| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* First Department of Internal Medicine and
Department of Dermatology, Johannes Gutenberg Universität, Mainz, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In the course of this study we investigated the activation of highly purified human monocytes and in vitro-generated monocyte-derived DC and the maturation of these DC by recombinant human HSP60, human inducible HSP72, and preparations of gp96 and HSP70 from human cells. To eliminate the influence of undefined serum factors on APC activation, we performed experiments in the presence or the absence of human serum and used FCS-free DC culture methods as previously described (13, 14). Under stringent conditions, in the absence of serum and with highly purified monocytes for DC generation, HSP60 and, to a lesser extent, HSP72 induced DC maturation. Under the same conditions HSP60 as well as HSP72 very efficiently induced the release of proinflammatory cytokines from monocytes and immature DC. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73 with minor amounts of inducible HSP72, induced only marginal cytokine release from monocytes. gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations are not susceptible to gp96 signaling under the stringent experimental conditions applied in this study.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recombinant human HSP60, HSP72, and recombinant bovine HSP73- ATPase fragment were obtained from StressGen (Victoria, Canada). HSP70 was purified from human spleen or liver as described for the HSP70 preparation from murine liver (15). Briefly, a 100,000 x g supernatant was prepared from tissue homogenate and applied to Blue Sepharose CL-6B (Amersham Pharmacia, Uppsala, Sweden) to remove albumin. The precleared lysate was applied to ADP-agarose affinity columns (Sigma, Taufkirchen, Germany). The ADP-binding protein fraction was eluted with 3 mM ADP and after buffer exchange applied to DEAE-Sepharose anion exchange columns (Amersham Pharmacia), which were equilibrated with 20 mM NaCl buffer in 20 mM sodium phosphate (pH 7.0). HSP70 was eluted with 150 mM NaCl in 20 mM sodium phosphate.
gp96 was purified from a human EBV-transformed B cell line, SK-EBV-29,
as described originally for murine gp96 (16) with slight
modifications as described later by the same group (17).
Briefly, cells were homogenized in hypotonic buffer (30 mM
NaHCO3 and 0.2 mM PMSF, pH 7.1) by Dounce
homogenization (Kontes, Vineland, NJ), and a 100,000 x
g supernatant was obtained. Proteins were fractionated by
50–70% ammonium sulfate precipitation and then applied to a Con
A-Sepharose column (Amersham Pharmacia). Glycoproteins were eluted by
10% methyl-
-D-mannopyranoside. The eluate was
then applied to DEAE-Sepharose anion exchange columns (Amersham
Pharmacia) equilibrated with 300 mM NaCl in 5 mM sodium phosphate
buffer (pH 7.0). Finally, gp96 was eluted by 700 mM NaCl in 5 mM sodium
phosphate buffer. The buffer of HSP70 and gp96 preparations was
exchanged to PBS by ultrafiltration (Biomax-30kDa and Biomax-50kDa;
Millipore, Bedford, MA), thereby achieving final protein concentrations
of 2 mg/ml.
Protein concentrations were determined by Bradford assays photometrically. The purity of the protein preparations was controlled by SDS-PAGE and silver staining of gels. The identity of the proteins was controlled by immunoblot analysis with rat anti-grp94, rat anti-HSP73 (constitutive form of HSP70), mouse anti-HSP72 (inducible form of HSP70), and mouse anti-HSP72/HSP73 (StressGen). The HSP72 concentration in HSP70 preparations was determined by sandwich ELISA (HSP70 enzyme immunoassay kit, StressGen).
The biological activities of gp96 and HSP70 preparations were confirmed by testing the peptide binding activity of both HSP as recently described (17). For these peptide binding studies the tyrosinase peptide Ag YMDGTMSQV, a human melanoma Ag (18), was tested for binding to HSP as previously described (17). Briefly, gp96 or HSP70 at 300 µg/ml was incubated with the radioiodinated peptide at a molar ratio of 1:100 in sodium phosphate buffer containing 0.7 M NaCl for 30 min at 50°C. This was followed by incubation at 21°C for 30 min. HSP-peptide complexes were visualized by SDS-PAGE, protein staining, and autoradiography.
The amount of endotoxin, present in HSP or LPS stock solutions (Escherichia coli 0111:B4 LPS; Sigma), was determined with the quantitative chromogenic Limulus amebocyte lysate assay (QCL-1000; BioWhittaker, Walkersville, MD) according to the manufacturer’s instructions. The mean endotoxin concentrations of HSP stock solutions were calculated from an E. coli 0111:B4 LPS standard curve and expressed in endotoxin units (EU) per milliliter. From these, the endotoxin concentrations of dilutions from these stock solutions were calculated as EU per milliliter. The endotoxin activities in relation to the amount of protein in the HSP stock solutions were low, with 0.006–0.14 EU/µg protein for HSP60 or HSP72 and 0.0002–0.015 EU/µg protein for HSP70 and gp96 preparations. The EU activity of LPS was 4.58 EU/ng LPS.
Monocytes and DC
PBMC were derived by Ficoll-Hypaque density gradient centrifugation from buffy coats of healthy blood donors obtained from the blood bank of University Hospital of Mainz. CD14-positive monocytes were enriched to >95% purity by immunomagnetic elimination of T cells, NK cells, B cells, DC, and basophils by a cocktail of CD3, CD7, CD19, CD45RA, CD56, and anti-IgE Abs (MACS Monocyte isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany).
DC were generated as previously described (13, 14).
Briefly, immature DC were generated by culture of highly purified
monocytes in X-VIVO-15 medium (BioWhittaker) supplemented with 800 U/ml
GM-CSF and 1000 U/ml IL-4 for 6 days, replacing 50% of the culture
medium every other day. If indicated, 10% human AB serum was included
in the medium. Mature DC were generated by transferring immature DC to
fresh GM-CSF and IL-4 supplemented X-VIVO-15 medium, with the addition
of IL-1
(10 ng/ml), IL-6 (1000 U/ml), TNF- (10 ng/ml), and
PGE2 (1 µg/ml) for 2 days.
Induction of cytokine release from monocytes and DC by HSP
Monocytes (65,000/well) or DC (50,000/well) were stimulated with
titrated amounts of HSP and LPS as an endotoxin background control for
the HSP in X-VIVO-15 in the presence or the absence of 10% human AB
serum in duplicate wells of 96-well, round-bottom plates for 24 h.
Concentrations of IL-6, TNF-, IL-12p40, IL-12p70, and IL-10 were
determined in culture supernatants by standard sandwich ELISA
techniques (BD PharMingen, Heidelberg, Germany). In blocking
experiments HSP or LPS was heated (95°C, 30 min), or cells were
preincubated for 1 h with 10 µg/ml polymyxin B sulfate (Sigma)
or 10 µg/ml anti-CD14 (Beckman Coulter, Krefeld, Germany).
Culture of monocytes and DC with HSP
Monocytes were cultured in X-VIVO-15 medium with HSP or with LPS as an endotoxin background control at the same endotoxin concentration (EU per milliliter) as the corresponding dilution of HSP. The cultures were supplemented every other day with half the amount of HSP or LPS as that used at the start of the culture. After 6 days the cultures were analyzed by flow cytometry.
Maturation of DC was tested by culturing immature DC in X-VIVO-15
medium conditioned with GM-CSF and IL-4 with or without additional
supplements: HSP, LPS, or the cytokine mixture of IL-1, IL-6,
TNF-, and PGE2, described above, as a positive
control. After 2 days DC cultures were analyzed by flow cytometry or
applied to allogeneic MLR experiments.
Flow cytometry
Viable leukocytes were gated by excluding cells stained with 7-amino-actinomycin D (Via-Probe; BD PharMingen) and including cells stained with anti-CD45-FITC (Beckman Coulter, Krefeld, Germany). Specific cell surface staining was performed using PE-conjugated Ab in saturating amounts: mouse IgG1 isotype control; anti-CD14, -CD1a, -CD83, and -CD80 Ab (Beckman Coulter); anti-CD86 Ab (Dianova, Hamburg, Germany); and anti-mannose receptor Ab (BD PharMingen). The relative median fluorescence intensity was calculated as the quotient of the median fluorescence intensities of the respective marker Ab and the IgG1 isotype control.
Allogeneic MLR experiments
Immature DC were incubated with HSP, with LPS containing the same endotoxin activity as the corresponding HSP preparation, or without additional supplements in GM-CSF- and IL-4-conditioned X-VIVO-15. After 48 h the DC were washed and added to 96-well, flat-bottom plates at concentrations of 2,500, 5,000, and 10,000/well in triplicate. Immunomagnetically purified CD4-positive allogeneic T cells (MACS CD4 Microbeads; Miltenyi Biotec) were added at a concentration of 0.2 x 106/well and were cocultured with the DC for 4 days in X-VIVO-15 medium without serum. [3H]thymidine (1 µCi/well) was added, and after 16 h [3H]thymidine incorporation was determined.
Statistical analysis
Statistical analysis of the data was performed with StatView (Abacus Concepts, Berkley, CA), employing descriptive statistics and nonparametric Mann-Whitney tests to determine the significance of differences between data.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The following HSP were tested for their capability to induce
cytokine release from highly purified human monocytes: recombinant
human HSP60, recombinant inducible HSP72, recombinant bovine HSP70
ATPase fragment, gp96 preparations from a human mycoplasma-free
EBV-transformed B cell line, and HSP70 preparations from human liver
and spleen. HSP70 preparations from the EBV-transformed B cell line
variably contained a low m.w. contamination and accordingly were not
used as a source of HSP70 in this study. The high purity of gp96 and
HSP70 preparations was checked by SDS-PAGE, silver staining of gels,
and immunoblotting (Fig. 1
). The protein
purification methods were employed exactly as described by Srivastava
and co-workers (15, 16, 17); they were highly reproducible
with consistent protein yields and without signs of protein
denaturation-like protein aggregation or insolubility in aqueous
buffers. All HSP70 preparations consisted mainly of constitutively
expressed HSP73 and contained only minor amounts of inducible HSP72, as
demonstrated by immunoblotting with specific mAb (Fig. 1). This was
confirmed by HSP72-ELISA, which showed that the HSP70 preparations
contained, on the average, 3.8% HSP72 (SD = 1.2). The biological
activities of gp96 and HSP70 preparations were confirmed by peptide
binding activity of both HSP as described recently (17, 19), and the biological activity of gp96 is demonstrated in Fig. 2.
|
|
Recombinant HSP60 and HSP72 had the highest activity in the
induction of IL-6 release from highly purified human monocytes under
serum-free conditions (Fig. 3). The
concentrations at which both HSP induced significant IL-6 release from
monocytes varied between individual tests within a range of 0.01–0.5
µg/ml, when different charges of HSP and different monocyte donors
were employed. The recombinant bovine HSP73 ATPase fragment served as a
specificity control for recombinant HSP60 and HSP72 and did not induce
any significant release of IL-6 from monocytes at concentrations up to
10 µg/ml (data not shown). HSP70 preparations started to induce
cytokine release only at concentrations of 10–30 µg/ml. gp96 did not
induce any cytokine release up to concentrations of 270 µg/ml. The
presence of human serum increased the cytokine release induced by HSP,
except for gp96 (data not shown). Since the endotoxin concentration
expressed as EU per milliliter of protein had been determined for each
HSP stock solution, the endotoxin concentration of each HSP dilution
could be calculated and used for a comparative analysis. HSP60 and
HSP72 induced IL-6 release at dilutions of <1% of the endotoxin
concentration at which LPS caused IL-6 release (Fig. 3B).
This strongly suggests the independence of HSP-induced cytokine release
from the low endotoxin concentrations of the HSP preparations. HSP60,
HSP72, and HSP70 preparations also induced the release of TNF- and
IL-12p40 with similar concentration-response relationships as those
observed for IL-6 release (Fig. 4, A and C). In contrast, HSP did not induce
significant IL-10 release from monocytes (Fig. 4B), which
indicates a predominantly proinflammatory cytokine response of
monocytes to HSP60 and HSP72. Neither LPS nor HSP induced release of
IL-12p70 from monocytes. The inactivity of gp96 in these tests was not
due to toxicity of the gp96 preparations, because gp96 in
concentrations up to 270 µg/ml did not block LPS-induced monocyte
stimulation (data not shown). Also, gp96 preparations from other
sources, a melanoma cell line and stomach cancer tissue, did not induce
any cytokine release by human monocytes at concentrations up to 270
µg/ml (data not shown).
|
|
, A and B), while
the activity of LPS in parallel experiments was resistant to heat, but
was completely blocked by polymyxin B (Fig. 5C). Anti-CD14
mAb blocked the effects of HSP and LPS as well.
|
and
IL-12p40 (Fig. 6) starting at
concentrations between 1–3 µg/ml. IL-10 release, and IL-12p70
release was not detected (data not shown). The activity was 300-fold
for HSP72 and 105-fold for HSP60 above the
endotoxin background (data not shown). In contrast to their immature
precursors, mature DC were not responsive to any of the tested HSP
(data not shown).
|
Highly purified human monocytes were cultured in X-VIVO-15 medium
alone or with one of the following supplements: 10 µg/ml of HSP60 or
HSP72, 400 µg/ml of gp96 or HSP70, LPS at the different background
endotoxin concentrations of each HSP, or GM-CSF/IL-4 as a positive
control for the generation of immature DC. After 6 days cells were
evaluated for the expression of surface markers by flow cytometry (Fig. 7). As expected, the cultures incubated
with GM-CSF, and IL-4 showed the phenotypic characteristics of immature
DC with up-regulation of the mannose receptor, indicating endocytic
activity, low expression of the costimulatory signal molecules CD80 and
CD86, and loss of CD14 expression. CD83 expression was not detected
(data not shown). HSP60 and HSP72 induced a slight up-regulation of
CD14 compared with medium and endotoxin background controls and a
slight decrease in the expression of the mannose receptor compared with
endotoxin background controls. In addition, HSP60 induced a slight
increase in CD80 expression compared with the endotoxin background
control. Preparations of HSP70 and gp96 did not induce significant
changes in marker expression compared with their endotoxin background
controls. The presence of 10% human serum in the cultures had no
significant effect on these results (data not shown). As a result it
can be stated that none of the HSP induced differentiation of human
monocytes into immature DC.
|
Immature DC were cultured in X-VIVO-15 medium containing GM-CSF
and IL-4 with or without further supplements: HSP as used in the
previous experiments, LPS at the different background endotoxin
concentrations of each HSP and at higher concentrations of 10, 100, and
1000 ng/ml, or a cytokine mixture consisting of IL-1, IL-6, TNF-,
and PGE2, which leads to DC maturation
(13). After 48 h cells were harvested and analyzed
for the expression of the costimulatory signal molecules CD80 and CD86
and of the DC maturation marker CD83 (Fig. 8). As expected, the cytokine mixture
induced DC maturation, as shown by the major increases in CD83, CD80,
and CD86 expression. HSP60 at 10 µg/ml almost induced a similar DC
maturation, although there was some variation between individual
experiments (Fig. 8). HSP72 at 10 µg/ml as well as HSP70 preparations
at 400 µg/ml induced a minor up-regulation of CD80, CD86, and CD83
compared with their endotoxin background control. gp96 preparations at
400 µg/ml did not induce any DC maturation. DC maturation caused by
100 ng/ml of LPS was less pronounced than that for HSP60 and the
cytokine mixture (Fig. 8). LPS at 100 ng/ml had a 6000-fold higher
endotoxin activity than HSP60 in this experiment, which clearly shows
the independence of HSP60-induced DC maturation from endotoxin.
Additionally, polymyxin B at 10 µg/ml did not block this HSP60 effect
(data not shown). The recombinant bovine HSP73 ATPase fragment served
as a specificity control for recombinant HSP60 and HSP72 and did not
induce maturation of DC at concentrations up to 10 µg/ml (data not
shown).
|
). Immature DC that had
been incubated with LPS at concentrations equivalent to the low
endotoxin activities of the HSP preparations induced, on the average,
15-fold T cell proliferation above background. This was not
significantly different from the proliferation induced by immature DC
without additional stimuli (data not shown). However, immature DC
incubated with HSP60 and HSP72 in contrast to DC incubated with LPS at
background concentrations enhanced this T cell proliferation
significantly (2-fold for HSP60 (p < 0.0003)
and 1.4-fold for HSP72 (p < 0.015)). DC
incubated with gp96 and HSP70 preparations did not show an enhanced T
cell stimulatory capacity compared with control DC (Fig. 9).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 7). In contrast to the observed DC maturation mediated by HSP60, none of the tested HSP induced differentiation of human monocytes to immature DC. We confirmed that human HSP60 and inducible HSP72 induce the release of proinflammatory cytokines from human monocytes (5, 6, 7). Human serum increased the cytokine release of monocytes caused by HSP60 and HSP72, which might be due to the presence of soluble CD14 in serum or of other as yet undefined serum components. As shown in recent studies and here, CD14 plays an important role in cytokine release by monocytes induced by HSP60 and HSP70 (5, 6, 7). However, the observed effects of HSP60 and HSP72 on CD14-negative dendritic cells under serum-free conditions make it obvious that HSP60 and HSP72 must also have CD14-independent signal functions. Comparison of the effects of the recombinant HSP72 and HSP70 protein preparations, which contained mainly constitutively expressed HSP73 with minor amounts of HSP72, indicate that the danger signal function of HSP70 molecules relies mainly on inducible HSP72. This is in accordance with the finding of Kupper et al. (12) referred to above; only the inducible HSP72, but not the constitutively expressed HSP73, induced DC maturation.
TNF-, IL-6, and IL-12p40 were released by monocytes and immature DC
in response to HSP60 and HSP72, whereas release of IL-10 and IL-12p70
was not observed. TNF- and IL-6 are pleiotropic proinflammatory
cytokines, both supporting T cell responses. TNF- as well as IL-6
support DC maturation (13). IL-6 supports T cell
proliferation and differentiation (20). Transcription of
the IL-12p40 promoter is a prerequisite of release of the bioactive
IL-12p70 heterodimer, the key cytokine for priming a Th1 response
(21). The observation that IL-12p70 was not released by
monocytes and DC in response to HSP is in accordance with previous
reports that two signals, a Toll-like receptor (TLR)-mediated signal
such as LPS together with IFN-
priming or CD40 ligation, are needed
for DC to release IL12p70 (22, 23). HSP60 provides TLR2-
and TLR4-mediated signals to innate immune cells (24), but
obviously does not provide the second signal needed for IL-12p70
release. Since we did not observe release of IL-10 by human monocytes
and DC in response to HSP, the cytokine response to HSP seems to be
mainly proinflammatory, in accordance with previous reports (5, 24).
We ruled out by extensive controls that endotoxin contaminations are responsible for the effects of HSP60 and HSP72. First, every experiment was performed with an LPS control either titrated or with LPS at a concentration corresponding to the endotoxin concentration of the respective HSP. HSP60 and HSP72 activated monocytes and DC at protein concentrations that had at least 100-fold lower endotoxin concentrations than the endotoxin concentrations of LPS dilutions necessary for inducing a similar APC activation. Secondly, monocyte activation by HSP60 and HSP72 could be blocked by heat treatment of the HSP, but not by polymyxin B, while the opposite results were obtained when monocytes were activated by LPS. Thirdly, DC maturation induced by HSP60 could not be blocked by polymyxin B. It is obvious that the most active HSP in our study, HSP60 and HSP72, were recombinant proteins, whereas the less active or inactive HSP, HSP70 and gp96, had been purified from tissues or cells biochemically. Therefore, we included a recombinant protein, bovine HSP73-ATPase fragment, as a specificity control to exclude a copurifying stimulating agent in the recombinant protein stock solutions. This recombinant control protein neither induced cytokine release from monocytes nor led to DC maturation.
The observed inactivity of gp96 to stimulate highly purified human monocytes and monocyte-derived DC was unexpected in view of recent reports describing cytokine release of murine peritoneal exudate cells and maturation of murine bone marrow-derived DC in response to murine gp96 (8, 25) and cytokine release and maturation of human DC in response to murine gp96 (11, 26). However, our results do not contradict these reports, but suggest that the capability of gp96 to activate monocytes and DC is not a stable characteristic, but may depend on specific conditions. For example, the maturation of murine DC was much more pronounced in response to cell surface-targeted gp96 (25) than for soluble gp96. The latter induced up-regulation only of CD86, not of CD80, at a high concentration of 400 µg/ml (8). Different experimental conditions may be responsible for the different results of two recent reports from one group concerning the response of human monocytes and DC to gp96 (11, 26). First, in these reports murine gp96 instead of human gp96 as in our work was employed for testing human DC. Despite the fact that gp96 is a highly conserved molecule, there is no complete homology between murine and human gp96 (27). Secondly, we used exactly the same method for gp96 purification as that described recently by Srivastava and co-workers (17) employing a sequential ammonium sulfate precipitation as a preclearing step before Con A chromatography and DEAE anion exchange chromatography, whereas in the recent report a method without sequential ammonium sulfate precipitation was cited (28). In our experience, the preclearing step is necessary to clearly separate human gp96 from a 110-kDa protein, even if anion exchange chromatography with a linear NaCl gradient is used as the last gp96 purification step (M. Heike, unpublished observations). Thirdly, in the recent reports plastic-adherent PBMC were used as a start-up population for the DC cultures (11), which contain monocytes and variable contaminations of other mononuclear cells, for example, DC, as discussed above (12, 29). Finally, in the previous report DC cultures were performed with FCS (11), which can contain undefined factors influencing DC maturation (29) in contrast to the serum-free conditions in the DC maturation experiments reported here. We can rule out biological inactivity of gp96 as a reason for the failure to activate monocytes and DC because gp96 as well as HSP70 preparations showed significant peptide binding activity as described previously (17). Additionally, in a parallel study complexes of gp96 and HSP70 with peptide Ags stimulated peptide-specific CTL clones in the presence of APC (M. Heike, manuscript in preparation).
Our results give some additional information about the mechanism
involved in HSP60-induced monocyte and DC activation. The activation of
macrophages or DC by HSP60 and gp96 has to be preceded by endocytosis
via a specific receptor and is mediated by TLR2 and TLR4 signaling
(24, 26). The endocytosis receptor of gp96 and other HSP,
HSP60 not included, has been identified as the
2-macroglobulin receptor CD91
(19), which is expressed by human monocytes and DC
(30). Obviously, our results on the difference between
gp96 and HSP60 in their ability to activate monocytes and DC support
the finding of Habich et al. (31) that the receptor for
HSP60 is distinct from the gp96 receptor.
It should be emphasized that our experiments and conclusions are restricted to the effects of HSP on highly purified human monocytes and monocyte-derived DC under serum-free conditions. It remains to be investigated whether other human APC populations, such as freshly isolated myeloid and plasmocytoid blood DC or DC derived from CD34-positive blood or bone marrow progenitor cells, show other response patterns to HSP. Nevertheless, our results extend previous work by showing that HSP can differ considerably in their capacities to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system. Together with the recently demonstrated receptor-mediated uptake of HSP by APC (19, 32, 33), this function renders HSP to powerful natural adjuvants with important implications for the development of vaccines against tumor and infectious diseases.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 This work represents essential parts of the Ph.D. thesis of K.B.
3 Address correspondence and reprint requests to Dr. Michael Heike, Medizinische Klinik Mitte, Klinikum Dortmund, Beurhausstrasse 40, D-44137 Dortmund, Germany. E-mail address: stkd.mheike{at}dokom.net
4 Abbreviations used in this paper: HSP, heat shock protein; DC, dendritic cells; EU, endotoxin unit; TLR, Toll-like receptor.
Received for publication November 5, 2001. Accepted for publication September 24, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and interleukin 6, reactive nitrogen intermediates, and toxoplasmastatic activity in murine peritoneal macrophages. Infect. Immun. 63:3454.[Abstract]
B pathway. Int. Immunol. 12:1539. in monocytic cells. J. Exp. Med. 183:147.This article has been cited by other articles:
|
|
 |
|
  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]
|
|
|
|
|
|
M.-F. Tsan and B. Gao Heat shock proteins and immune system J. Leukoc. Biol., June 1, 2009; 85(6): 905 - 910. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q.-Q. Huang, R. Sobkoviak, A. R. Jockheck-Clark, B. Shi, A. M. Mandelin II, P. P. Tak, G. K. Haines III, C. V. Nicchitta, and R. M. Pope Heat Shock Protein 96 Is Elevated in Rheumatoid Arthritis and Activates Macrophages Primarily via TLR2 Signaling J. Immunol., April 15, 2009; 182(8): 4965 - 4973. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Chen, J. Guo, C. Han, M. Yang, and X. Cao Heat Shock Protein 70, Released from Heat-Stressed Tumor Cells, Initiates Antitumor Immunity by Inducing Tumor Cell Chemokine Production and Activating Dendritic Cells via TLR4 Pathway J. Immunol., February 1, 2009; 182(3): 1449 - 1459. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lachance, J. L. Bailey, and P. Leclerc Expression of Hsp60 and Grp78 in the human endometrium and oviduct, and their effect on sperm functions Hum. Reprod., October 1, 2007; 22(10): 2606 - 2614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. Johansson, L. Walther-Jallow, A. Smed-Sorensen, and A.-L. Spetz Triggering of Dendritic Cell Responses after Exposure to Activated, but Not Resting, Apoptotic PBMCs J. Immunol., August 1, 2007; 179(3): 1711 - 1720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kurotaki, Y. Tamura, G. Ueda, J. Oura, G. Kutomi, Y. Hirohashi, H. Sahara, T. Torigoe, H. Hiratsuka, H. Sunakawa, et al. Efficient Cross-Presentation by Heat Shock Protein 90-Peptide Complex-Loaded Dendritic Cells via an Endosomal Pathway J. Immunol., August 1, 2007; 179(3): 1803 - 1813. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Semnani, P. B. Keiser, Y. I. Coulibaly, F. Keita, A. A. Diallo, D. Traore, D. A. Diallo, O. K. Doumbo, S. F. Traore, J. Kubofcik, et al. Filaria-Induced Monocyte Dysfunction and Its Reversal following Treatment. Infect. Immun., August 1, 2006; 74(8): 4409 - 4417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B Dybdahl, S A Slordahl, A Waage, P Kierulf, T Espevik, and A Sundan Myocardial ischaemia and the inflammatory response: release of heat shock protein 70 after myocardial infarction Heart, March 1, 2005; 91(3): 299 - 304. [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]
|
|
|
|
 |
|
 A. Osterloh, F. Meier-Stiegen, A. Veit, B. Fleischer, A. von Bonin, and M. Breloer Lipopolysaccharide-free Heat Shock Protein 60 Activates T Cells J. Biol. Chem., November 12, 2004; 279(46): 47906 - 47911. [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]
|
|
|
|
 |
|
 B. Dybdahl, A. Wahba, R. Haaverstad, I. Kirkeby-Garstad, P. Kierulf, T. Espevik, and A. Sundan On-pump versus off-pump coronary artery bypass grafting: more heat-shock protein 70 is released after on-pump surgery Eur. J. Cardiothorac. Surg., June 1, 2004; 25(6): 985 - 992. [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]
|
|
|
|
|
|
K. Fleischer, B. Schmidt, W. Kastenmuller, D. H. Busch, I. Drexler, G. Sutter, M. Heike, C. Peschel, and H. Bernhard Melanoma-Reactive Class I-Restricted Cytotoxic T Cell Clones Are Stimulated by Dendritic Cells Loaded with Synthetic Peptides, but Fail to Respond to Dendritic Cells Pulsed with Melanoma-Derived Heat Shock Proteins In Vitro J. Immunol., January 1, 2004; 172(1): 162 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Goulhen, D. Grenier, and D. Mayrand ORAL MICROBIAL HEAT-SHOCK PROTEINS AND THEIR POTENTIAL CONTRIBUTIONS TO INFECTIONS Critical Reviews in Oral Biology & Medicine, November 1, 2003; 14(6): 399 - 412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Gao and M.-F. Tsan Recombinant Human Heat Shock Protein 60 Does Not Induce the Release of Tumor Necrosis Factor {alpha} from Murine Macrophages J. Biol. Chem., June 13, 2003; 278(25): 22523 - 22529. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
