that the TNF
-inducing
activity of rhHsp70 was heat-sensitive, whereas that of LPS was
heat-resistant (12, 15-17). It was concluded that the TNF
-inducing
activity of rhHsp70 could not have been due to the effect of
contaminating LPS. As shown in Fig. 6, we
also demonstrated that the TNF
-inducing activity of rhHsp70-2 at 1 µg/ml, but not that of LPS at 100 ng/ml, was sensitive to prior
heating in a boiling water bath for 60 min.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of heat inactivation on
TNF -inducing activities of rhHsp70-2 and
LPS. Stock solutions of rhHsp70-2 at 100 µg/ml and LPS at 10 µg/ml were placed in boiling water for 1 h. Macrophages were
treated with or without rhHsp70-1, rhHsp70-2, heated rhHsp70-2, LPS,
or heated LPS at the indicated concentrations for 4 h. TNF
concentrations in media were then determined. Values represent the
means ± S.D. of three experiments. *, p < 0.01 (versus rhHsp70-2).
|
|
The demonstrated heat sensitivity of rhHsp70-2 in inducing TNF
release appeared in contradiction to our above conclusion that the
TNF
-inducing activity of rhHsp70-2 was due to the contaminating LPS. However, the following evidence suggests that LPS at low concentrations, such as 20 ng/ml, is heat-sensitive and that heat sensitivity by itself is not sufficient to distinguish whether an
observed effect is due to LPS.
As shown in Fig. 7A, the
addition of 0.2 ng/ml LPS to rhHsp70-1 (1 µg/ml) resulted in a
TNF
-inducing effect similar to that of 0.2 ng/ml LPS or 1 µg/ml
rhHsp70-2 (as previously demonstrated in Fig. 5). However, the
TNF
-inducing effect of the combination of LPS and rhHsp70-1 was as
heat-sensitive as that of the rhHsp70-2. Analysis of rhHsp70 using
SDS-PAGE revealed that heat inactivation removed most of Hsp70 from the
rhHsp70-1 and rhHsp70-2 solutions (Fig. 7B). Thus, it was
possible that heat inactivation removed the rhHsp70 along with LPS from
the solution, resulting in the observed lost of TNF
-inducing
activity.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of heat inactivation on the
TNF -inducing activity of LPS added in
rhHsp70-1. Stock solutions of 100 µg/ml rhHsp70-2 and 20 ng/ml
LPS added to 100 µg/ml rhHsp70-1 were heated in boiling water for
1 h. A, macrophages were treated with either rhHsp70 or
LPS at the indicated concentrations for 4 h. TNF concentrations
in media were determined. B, 2 µg of each rhHsp70
preparation before and after heat inactivation were analyzed on SDS
gels stained with Coomassie Blue. Lane 1, rhHsp70-1;
lane 2, heated rhHsp70-1; lane 3, rhHsp70-2;
lane 4, heated rhHsp70-2. Values in A represent
the means ± S.D. of three experiments. *, p < 0.05 (versus non-heated rhHsp70).
|
|
To eliminate the potential confounding effect of rhHsp in the
above-observed heat sensitivity of LPS, we heated LPS alone at two
concentrations, 20 ng/ml and 20 µg/ml, and then determined the
endotoxin activity and TNF
-inducing activity of 0.2 ng/ml LPS before
and after heating. As shown in Fig. 8,
heating 20 ng/ml LPS in a boiling water bath for 60 min markedly
reduced its endotoxin activity as well as its TNF
-inducing activity.
Likewise, heating LPS at 20 µg/ml also reduced its endotoxin
activity and TNF
-inducing activity, but to a lesser degree. Thus,
LPS at low concentrations is highly heat-sensitive.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of heat inactivation on the endotoxin
activity and TNF -inducing activity of
LPS. LPS stock solution at 20 µg/ml or 20 ng/ml was heated in
boiling water for 1 h. Both heated and non-heated LPS were tested
for endotoxin activity using LAL assay (A) and for TNF
induction in macrophages (B) at the same concentration (0.2 ng/ml). Values represent the means ± S.D. of three experiments.
*, p < 0.05 (versus non-heated
controls).
|
|
 |
DISCUSSION |
Results presented in the current study demonstrated that rhHsp-70
did not induce TNF
release from murine macrophages and that the
TNF
-inducing activity of the rhHsp70-2 preparation was entirely due
to the contaminating LPS. This conclusion was derived from the
following evidence. First, the highly purified rhHsp70 preparation
(rhHsp70-1) with an LPS content of 1.4 pg/µg was unable to induce
TNF
release from macrophages at concentrations up to 5 µg/ml,
whereas the less purified rhHsp70 preparation (rhHsp70-2), at 1 µg/ml, with a LPS content of 0.2 ng/µg was able to induce TNF
release to the same extent as that induced by 0.2 ng/ml LPS (Figs. 1
and 2D). Second, failure of rhHsp70-1 to induce TNF
release was not due to defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical Hsp70 content as determined by SDS
gels stained with Coomassie Blue and Western blots probed with an
anti-rhHsp70 antibody (Fig. 2, A and B). Both
rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles (Fig.
2C). Third, removal of LPS from rhHsp70-2 by polymyxin
B-agarose column or direct addition of polymyxin B to the incubation
medium essentially eliminated the TNF
-inducing activity of
rhHsp70-2 (Figs. 3 and 4). Fourth, the addition of LPS at the
concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same
TNF
-inducing activity as observed with rhHsp70-2 (Fig. 5).
Using rhHsp70 obtained from the same source as the one we used in the
current study (StressGen Biotechnologies Corp.), a number of
laboratories reported that rhHsp70 markedly induced TNF
production by monocytes and macrophages (12, 15-17). However, with the exception of one report by Dybdahl et al. (12), none of the reports
indicated which rhHsp70 preparation from StressGen was used in the
studies. Using the less purified preparation of rhHsp70
(NSP-555/SPP-755 or rhHsp70-2), Dybdahl et al. (12) report
that their rhHsp70 preparation had an endotoxin activity of about 100 EU/mg. They demonstrated that the rhHsp70 preparation could induce
TNF
and interleukin-6 production by murine macrophages and human
monocytes at rhHsp70 concentrations of 3-10 µg/ml. The endotoxin
activities present in 3-10 µg/ml rhHsp70, e.g. 0.3-1
EU/ml (equivalent to that of 0.1-0.3 ng/ml LPS), were sufficient to
induce the cytokine production as demonstrated in the current study.
Other studies (15-17), although not specifying which rhHsp70
preparation was studied, used two criteria to rule out the possibility
that the observed cytokine-inducing effect was due to the contaminating LPS. These included the differential sensitivity of rhHSP70 and LPS to
heat inactivation and the specific LPS inhibitory effect of polymyxin
B. However, as demonstrated in the current study, these criteria are
insufficient to rule out the possibility of LPS being responsible for
the observed effects.
The current study demonstrates that LPS is heat-sensitive, particularly
at low concentrations, e.g. 20 ng/ml. This appeared to be
contrary to the popular belief that LPS is heat-resistant (12, 15-17).
However, the immunosuppressive activity of Shigella sonnei
LPS has also been shown to be heat-sensitive (28). In the current
study, we show that the results of heat sensitivity experiments may
vary depending on the concentrations of LPS used to activate cells. As
shown in Fig. 6, when 100 ng/ml LPS was used to stimulate TNF
release from murine macrophages, heat inactivation of LPS appeared to
have no effect on TNF
induction. Results in Fig. 1C
showed, however, LPS at 0.1 ng/ml was sufficient to induce significant
TNF
release from murine macrophages. Thus, even if heating in
boiling water for 60 min inactivated more than 99% of LPS at 100 ng/ml, there would still be sufficient active LPS left to induce TNF
release, giving the impression that TNF
-inducing activity of LPS was
heat-resistant. Results in Fig. 8 showed that heating LPS at 20 ng/ml,
the same concentration found in 100 µg/ml rhHsp70-2 when it was
heated, resulted in an 80% decrease in TNF
-inducing activity. This
explains why the observed TNF
-inducing activity of rhHsp70-2
was heat-sensitive.
In the current study, we demonstrated that removal of the contaminating
LPS from rhHsp70-2 by polymyxin B-agarose column or by preincubation
of macrophages with polymyxin B almost completely eliminates the
TNF
-inducing activity of rhHSP70-2. In contrast, others observed no
significant effect of preincubation or co-incubation with polymyxin B
on cytokine-inducing activity of the rhHsp70 (12, 15, 17). The reason
for this discrepancy is not clear. The effect of polymyxin B may depend
on the concentrations of polymyxin B and rhHsp70 and the amount of
contaminating LPS present in the final incubation medium. Further
studies are necessary to clarify this discrepancy.
In addition to rhHsp70, recombinant chlamydial Hsp60 and recombinant
human Hsp60 have all been shown to have cytokine-inducing effects
(29-32). Hsp70, Hsp90, and gp96 isolated from mouse liver are also
shown to induce cytokine production (33), although at concentrations
1-2 orders of magnitude higher than the recombinant Hsp70s. In view of
the current study, further investigation is required to determine
whether these observed cytokine-inducing effects of heat shock proteins
(Hsps) was indeed due to the Hsps themselves or the contamination of
LPS and/or bacterial lipoproteins in heat shock protein preparations.