| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
German Diabetes Research Institute at the Heinrich-Heine-University of Düsseldorf, Düsseldorf, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2-macroglobulin receptor as binding site. Hsp60 binding
occurred in the absence of surface TLR4. However, no cytokine response
was induced by hsp60 in TLR4-deficient macrophages. We conclude that
hsp60 binds to a stereo-specific receptor on macrophages, and that
different surface molecules are engaged in binding and signal
transduction. Furthermore, the binding site for hsp60 is separate from
the common receptor for hsp70, hsp90, and gp96, which suggests an
independent role of hsp60 as danger Ag and in
immunoregulation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previous studies have shown that human recombinant hsp60 induces a
proinflammatory response in mouse as well as in human macrophages
(17, 18). This response includes the release of
inflammatory mediators such as IL-6 and TNF-. In addition, human
hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and
IL-15 (18). These findings suggest a role of hsp60 as a
danger signal for the innate immune system. At present little is known
about the hsp60-macrophage interactions, except that Toll-like receptor
(TLR) 2 and TLR4 appear to be involved in signal transduction
(19, 20). However, since TLR2 and TLR4 act as signaling
receptors to a broad variety of microbial constituents and other
immunostimulatory agents (21, 22), a direct interaction
between hsp60 and TLRs is improbable. Recently, a potential receptor
for gp96 and the related cytosolic hsp90 has been identified by Binder
et al. (23, 24). In this study, a receptor, which was
previously known as 2-macroglobulin
(2M) receptor, has been shown to be a cell
surface receptor for gp96 and hsp90 on macrophages. Meanwhile, other
studies provided evidence for the existence of a proteinaceous receptor
for hsp70 on macrophages (25, 26), which was recently also
identified as the 2M receptor
(26). These findings indicate that the
2M receptor serves as a common receptor for
gp96, hsp90, and hsp70.
Based on these findings, the present study was designed to identify a potential receptor for hsp60 on macrophage cell surfaces. Therefore, a flow cytometry-based assay was established to analyze the specific binding of human recombinant hsp60 to cell surfaces of the macrophage cell lines J774 A.1 and RAW264.7. By the use of this experimental system, we were able to demonstrate for the first time a direct binding of hsp60 to macrophages. The binding of hsp60 was found to be saturable and occurred with submicromolar affinity. Furthermore, our data indicate that the receptor for hsp60 on macrophages is different from the receptor for hsp70, hsp90, or gp96. In addition, binding studies with TLR4-deficient macrophages revealed that the receptor structure for hsp60 involves at least two functionally different components for binding and signaling.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The mouse macrophage cell line J774 A.1 was purchased from the German Collection of Microorganism and Cell Culture (Braunschweig, Germany). J774 A.1 cells were cultured in RPMI 1640 medium (PAA Laboratories, Linz, Austria) supplemented with 10% (v/v) FCS (Life Technologies, Rockville, CA), ampicillin (25 mg/L), penicillin (120 mg/L), streptomycin (270 mg/L), 1 mM sodium pyruvate, 2 mM L-glutamine, nonessential amino acids (10 ml/L, 100x), 24 mM NaHCO3, and 10 mM HEPES. The mouse macrophage cell line RAW264.7 was purchased from American Type Culture Collection (Manassas, VA) and cultured in DMEM (PAA Laboratories GmbH) supplemented with 4 mM L-glutamine, 1.4 g/L sodium bicarbonate, 4.5 g/L glucose, 1 mM sodium pyruvate, and 10% (v/v) FCS.
Mouse bone marrow-derived macrophages
C57BL/10ScCr mice, lacking the complete TLR4 protein, and mice of the normal TLR4 protein, expressing control strain C57BL/10ScSn (27), were kindly provided by M. Freudenberg (Max Planck Institute for Immunology, Freiburg, Germany). C57BL/6JBom mice, purchased from the Breeding and Research Center (Bomholtgård, Ry, Denmark), were used as a further control. Bone marrow cells were obtained by flushing femurs and tibias with ice-cold PBS and washed by centrifugation (500 x g, 5 min). A total of 3.5 x 106 bone marrow cells was incubated in tissue culture dishes with Pluznik medium (19, 28). After 7–8 days, adherent bone marrow-derived macrophages (BMM) were detached by incubation with ice-cold Ca2+-, Mg2+-free HBSS for 10 min, followed by washing with HBSS (500 x g, 5 min), and were used for binding studies.
Reagents
Recombinant human hsp60 was obtained from Peptor (Rehovot,
Israel) or StressGen Biotechnologies (Victoria, BC, Canada).
Recombinant human hsp70 and hsp90 were purchased from StressGen
Biotechnologies, and recombinant mouse gp96 from IMMATICS
Biotechnologies (Tübingen, Germany). Escherichia coli
O36:B6 LPS, BSA, OVA, alcohol dehydrogenase
(ADH), and 2M were from Sigma (Deisenhofen,
Germany), and transferrin was from Molecular Probes (Leiden, The
Netherlands).
All proteins used in this study were tested for their endotoxin
contents by quantitative Limulus amebocyte lysate assay
(BioWhittaker, Verviers, Belgium). Endotoxin contents of hsp70, hsp90,
and OVA were 
0.25 EU/µg protein. The endotoxin amount of
2M was 
0.025 EU/100 ng protein, whereas
gp96 was 1 EU/µg protein. For the various hsp60 preparations, an
endotoxin content in the range of 0.01–2 EU/µg protein was
determined. No difference in binding or competition for binding was
observed for preparations varying in LPS content.
Protein labeling
Labeling of hsp60 and ADH with fluorescent dye was performed using the Alexa Fluor 488 Protein Labeling kit (Molecular Probes), according to the manufacturer’s recommendations. Briefly, 1 mg hsp60 or ADH was incubated with Alexa Fluor 488 in 0.1 M sodium bicarbonate for 1 h at room temperature, followed by incubation for 3 h at 4°C. Unconjugated dye was removed by extensive dialysis in PBS. The number of dye molecules bound per protein molecule was determined by measuring the OD at 280 and 494 nm. It was calculated that six to nine Alexa Fluor molecules bind to each protein molecule. Alexa Fluor 488-labeled transferrin was purchased from Molecular Probes, and gp96-FITC was supplied by IMMATICS Biotechnologies.
Hsp60 binding and uptake
After 2 days of continuous culture, J774 A.1 and RAW264.7 cells were gently washed off the culture flask. BMM were generated as described above. The cells were centrifuged at 500 x g for 5 min (4°C) and resuspended in PBS with 1% BSA for the binding assay (4°C) or in RPMI medium with 10% FCS for the uptake experiments (37°C). Cells of either macrophage line (1 x 106 cells/ml) were incubated in a total volume of 100 µl with various concentrations of Alexa488-labeled hsp60, transferrin, ADH, or FITC-labeled gp96 for 45 min on ice for the binding studies or for 15 min on ice, and subsequently for 30 min at 37°C for the uptake studies. For the competition assays, the macrophages were preincubated with the unlabeled ligands for 30 min on ice. Then the labeled ligand was added and the incubation was continued for another 45 min on ice. Subsequently, cells were washed with PBS/1% BSA and resuspended in PBS containing 1% paraformaldehyde. The samples were evaluated using a FACScan flow cytometer (BD Biosciences, Rockville, CA). Cell surface binding of fluorescent-labeled proteins was calculated using the geometric mean fluorescence value after subtracting the autofluorescence of the cells.
Confocal microscopy
J774 A.1 macrophages were adjusted to a density of 1 x 105 cells/ml and seeded on glass slides in a volume of 200 µl (2 x 104 cells). After incubation for 24 h (37°C, 5% CO2), the adherent cells were treated with hsp60-Alexa488 or transferrin-Alexa488 in the absence or presence of the unlabeled ligands at 4°C, as described above. Then the cells were washed with PBS, followed by fixation with 1% paraformaldehyde for 15 min. Finally, the cells were washed three times with PBS and resuspended in 100 µl medium. Labeled cells were visualized using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Jena, Germany).
Stimulation of macrophages for TNF- and NO production
For the stimulation of TNF- or NO production, mouse
macrophages were adjusted to a density of 1 x
106 cells/ml and seeded in the wells of
flat-bottom 96-well plates (200 µl/well). After cultivation at 37°C
and 5% CO2 for 18 h, macrophages were
incubated with hsp60 (Peptor and StressGen Biotechnologies) or LPS.
After 6 h (TNF-) and 24 h (NO), culture supernatants were
collected and stored at -20°C until analysis.
TNF- measurements
The amounts of TNF- in culture supernatants were quantified
by sandwich ELISA using an OptEIA mouse TNF- Set (BD PharMingen, San
Diego, CA). A 96-well ELISA plate (BD Falcon, San Diego, CA) was coated
with a capture Ab anti-mouse TNF- mAb diluted in coating buffer
(PBS, pH 6.5) overnight at room temperature. After washing (PBS/0.05%
Tween 20, pH 7.4) and blocking (PBS/10% FCS, 1 h, room
temperature), binding of TNF- was performed by incubation of 50 µl
culture supernatants diluted 1/10-fold with assay diluent or serial
standard dilution of mouse rTNF- for 2 h at room temperature.
Subsequently, the wells were washed, and a total of 50 µl
biotinylated mouse TNF- mAb and avidin-HRP conjugate was placed in
each well (1 h, room temperature). After extensive washing, the samples
were incubated with 50 µl substrate solution (tetramethylbenzidine
and hydrogen peroxide, 20 min, room temperature). After addition of 50
µl stop solution (2 N
H2SO4), the OD was measured
at 450/550 nm on a microplate reader. The TNF- content was
calculated by using a standard curve of mouse rTNF- with substrate
solution as a blank.
Measurement of NO production
The amount of NO released by macrophages was assessed by the determination of accumulated nitrite (NO2-) in cell-free supernatants detected by the colorimetric Griess reaction, as described previously (29). Briefly, 50 µl supernatant and serial dilutions of NaNO2 standard solution were placed in a 96-well microtiter plate (Greiner, Frickenhausen, Germany) and then mixed with an equal volume of Griess reagent containing 1% sulfanilamide, 0.1% naphthylethylenediamide dihydrochloride, and 2.5% H3PO4. After incubation for 10 min at room temperature, the OD of the reaction products was measured at 550 nm on a microplate reader, and the amount of accumulated nitrite in the samples was quantified using the standard curve obtained with NaNO2.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A flow cytometry-based assay was established to investigate the interaction of human hsp60 with cells of the mouse macrophage lines J774 A.1 and RAW264.7. Therefore, human recombinant hsp60 was labeled with Alexa488 fluorescence dye under physiological conditions in PBS, resulting in the coupling of six to nine molecules of Alexa488 per molecule of hsp60. We used different hsp60 preparations for these binding studies and obtained reproducible results. The cells were incubated in the presence of hsp60-Alexa488 or for inhibition studies, the cells were preincubated with unlabeled proteins, followed by the incubation with hsp60-Alexa488. Throughout the assay, the temperature was kept at 4°C to minimize nonspecific endocytotic events. After washing and fixation of the cells, quantitative analysis of the binding was performed by measuring the fluorescence intensity of the cells.
In a first series of experiments, the intensity and specificity of
hsp60 binding to J774 A.1 and RAW264.7 were investigated (Fig. 1
). Binding of hsp60 to J774 A.1 (Fig. 1A) and RAW264.7 (Fig. 1B) cells was demonstrable
as shown by the increasing mean fluorescence after hsp60 binding to 10
and 15 compared with the mean autofluorescence values of 2.5 and 2. The
specificity of the hsp60 binding was proved by strong inhibition of
hsp60-Alexa488 binding after preincubation with a 10-fold excess of
unlabeled hsp60, whereas preincubation with the same molar excess of
OVA was without effect. OVA was used as control because its binding
should not interfere with binding sites for hsp60. To exclude the
possibility of nonspecific binding of Alexa488-labeled proteins,
similar binding assays were performed with Alexa488-labeled ADH, a
cytosolic enzyme that should not bind to cell surface receptors. As
expected, no significant binding of ADH-Alexa488 to the investigated
macrophages could be observed (data not shown).
|
A, the mean
autofluorescence of the cells was 3.6, and with increasing
concentrations of hsp60 up to 1.2 µM, we observed higher mean
fluorescence intensities in the cell preparations (8.8–15.8 mean
fluorescence). Saturable binding of hsp60 to J774 A.1 cells was reached
at 700 nM.
|
B). J774 A.1 cells were preincubated with increasing
doses of unlabeled hsp60, followed by the addition of 350 nM
hsp60-Alexa488. The mean fluorescence after hsp60-Alexa488 binding was
8.2. Preincubation with increasing doses of unlabeled hsp60 in the
range of 350 nM up to 1.75 µM (5-fold excess) resulted in an
increased inhibition of hsp60 binding (6.8–3.9 mean fluorescence). At
concentrations higher than 1.75 µM unlabeled hsp60, the inhibition
remained at a range of >70%, corresponding to a mean fluorescence
signal of 3.9–3. Binding and uptake of hsp60
To investigate, if binding of hsp60 is followed by the uptake of
the bound ligand, we performed a further series of experiments with
J774 A.1 cells incubated at 4°C and 37°C. In these experiments,
Alexa-labeled transferrin was used as a positive control since the
transferrin receptor is known to be expressed on the surface of all
cell types (30). Furthermore, receptor-mediated uptake has
been described for transferrin (31). At 4°C,
preincubation with a 10-fold excess of unlabeled transferrin inhibited
the binding of the fluorescent-labeled transferrin by 90%, whereas the
same concentration of OVA did not affect transferrin binding (Fig. 3A). As shown before,
preincubation with a 10-fold excess of unlabeled hsp60 strongly
inhibited the binding of hsp60-Alexa488 at 4°C (Fig. 3C).
Moreover, when we tested for competition of hsp60 binding by
transferrin and vice versa, no inhibitory effects could be detected
(Fig. 3, A and C). This finding underlines the
specificity of hsp60 and transferrin binding to J774 A.1 cells.
|
B) and hsp60 (Fig. 3D), as suggested by the
shift of the mean fluorescence from 8 to 60 for transferrin and from 10
to 50 for hsp60. The specificity of the uptake was proved by the
competition of the labeled proteins with the corresponding unlabeled
ligand. Inhibition of the uptake was about 80% for both proteins (Fig. 3, B and D). Localization of bound hsp60 by confocal microscopy
The specific binding of hsp60 to the cell surface of J774 A.1
cells was confirmed by the use of confocal microscopy. J774 A.1 cells
were incubated either with hsp60-Alexa488 or transferrin-Alexa488 at
4°C for 45 min (Fig. 4, C
and D). ADH-Alexa488 served as a negative control (Fig. 4B). Cells clearly showed surface binding of hsp60 and
transferrin, but no binding of ADH.
|
To further characterize the receptor for hsp60, we tested for an
involvement of the receptor(s) for hsp70, hsp90, or gp96. J774 A.1
cells were preincubated with a 10-fold excess of either unlabeled
hsp70, hsp90, gp96, 2M, or OVA at 4°C for 30
min, followed by the addition of 350 nM hsp60-Alexa488 (Fig. 5A). Preincubation with hsp70,
hsp90, gp96, or 2M led to negligible
inhibition of hsp60 binding. OVA, which was used as control, did not
compete hsp60 binding. The specificity of hsp60 binding was confirmed
again by preincubation with unlabeled hsp60, resulting in an 80%
inhibition of the binding of hsp60-Alexa488.
|
B). Specific
binding of gp96 to J774 A.1 cells was observed, which could be
inhibited by a 10-fold excess of unlabeled gp96. Preincubation with
hsp60 did not reduce gp96 binding. Gp96 binding was also not inhibited
by an excess of hsp90 or hsp70, whereas effective competition for gp96
binding was observed in the presence of unlabeled
2M (65% inhibition) or gp96 (56%
inhibition). Taken together, these results clearly indicate that the
receptor for hsp60 on macrophages is not identical to the receptors for
gp96, hsp90, and hsp70. TLR4 as a candidate receptor for hsp60
Since previous observations (19, 20) indicate that
TLR4 may mediate the inflammatory signaling of hsp60, we investigated
the role of TLR4 as a potential binding receptor for hsp60. We
therefore compared the binding of hsp60 to BMM of C57BL/10ScSn mice,
which express the complete functionally active TLR4 protein (Fig. 6A), with the hsp60 binding to
C57BL/10ScCr-derived BMM (Fig. 6B), which completely lack
the TLR4 protein (27). Our experiments showed binding of
hsp60 to the primary cultures of mouse BMM in a similar range
as to J774 A.1 and RAW264.7 cells, thus demonstrating that hsp60
binding is not a property restricted to cells of macrophage lines.
Binding of hsp60 to BMM of C57BL/10ScSn and C57BL/10ScCr mice occurred
with comparable intensity and showed similar dose dependency.
Incubation of BMM of C57BL/10ScSn mice (Fig. 6A) at 4°C
with hsp60-Alexa488 in the indicated concentrations resulted in
an increase of the mean fluorescence values to 13 and 17 compared with
the mean autofluorescence value of 6.5. The binding of hsp60 to BMM of
C57BL/10ScCr (Fig. 6B) for the tested hsp60 concentrations
was in a similar range (14 and 18) as the binding to BMM of
C57BL/10ScSn mice. As a further control, we tested hsp60 binding to
C57BL/6JBom mouse-derived BMM and observed binding in a similar range
(data not shown). These results indicate that TLR4 does not make a
major contribution to the binding of hsp60-binding receptor, although
TLR4 is involved in hsp60 signaling.
|
To confirm that the preparations of hsp60, used for the binding
assays, were functionally competent, their stimulatory activity was
tested on cells of the macrophage lines J774 A.1 (Fig. 7, A and B) and
RAW264.7 (Fig. 7, C and D) by measuring their
TNF- and NO production. LPS (10 ng/ml), which was used as a positive
control in these assays, induced the release of 16–22 ng/ml TNF- in
both cell lines. When J774 A.1 cells were exposed to hsp60 (Peptor or
StressGen Biotechnologies), substantial amounts of TNF- in
the range of 12–19 ng/ml were detectable (Fig. 7A). In
parallel, we determined TNF- production in RAW264.7 cells (Fig. 7C) after incubation with hsp60 (Peptor or StressGen
Biotechnologies). As shown in Fig. 7C, cells treated with
hsp60 released TNF- in a range of 11–18 ng/ml. We further analyzed
the level of NO release by measuring the accumulated nitrite in
cell-free supernatants. LPS (10 ng/ml), which served again as a
positive control in these assays, stimulated the production of 61–79
µM nitrite in both cell lines. In the supernatants of J774 A.1 cells
treated with hsp60 (Peptor or StressGen Biotechnologies), nitrite
levels in the range of 29–47 µM (Fig. 7B) were
detectable. In parallel, we determined NO production in RAW264.7 cells
(Fig. 7D) after incubation with hsp60 (Peptor or StressGen
Biotechnologies). As shown in Fig. 7D, cells exposed to
hsp60 accumulated nitrite concentrations in a range of 72–81 µM in
their supernatants. Taken together, the results clearly indicate that
the tested hsp60 preparations were competent to induce a biological
response in J774 A.1 and RAW264.7 cells, as shown by the stimulation of
TNF- and NO production.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
300 nM. When macrophages were incubated with hsp60 at 37°C for 30
min, about 5 times more labeled ligand was seen per cell in comparison
with binding studies at 4°C. This indicates that binding of hsp60 to
the macrophage cell surface is followed by rapid endocytosis. The
latter process was also inhibitable by excess unlabeled hsp60, and
therefore involves prior specific binding to a hsp60 receptor on the
cell surface. Binding and uptake of hsp60 therefore share the
characteristics of receptor-mediated endocytosis with properties
described for gp96 and heat shock cognate protein 70
(32).
We therefore analyzed whether hsp60 would make use of the receptor
system described for other hsps. Recently, Binder et al.
(23) have identified the 2M
(CD91) receptor as a receptor for gp96. In this study, it was shown
that gp96 as well as the phylogenetically related hsp90 and
2M effectively compete with labeled gp96 for
binding to macrophages, and a common receptor for gp96 and hsp90 has
been proposed (23, 24). Specific binding of hsp70 to
macrophages and monocytes has also been recently shown (25, 33). Most recently, it has been demonstrated that gp96, hsp90,
as well as hsp70 utilize the 2M receptor as a
common receptor (26).
Our data provide evidence that the recognition unit for hsp60 is
different from that used by hsp70, hsp90, or gp96. In a first
experimental approach, we studied whether excess hsp70, hsp90, or gp96
would compete with binding of hsp60 to macrophages. We did not observe
any cross-reactivity. 2M, a potent inhibitor
of the gp96 receptor, also did not decrease hsp60 binding. A second
approach was to use labeled gp96 and attempt inhibition of binding by
hsp60. gp96 bound to the cell surface with similar fluorescence
intensity as observed for hsp60. Furthermore, we confirmed the
specificity of binding in that unlabeled gp96, but not OVA was
inhibitory. The involvement of the 2M receptor
was also confirmed by displacement of gp96 binding by excess
2M. By contrast, excess hsp60 did not affect
gp96 binding. Taken together, the experiments show that hsp60 makes use
of a surface receptor that is different from binding sites for gp96 and
2M as well as from binding sites for hsp70 and
hsp90.
Interestingly, a 10-fold molar excess of hsp70 or hsp90 was not able to significantly decrease binding of gp96. This does not exclude that hsp70 or hsp90 may interact with the gp96 receptor, albeit at much lower affinity. Indeed, in the binding studies published, much higher concentrations of hsp70 were required for displacement of gp96 (24). By contrast, Sondermann et al. (25) reported that effective displacement of labeled hsp70 by unlabeled hsp70 required inhibitor concentrations in a similar range as used in this study. These findings suggest the existence of a high affinity receptor for hsp70 in addition to the gp96 receptor.
Both preparations of human hsp60 used in this study showed
proinflammatory activity, as evident from the induction of TNF- and
NO release in both macrophage lines. The biological activity of hsp60
is dose dependent, and a cytokine response requires concentrations of
1–3 µg protein/ml (18, 19). These concentrations
correlate well with the dose dependency of hsp60 reported in this work.
Since 50% saturation of binding sites is seen at about 0.3 µM, an
inflammatory response may require binding to 10–20% of hsp60
receptors.
We and others have reported previously that CD14, MD-2, TLR2, and TLR4 may mediate the inflammatory signaling of hsp60 (19, 20, 34). However, these studies were confined to functional assays and did not determine actual binding. When considering the physicochemical variety of lipids, glycoconjugates, and peptides signaling via the TLR complex (21, 22), direct interaction with all of these potential ligands, including hsp60, seems improbable. Indeed, in a preliminary experimental approach, BMM of C57BL/10ScCr mice completely lacking the TLR4 protein showed similar hsp60-binding intensity and dose dependency as BMM of a control mouse strain with normal TLR4 expression. In addition, exogenous hsp60 could not provoke a functional response in BMM of TLR4-deficient C57BL/10ScCr mice and in BMM of C3H/HeJ mice that express a mutated TLR4 molecule unable to transduce a binding signal (Ref. 19 , unpublished observations). We therefore assume that the CD14-TLR4 receptor complex is not involved in binding of hsp60, but mediates the further signal transduction of hsp60 in macrophages, downstream of ligand binding.
Taken together, our results provide evidence for the existence of a specific receptor for hsp60 binding and endocytosis, which is distinct from receptors for hsp70, hsp90, or gp96. We conclude that at least two different pathways for the recognition of hsp by innate immune cells have evolved, suggesting different roles of hsp60 vs hsp70, hsp90, and gp96 as danger Ags and in immunoregulation. Moreover, our results indicate that the cell surface structure interacting with hsp60 involves separated binding and signaling components, suggesting a more complex structure of hsp receptors than hitherto supposed.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Christiane Habich, Clinical Department, German Diabetes Research Institute, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany. E-mail address: christiane.habich{at}ddfi.uni-duesseldorf.de
3 Abbreviations used in this paper: hsp, heat shock protein; ADH, alcohol dehydrogenase; 2M, 2-macroglobulin; BMM, bone marrow-derived macrophage; TLR, Toll-like receptor.
Received for publication July 16, 2001. Accepted for publication November 15, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-cell secretory granules and its altered distribution in non-obese diabetic mice. Diabetologia 35:316.[Medline]
-cells correlates with induction of HSP60 autoantibodies. Diabetes 42:908.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  T. B. M. Hickey, L. M. Thorson, D. P. Speert, M. Daffe, and R. W. Stokes Mycobacterium tuberculosis Cpn60.2 and DnaK Are Located on the Bacterial Surface, Where Cpn60.2 Facilitates Efficient Bacterial Association with Macrophages Infect. Immun., August 1, 2009; 77(8): 3389 - 3401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Osterloh, F. Geisinger, M. Piedavent, B. Fleischer, N. Brattig, and M. Breloer Heat shock protein 60 (HSP60) stimulates neutrophil effector functions J. Leukoc. Biol., August 1, 2009; 86(2): 423 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-E. Alard, M. Dueymes, R. A. Mageed, A. Saraux, P. Youinou, and C. Jamin Mitochondrial heat shock protein (HSP) 70 synergizes with HSP60 in transducing endothelial cell apoptosis induced by anti-HSP60 autoantibody FASEB J, August 1, 2009; 23(8): 2772 - 2779. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cohen-Sfady, M. Pevsner-Fischer, R. Margalit, and I. R. Cohen Heat Shock Protein 60, via MyD88 Innate Signaling, Protects B Cells from Apoptosis, Spontaneous and Induced J. Immunol., July 15, 2009; 183(2): 890 - 896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Lalancette-Hebert, D. Phaneuf, G. Soucy, Y. C. Weng, and J. Kriz Live imaging of Toll-like receptor 2 response in cerebral ischaemia reveals a role of olfactory bulb microglia as modulators of inflammation Brain, April 1, 2009; 132(4): 940 - 954. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Hirsh and W. G. Junger Roles of Heat Shock Proteins and {gamma}{delta}T Cells in Inflammation Am. J. Respir. Cell Mol. Biol., November 1, 2008; 39(5): 509 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-F. Cheng, J. Fan, M. Fedesco, S. Guan, Y. Li, B. Bandyopadhyay, A. M. Bright, D. Yerushalmi, M. Liang, M. Chen, et al. Transforming Growth Factor {alpha} (TGF{alpha})-Stimulated Secretion of HSP90{alpha}: Using the Receptor LRP-1/CD91 To Promote Human Skin Cell Migration against a TGF{beta}-Rich Environment during Wound Healing Mol. Cell. Biol., May 15, 2008; 28(10): 3344 - 3358. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Schmitt, M. Gehrmann, M. Brunet, G. Multhoff, and C. Garrido Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy J. Leukoc. Biol., January 1, 2007; 81(1): 15 - 27. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Nussbaum, A. Zanin-Zhorov, F. Quintana, O. Lider, and I. R. Cohen Peptide p277 of HSP60 signals T cells: inhibition of inflammatory chemotaxis Int. Immunol., October 1, 2006; 18(10): 1413 - 1419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Hussain, D. Yang, D. Suki, K. Aldape, E. Grimm, and A. B. Heimberger The role of human glioma-infiltrating microglia/macrophages in mediating antitumor immune responses Neuro-oncol, July 1, 2006; 8(3): 261 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Mandal, A R Afzal, S J D Brecker, J Poloniecki, Q Xu, and M Jahangiri Association of serum soluble heat shock protein 60 with toll-like receptor 4 polymorphism and severity of coronary artery disease. Heart, May 1, 2006; 92(5): 683 - 685. [Full Text] [PDF]
|
|
|
|
 |
|
 G. Foteinos, A. R. Afzal, K. Mandal, M. Jahangiri, and Q. Xu Anti-Heat Shock Protein 60 Autoantibodies Induce Atherosclerosis in Apolipoprotein E-Deficient Mice via Endothelial Damage Circulation, August 23, 2005; 112(8): 1206 - 1213. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Pfister, C. M. Stroh, H. Perschinka, M. Kind, M. Knoflach, P. Hinterdorfer, and G. Wick Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy J. Cell Sci., April 15, 2005; 118(8): 1587 - 1594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Habich, K. Kempe, R. van der Zee, R. Rumenapf, H. Akiyama, H. Kolb, and V. Burkart Heat Shock Protein 60: Specific Binding of Lipopolysaccharide J. Immunol., February 1, 2005; 174(3): 1298 - 1305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lang, D. Benke, F. Eitner, D. Engel, S. Ehrlich, M. Breloer, E. Hamilton-Williams, S. Specht, A. Hoerauf, J. Floege, et al. Heat Shock Protein 60 Is Released in Immune-Mediated Glomerulonephritis and Aggravates Disease: In Vivo Evidence for an Immunologic Danger Signal J. Am. Soc. Nephrol., February 1, 2005; 16(2): 383 - 391. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zheng, M. He, M. Long, R. Blomgran, and O. Stendahl Pathogen-Induced Apoptotic Neutrophils Express Heat Shock Proteins and Elicit Activation of Human Macrophages J. Immunol., November 15, 2004; 173(10): 6319 - 6326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fleshner and M. L. Laudenslager Psychoneuroimmunology: Then and Now Behav Cogn Neurosci Rev, June 1, 2004; 3(2): 114 - 130. [Abstract] [PDF]
|
|
|
|
|
|
W.-F. Lo, C. D. Dunn, H. Ong, E. S. Metcalf, and M. J. Soloski Bacterial and Host Factors Involved in the Major Histocompatibility Complex Class Ib-Restricted Presentation of Salmonella Hsp 60: Novel Pathway Infect. Immun., May 1, 2004; 72(5): 2843 - 2849. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. J. Binder and P. K. Srivastava Essential role of CD91 in re-presentation of gp96-chaperoned peptides PNAS, April 20, 2004; 101(16): 6128 - 6133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-F. Tsan and B. Gao Cytokine function of heat shock proteins Am J Physiol Cell Physiol, April 1, 2004; 286(4): C739 - C744. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P. Gobert, J.-C. Bambou, C. Werts, V. Balloy, M. Chignard, A. P. Moran, and R. L. Ferrero Helicobacter pylori Heat Shock Protein 60 Mediates Interleukin-6 Production by Macrophages via a Toll-like Receptor (TLR)-2-, TLR-4-, and Myeloid Differentiation Factor 88-independent Mechanism J. Biol. Chem., January 2, 2004; 279(1): 245 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
|
|
|
|
|
|
S. B. Flohe, J. Bruggemann, S. Lendemans, M. Nikulina, G. Meierhoff, S. Flohe, and H. Kolb Human Heat Shock Protein 60 Induces Maturation of Dendritic Cells Versus a Th1-Promoting Phenotype J. Immunol., March 1, 2003; 170(5): 2340 - 2348. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Bethke, F. Staib, M. Distler, U. Schmitt, H. Jonuleit, A. H. Enk, P. R. Galle, and M. Heike Different Efficiency of Heat Shock Proteins (HSP) to Activate Human Monocytes and Dendritic Cells: Superiority of HSP60 J. Immunol., December 1, 2002; 169(11): 6141 - 6148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Xu Role of Heat Shock Proteins in Atherosclerosis Arterioscler Thromb Vasc Biol, October 1, 2002; 22(10): 1547 - 1559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Billack, D. E. Heck, T. M. Mariano, C. R. Gardner, R. Sur, D. L. Laskin, and J. D. Laskin Induction of cyclooxygenase-2 by heat shock protein 60 in macrophages and endothelial cells Am J Physiol Cell Physiol, October 1, 2002; 283(4): C1267 - C1277. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
