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* Peter Gorer Department of Immunobiology, and
Department of Oral Medicine and Pathology, Guy’s, King’s, & St. Thomas’ Hospital Medical and Dental Schools, London, United Kingdom;
Lionex Diagnostics and Therapeutics, Braunschweig, Germany
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, NO, and C-C chemokines. The N-terminal, ATPase portion
(HSP701–358) failed to stimulate any of these cytokines or
chemokines. Both native and the truncated HSP70359–610
stimulation of chemokine production is mediated by the CD40
costimulatory molecule. Maturation of dendritic cells was induced by
stimulation with native HSP70, was not seen with the N-terminal
HSP701–358, but was enhanced with
HSP70359–610, as demonstrated by up-regulation of CD83,
CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed
that immunization with HSP70359–610 enhances the
production of IL-12 and RANTES. Immunization with peptide-bound
HSP70359–610 in mice induced higher serum IgG2a and IgG3
Abs than the native HSP70-bound peptide. This study suggests that the
C-terminal, peptide-binding portion of HSP70 is responsible for
stimulating Th1-polarizing cytokines, C-C chemokines, and an adjuvant
function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 macroglobulin receptor CD91
(13, 14). HSP70 is also able to translocate across
cellular membranes and gains cytoplasmic entry (15), which
allows the protein Ags to be processed by cytoplasmic proteasomes, and
subsequently, to enter the MHC class I pathway.
In addition to peptide binding and delivery, HSP70 can serve as a
carrier of peptides or proteins that are effective immunogens for B
cells, CD4+, and CD8+ T
cells without requiring an adjuvant (8, 16, 17). Several
studies have shown that mycobacterial HSP can induce inflammatory
cytokines (18, 19, 20) and C-C chemokines (17).
The CD40R mediates mycobacterial HSP70 stimulation of monocytes or
dendritic cells (DCs), generating the C-C chemokines RANTES, macrophage
inflammatory protein (MIP)-1, and MIP-1
(21).
Stimulation of CD40 on APCs may account for HSP70 functioning as an
effective Ag carrier that elicits MHC class I-restricted CTL responses
without an additional adjuvant.
In this study, we generated two major fragments of HSP70: an N-terminal
44-kDa ATPase portion (HSP701–358), and a
C-terminal 28-kDa portion (HSP70359–610), which
contains the 18-kDa peptide-binding region (aa 359–540). The data
suggest that stimulation of human monocytes with HSP70 is mediated by
the C-terminal HSP70359–610, which binds CD40
and elicits IL-12, TNF-, NO, and C-C chemokines. Maturation of human
DC was induced by stimulation with native HSP70, but was enhanced
by HSP70359–610 and was comparable with that
stimulated by CD40 ligand trimer (CD40LT). Immunization with
HSP70359–610 elicits Th1-polarizing cytokines,
C-C chemokines, and adjuvant function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recombinant Mycobacterium tuberculosis HSP70,
HSP70359–610, and
HSP701–358 were prepared from the
Escherichia coli pop strain. DNA encoding the N-terminal
ATPase (aa 1–358) domain of HSP70 was cloned and expressed in E.
coli using the pET 22b vector. DNA encoding the C-terminal
peptide-binding domain (aa 359–610) was expressed using pJLA603 vector
(22). Cloned inserts were verified by DNA sequence
analysis. In both cases, recombinant polypeptides were prepared by
affinity chromatography using Ni2+-chelating
resin, and identity of the polypeptides was confirmed by N-terminal
sequence analysis (10 cycles for each). The proteins were purified by
Q-Sepharose followed by ATP affinity chromatography. The HSP
preparations were further treated with polymixin B-coated beads
(Sigma-Aldrich, Dorset, U.K.) to remove LPS. The LPS content of the HSP
preprations was determined by the Limulus amebocyte lysate
assay (Sigma-Aldrich), and showed <0.006 U/µg of HSP70 or 5 pg/1
µg of the HSP preparations. Soluble CD40LT was kindly donated by Dr.
F. Villinger (Emory University, Atlanta, GA). Synthetic peptides
corresponding to CCR5 residues 1–20 (N-terminal: MDYQVSSPIYDINYYTSEPC)
and 88–102 (first loop: HYAAAQWDFGNTMCQ); were purchased from
Neosystem (Strasbourg, France). Adenosine 5'-O-(3-thiotriphosphate
(ATP-
-S), LPS, and polymixin B were obtained from Sigma-Aldrich.
FITC-conjugated mouse mAbs to CD14 (IgG2a) and CD83 (IgG2b) were
obtained from Immunotech (Oxford, U.K.). FITC-conjugated Abs to
CD80(IgG1), CD86(IgG1), CCR7(IgG1), and HLA-DR (IgG2a) and mouse
control IgG isotypes were purchased from Serotech (Oxford, U.K.).
Cell lines
Monocytic THP1 cell lines were obtained from MRC (National Institute for Biological Standards and Control, Potters Bar, U.K.). The nonadherent THP1 cell lines were cultured in RPMI 1640 medium supplemented with 10% FCS, 100 µg/ml of penicillin and streptomycin, and 2 mM of glutamine; and the medium was replaced every 3–4 days. Human embryonic kidney (HEK) cell line 293 cells acquired from National Institutes of Health (Rockville, MD) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 µg/ml of penicillin and streptomycin, and passaged every 3–4 days.
Transfection of HEK 293 cells with CD40
Full-length human CD40 cDNA in the pCDM8 plasmid vector
(Invitrogen, San Diego, CA) was a kind gift from Dr. B. Seed
(23). pCDM8 encoding the E. coli
-galactosidase (lacZ) was used as a control. The cells
were cultured in 25-cm2 flasks or 24-well plates
until 30–50% confluence, and transfected using LipofectAmine Plus
(Life Technologies, Paisley, U.K.). Transfection was performed
according to the manufacturer’s protocol, and CD40 was detected by
flow cytometry using PE-conjugated CD40 mAb. The assays were conducted
sequentially at days 2, 3, 4, and 5 after transfection, and the
percentage of CD40+ cells was consistently
>65%.
Preparation of human DCs
PBMC were isolated from healthy donors by centrifugation on a Ficoll-Hypaque density gradient (Amersham Biosciences, Little Chalfont, U.K.). The CD14+ monocytes were enriched by depletion of CD14- cells using Monocytes Isolation kit (MACS; Miltenyi Biotec, Surrey, U.K.). Human DCs were generated by culturing monocytes with GM-CSF (400 U/ml) and IL-4 (100 U/ml) for 5 days in RPMI 1640 medium, supplemented with 10% FCS, 2 mM glutamine, and 100 µg/ml of penicillin and streptomycin (24). To stimulate human DC, 0.5 µM HSP70, HSP70359–610, HSP701–358, CD40LT (10 µg/ml), or LPS (1000 ng/ml) were added and after further 2 days culture, and the cells were analyzed for expression of surface markers by flow cytometry.
Induction of C-C chemokines and cytokines
THP1 cells (2 x 105 ml), DC, or
transfected 293 cells (80–90% confluent) were cultured in 24-well
plates and incubated with HSP70, HSP70359–610,
and HSP701–358 at concentrations of 0–0.5 µM,
or CD40LT (0.1–10 µg/ml). After 3–5 days, the supernatant was used
to assay C-C chemokines, or the cytokines IL-12 and TNF-.
Assay of RANTES, MIP-1, IL-12, and TNF-
The supernatants collected from THP1 cells were diluted 10 times
and HEK 293 cell culture supernatant two times. Specific ELISA-paired
Ab kits were used for C-C chemokines (R&D Systems, Oxon, U.K.) or IL-12
and TNF- (BD PharMingen, Oxford, U.K.). The results were
expressed in picograms per milliliter.
Flow cytometry analysis
Aliquots of 2 x 104 cells were incubated with 10 µl of a panel of FITC-conjugated Abs to DC markers. For unconjugated primary Ab, the cells were further incubated with FITC-conjugated goat anti-mouse Fab. After washing twice, the cells were fixed in 1% formaldehyde PBS solution before flow cytometry analysis. The cells were analyzed on an Epics II flow cytometer (Coulter, High Wycombe, U.K.) and the data was analyzed on a Software WinMID.
Induction of RANTES in nonhuman primates
Simian PBMC were obtained from either HSP70, HSP70359–610-immunized, or naive rhesus macaques, and were stimulated in vitro with 0–0.5 µM of native HSP70, HSP70359–610, or HSP701–358. After a 3-day culture, the supernatant was collected for assaying RANTES.
Immunization of mice
HSP70 or HSP70359–610 was loaded with synthetic peptides by incubation with 20-fold molar excess of peptide, in PBS supplemented with 2 mM MgCl2, for 1–2 h at 37°C. Unbound peptide was removed by washing in a centrifugal concentrator with PBS. Groups (n = 4) of C57BL/6J mice were immunized i.p. with 50 µg equivalent of HSP70 or HSP70359–610 complexed with peptide in PBS. A second identical immunization was given after 4 wk, and mice were bled after a further 3 wk. Serum Abs were determined by ELISA.
ELISA for serum Abs
Proteins (HSP70 or HSP70359–610, 2 µg/ml) or peptides (10 µg/ml) in PBS were adsorbed to wells of polystyrene microtiter plates (Dynatech Laboratories, Chantilly, VA), which were then blocked by treatment with 1.5% BSA (1 h at room temperature). Bound Ags were incubated with serially diluted sera in duplicate. Bound IgG Abs were determined by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma-Aldrich), and subsequent reaction with paranitrophenol phosphate. Plates were read at 405 nm with the microplate reader model 450 (Bio-Rad, Hercules, CA). Each serum was analyzed three times. A synthetic peptide derived from the N-terminal (residues 1–20) of CCR5 was made as a control of specificity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that mycobacterial HSP70 stimulates the
human monocytic (THP1 cells), dendritic-like (KG1) cell lines, and
monocytes isolated from PBMC to produce RANTES, MIP-1, and MIP-1
by a mechanism dependent on the cell surface expression of CD40
(21). In an attempt to identify the C-C
chemokine-stimulating domains of HSP70, we have generated two
subdomains of the N-terminal ATPase portion of
HSP701–358 and the C-terminal portion of
HSP70359–610, which retains the peptide binding
activity (25). THP1 cells were incubated with these two
fragments as well as native HSP70, and after 3 days of stimulation, the
supernatant was assayed for the C-C chemokines RANTES and MIP-1.
HSP70359–610 or native HSP70 stimulates a
dose-dependent increase in RANTES and MIP-1 at concentrations of
0–0.1 µM, and a plateau was reached at a dose of 
0.2 µM (Fig. 1
). In comparison with native HSP70,
stimulation of RANTES and MIP-1 was greatly enhanced by
HSP70359–610, with a 20-fold increase in RANTES
(Fig. 1A) and 40-fold increase in MIP-1 (Fig. 1B). In contrast, treatment with
HSP701–358 (up to 1 µM) elicited a negligible
rise in the concentration of RANTES (Fig. 1A) or MIP-1
(Fig. 1B). It is noteworthy that <1 µM of the native or
C-terminal HSP70 elicited most of the chemokine production.
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A). This was 2- to 3-fold
greater than that found with native HSP70, but was similar to that
elicited with the control CD40LT (Fig. 2A). Native HSP70,
HSP70359–610, and CD40LT were not able to
stimulate the control lacZ transfected 293 cells to generate
RANTES.
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B). The maximum amount of LPS
in 10 µg of the HSP preparations was 50 pg, whereas the minimum
amount of LPS necessary for stimulation of RANTES production was
>10,000 pg/ml, as demonstrated previously and in this study
(21).
Induction of IL-12, TNF-, and NO by HSP70359–610
Production of the proinflammatory cytokines IL-12 and
TNF- was analyzed in the culture supernatants of monocytic THP1
cells, following treatment with native HSP70,
HSP701–358, or
HSP70359–610 fragment. Unstimulated THP1 cells
produced little IL-12 (<50 pg/ml), and stimulation with increasing
concentrations of either native HSP70 or
HSP701–358 fragment failed to induce a
significant level of IL-12 (Fig. 3A). However, a dose-dependent
increase in the concentration of IL-12 was generated by stimulating
THP1 cells with the HSP70359–610 fragment. An
increase in IL-12 was detected with 0.01 µM followed by a
marked increase at concentrations between 0.05 and 0.1 µM of
HSP70359–610. IL-12 was detected as early as
18 h after stimulation (data not shown), and maximal production of
3329 ± 469 pg/ml of IL-12 was reached after 3 days
treatment with 0.2–1 µM of HSP70359–610 (Fig. 3A).
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(10.5
± 4.4 pg/ml), and stimulation with the
HSP701–358 again failed to increase the
concentration of TNF- (Fig. 3B). Stimulation with
increasing concentrations of native HSP70 led to a small increase in
production of TNF- from 6.6 ± 7.9 pg/ml to 129.5 ± 72.6
pg/ml with 0.5 µM HSP70 (Fig. 3B). However, treatment of
THP1 cells with HSP70359–610 elicited a
dose-dependent increase in TNF- production. TNF- increased
>100-fold to 1045.1 ± 237.5 pg/ml after stimulation with 0.1
µM, as compared with the unstimulated control (Fig. 3B).
Production of NO was determined by an assay of nitrite production by
monocytic THP1 cells following activation with native HSP70,
HSP70359–610, and
HSP701–358 fragments. After a 3-day culture, the
supernatant was taken for analysis of nitrite. Resting cultures of THP1
cells produced low levels of nitrite, and this was unchanged with
increasing concentration of HSP701–358 fragment
(Fig. 3C). However, native HSP70, and to a greater extent
HSP70359–610 fragment, induced a dose-dependent
increase in the concentration of nitrite. This was comparable with that
of LPS, known to be a potent inducer of NO, although the dose response
was different from that of HSP70 or
HSP70359–610. The production of NO induced by
native HSP70 or HSP70359–610 was specific and
dependent on inducible NO synthase as
L-NG-monomethyl-L-arginine
monoacetate, the inducible NO synthase-specific inhibitor
suppressed NO production (data not shown).
Production of RANTES and IL-12 by mature DC was also determined
following native HSP70, HSP70359–610, or
HSP701–358 stimulation. Unstimulated immature DC
or those stimulated by HSP701–358 produced
negligible amounts of RANTES and some IL-12 (174.1 ± 110 and
48.5 ± 9.6 pg/ml; Fig. 2, C and D). Native
HSP70 induced RANTES production (2432.5 ± 243 pg/ml) as
well as low levels of IL-12 (1111.7 ± 94.6 pg/ml) by DC.
This is consistent with HSP70-induced DC maturation as defined by cell
surface expression of CD83, CD80, and CD86 (Fig. 4). However,
HSP70359–610 showed greatly enhanced stimulation
of both RANTES (8090.4 ± 845 pg/ml) and IL-12 (4974.1
± 203.1 pg/ml) in comparison with native HSP70 or
HSP701–358. The control CD40LT stimulated DC to
produce higher levels of RANTES or IL-12 than the native HSP70, but not
HSP70359–610. LPS at 500 ng/ml gave similar
results (Fig. 2, C and D).
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Culture of CD14+ monocytes with GM-CSF
and IL-4 differentiated the cells into nonadherent, immature DC with
typical DC morphology. These cells expressed 13.5%
CD83+, 3.0% CCR7, 83.7% CD80, and 15.7% CD86
(Fig. 4). Stimulation with HSP701–358
showed no increase in of any of the five maturation phenotypes;
CD86 showed a slight increase to 25.9% (but not in MFI), compared with
95 and 93.7% stimulated by HSP70359–610 and
native HSP70, respectively (Fig. 4). Treatment with HSP70 (5.0 µM)
up-regulated the expression of CD83 to 85.3%, CCR7 to 20.7%, CD80 to
92.3%, and CD86 to 93.7% (Fig. 4). However,
HSP70359–610 (0.3 µM) showed an enhanced
effect on the expression of CD83, CCR7, CD80, and CD86, which was
similar to that found with the C-C chemokines and cytokines.
Up-regulation of these phenotypic markers was very similar to those
found on treatment with CD40LT (or LPS; data not shown), except that
the CCR7 was up-regulated to 61.7% (LPS showed 26.9%). The HLA class
II remained unchanged at 96–99%, but the median fluorescence
intensity increased 2- to 3-fold after stimulation with native HSP70,
HSP70359–610, or CD40LT.
The effect of ATPase on native HSP70 and HSP70359–610 stimulation of chemokines and cytokines
A potential mechanism to account for the enhanced capacity
of HSP70359–610 to stimulate monocytic THP1
cells after removal of the N-terminal ATPase is that the latter may
have suppressed HSP70 stimulation of C-C chemokines and cytokines. We
tested this possibility by adding the ATPase fragment of
HSP701–358 to
HSP70359–610, but this failed to suppress
the production of RANTES, TNF-, or IL-12 (Table I). The role of ATPase in regulating
HSP70 stimulation was further studied by treatment of native or the
C-terminal fragment of HSP70 with 0.5 mM of the nonhydrolysable ATP
analog ATP--S. ATP--S failed to affect significantly the
concentration of RANTES stimulated by either native HSP70 or
HSP70359–610 (Table I).
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To compare the effect of immunization with
HSP70359–610 with that of the native HSP70, one
group of rhesus monkeys was immunized (three times) with HSP70, a
second group with HSP70359–610, and a third
group was not immunized. The concentration of RANTES was assayed
after specific stimulation of PBMC in vitro for 3 days with the native
HSP70, HSP70359–610, or
HSP701–358. A small dose-dependent increase in
the concentration of RANTES was elicited by the PBMC from naive
macaques, which was higher when stimulated with the C-terminal than the
native HSP70, but the N-terminal fragment failed to elicit any change
(Fig. 5A). Macaques immunized
with native HSP70 elicited an increased production of RANTES when
stimulated with native HSP70 (Fig. 5B), and this response
was further enhanced in HSP70359–610-immunized
macaques when stimulated by either native or
HSP70359–610 (Fig. 5C). Stimulation
with 0.5 µM of HSP70359–610 induced a 12-fold
increase in RANTES in naive PBMC (222.1 ± 33.2 pg/ml) compared
with a 7-fold increase when stimulated by native HSP70 (132.8 ±
25.3 pg/ml; Fig. 5A). However, PBMC from HSP70 primed
macaques stimulated with HSP70359–610 elicited a
30-fold increase of RANTES (525.6 ± 142.4 pg/ml) compared with a
7-fold increase stimulated by the native HSP70 (284 ± 35.3
pg/ml). Surprisingly, the HSP70359–610-immunized
macaques showed a 5-fold increase in RANTES in the unstimulated PBMC
(81.9 pg/ml) as compared with the native HSP70 (17.3 ± 3.0 pg/ml)
or unimmunized (18.3 ± 17.0 pg/ml) macaques (Fig. 5). In vitro
stimulation with HSP70 or HSP70359–610 induced
comparable levels of RANTES (1457.2 and 1458 pg/ml), but these were
12-fold and 3-fold higher than those immunized by the native HSP70,
respectively (Fig. 5).
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IL-12 production in PBMC was also studied in naive and immunized
macaques (Fig. 5D). No significant levels of IL-12 were
detected in naive macaque PBMC, whether unstimulated or following in
vitro stimulation with HSP70 and fragments. However, HSP70 and
HSP70359–610, but not
HSP701–358, stimulated PBMC from HSP70-immunized
macaques to produce a significant amount of IL-12 in comparison
with naive macaques. Increased levels of IL-12 production were found in
PBMC from HSP359–610-immunized macaques when
stimulated in vitro with HSP70, and to a greater extent when stimulated
with HSP70359–610 (Fig. 5D). We
failed to detect IL-4 in either unstimulated or HSP70-stimulated PBMC
in all three groups of macaques (data not shown), consistent with HSP70
or HSP70359–610 eliciting Th1-polarized immune
responses.
The effect of immunization with native HSP70 compared with the HSP70359–610 fragment loaded with CCR5 peptide on stimulating Abs in mice
To compare the adjuvanticity of
HSP70359–610 with that of intact HSP70 in
stimulating serum Ab responses, mice were immunized with a synthetic
peptide corresponding to the first extracellular loop of the
chemokine receptor CCR5 bound noncovalently to
HSP70359–610 or to native HSP70. Groups of four
C57BL/6J mice were immunized i.p. with a boost after 4 wk, and the
serum Ab response was determined by ELISA. Immunization with HSP70
noncovalently associated with the first loop peptide induced responses
to the peptide (1 in 8000), native HSP70 (1 in 32,000), and
HSP70359–610 (1 in 8000). However, immunization
with HSP70359–610 linked to the same peptide
resulted in an increased serum Ab response to the first loop peptide (1
in 32,000), but no detectable responses (<100) to either HSP70 or
HSP70359–610 (Table IIA). The IgG subclass of Ab induced to
the first loop peptide showed with both HSP70 and
HSP70359–610 that IgG2a titers were higher than
those of IgG1, the IgG2a:IgG1 ratio was greater for
HSP70359–610 (32:1) compared with HSP70 (4:1;
Table IIB), consistent with a Th1 Ab response. The higher
titers of total IgG in this assay reflect the inclusion of an extra
(IgG subclass-specific) Ab in the assay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
NO production, as well as enhancing the production of C-C chemokines.
In contrast, the ATPase domain of HSP70 (aa 1–358) lacked the capacity
to stimulate chemokines or cytokines and the native HSP70 either failed
or stimulated production of very low concentrations of IL-12, TNF-,
and NO.
TNF- is an important factor mediating innate immunity and is
involved in inflammation (27). NO has a broad
antimicrobial activity and also plays a role in regulation of adaptive
immune responses (28, 29). IL-12 plays an essential role
in controlling and maintaining a long-lasting protective immunity
against viral and intracellular bacterial infections (30).
Because IL-12 is one of the most potent cytokines inducing type 1
polarization (31), these findings may have important
implications in using the HSP70359–610 fragment
as a Th1-polarizing adjuvant. Indeed,
HSP70359–610-linked peptide elicited higher
serum IgG2a and IgG3 subclasses of Abs than native HSP70-bound peptide,
consistent with a Th1-polarizing response. Furthermore, the Th2 type of
cytokine (IL-4) was not produced in immunized macaques. Thus,
HSP70359–610 might be used as a microbial
adjuvant that attracts the entire immunological repertoire of cells by
virtue of stimulating the production of C-C chemokines and elicits a
Th1 response by generating IL-12. However, the peptide/protein binding
properties of HSP70 have to be first determined, for if they do not
have the hydrophobic motif necessary for noncovalent binding with the
HSP70 binding groove, the peptide or protein has to be covalently
linked to HSP70. The possibility that HSP70 may undergo intracellular
enzyme digestion to the C-terminal fragment will need to be
investigated.
The peptide binding function of the C-terminal portion of constitutive
HSP70 has been documented previously, especially as removal of ATPase
retained high-affinity binding of peptides (25). In this
study, we demonstrate that removal of the ATPase fragment of
mycobacterial HSP70 is required for effective HSP70 stimulation of
monocytes to produce IL-12, TNF-, and NO, and to enhance C-C
chemokine production. Indeed, a suppressive role of mycobacterial HSP70
ATPase domain has been demonstrated in rats with the production of
IL-10 and TGF- (32, 33). A cytotoxic cell-inducing
function was also demonstrated in the ATPase portion (aa 161–370) of
mycobacterial HSP70 when fused to a protein (9).
Other studies suggest that the native HSP70 might conceal the
stimulating epitope (7). We have failed to demonstrate an
inhibitory role of the ATPase fragment of
HSP701–358 by directly adding it to the native
HSP70 or C-terminal HSP70359–610 fragment.
Furthermore, addition of an ATPase inhibitor (ATP--S) also failed to
affect stimulation by native HSP70 or the C-terminal
HSP70359–610 fragment.
In vivo administration of HSP70359–610 in nonhuman primates also elicited IL-12 produced by PBMC when stimulated in vitro by either native HSP70 or HSP70359–610. Immunization with native HSP70 also elicited IL-12, although to a lesser extent. This is in contrast with the in vitro stimulation of human monocytic THP1 cells or peripheral blood monocytes, that HSP70359–610, but not the native HSP70, was capable of eliciting IL-12. This difference suggests that presensitized T cells in vivo were involved in producing IL-12, following in vitro stimulation with either HSP70 or HSP70359–610, as was found previously with HSP70 (17). Enhanced adjuvant function of HSP70359–610, compared with native HSP70, was also demonstrated in mice immunized with a peptide (aa 88–102) derived from the first loop of CCR5, which was noncovalently linked to HSP70. Surface plasmon resonance was used to demonstrate that the peptide could bind to both HSP70 and HSP70359–610. However, HSP70359–610 induced a significantly lower Ab titer to intact HSP70 or HSP70359–610 than that induced by HSP70. Using a truncated form of HSP70, rather than the native HSP70 as an adjuvant, appears to reduce the likelihood of inducing responses to self HSP70, which is desirable for a vaccine carrier.
DCs are important cells of the innate immune system and play a primary role in regulation of the adaptive immune responses (34). Immature DCs reside in epithelia and can terminally differentiate into mature DCs by various stimuli, such as microbial pathogens, inflammatory cytokines, or other "danger" signals (35). Maturation of DC is critical in the initiation of the immune response. HSP70 derived from necrotic cells (36) or recombinant human HSP70 induced DC maturation (37). In this study, we demonstrate that native mycobacterial HSP70 induces DC maturation, as assayed for the cell surface expression of CD83, CCR7, and HLA-DR, and the expression of the costimulatory molecules (CD80 and CD86). However, maturation of DC was enhanced by stimulation with the HSP70359–610 fragment, which up-regulated the maturation markers CD83, CCR7 HLA-DR, and CD80, CD86. These phenotypic changes were comparable with those elicited by CD40LT or LPS, so HSP70359–610 is a potent inducer of DC maturation.
In summary, we demonstrate that the cytokine and chemokine stimulatory
domain of HSP70 resides within the C-terminal fragment which elicits
IL-12, TNF-, NO, and C-C chemokines, and functions as a Th1 type
adjuvant.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas Lehner, Department of Immunobiology, Guy’s Hospital, 3rd Floor New Guy’s House, London SE1 9RT, U.K. E-mail address: thomas.lehner{at}kcl.ac.uk
3 Abbreviations used in this paper: HSP, heat shock protein; MIP, macrophage inflammatory protein; DC, dendritic cell; CD40LT, CD40 ligand trimer; HEK, human embryonic kidney; ATP--S, adenosine 5'-O-(3-thiotriphosphate).
Received for publication April 29, 2002. Accepted for publication June 26, 2002.
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  T. Mukai, Y. Maeda, T. Tamura, M. Matsuoka, Y. Tsukamoto, and M. Makino Induction of Cross-Priming of Naive CD8+ T Lymphocytes by Recombinant Bacillus Calmette-Guerin That Secretes Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein J. Immunol., November 15, 2009; 183(10): 6561 - 6568. [Abstract] [Full Text] [PDF]
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B. Valentinis, A. Capobianco, F. Esposito, A. Bianchi, P. Rovere-Querini, A. A. Manfredi, and C. Traversari Human recombinant heat shock protein 70 affects the maturation pathways of dendritic cells in vitro and has an in vivo adjuvant activity J. Leukoc. Biol., July 1, 2008; 84(1): 199 - 206. [Abstract] [Full Text] [PDF]
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 P. Gao, X. Sun, X. Chen, Y. Wang, B. A. Foster, J. Subjeck, P. B. Fisher, and X.-Y. Wang Secretable Chaperone Grp170 Enhances Therapeutic Activity of a Novel Tumor Suppressor, mda-7/IL-24 Cancer Res., May 15, 2008; 68(10): 3890 - 3898. [Abstract] [Full Text] [PDF]
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R. M. Srivastava, C. Varalakshmi, and A. Khar The Ischemia-Responsive Protein 94 (Irp94) Activates Dendritic Cells through NK Cell Receptor Protein-2/NK Group 2 Member D (NKR-P2/NKG2D) Leading to Their Maturation J. Immunol., January 15, 2008; 180(2): 1117 - 1130. [Abstract] [Full Text] [PDF]
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M. A. Arias, G. Jaramillo, Y. P. Lopez, N. Mejia, C. Mejia, A. E. Pantoja, R. J. Shattock, L. F. Garcia, and G. E. Griffin Mycobacterium tuberculosis Antigens Specifically Modulate CCR2 and MCP-1/CCL2 on Lymphoid Cells from Human Pulmonary Hilar Lymph Nodes J. Immunol., December 15, 2007; 179(12): 8381 - 8391. [Abstract] [Full Text] [PDF]
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M. A. Chase, D. S. Wheeler, K. M. Lierl, V. S. Hughes, H. R. Wong, and K. Page Hsp72 Induces Inflammation and Regulates Cytokine Production in Airway Epithelium through a TLR4- and NF-{kappa}B-Dependent Mechanism J. Immunol., November 1, 2007; 179(9): 6318 - 6324. [Abstract] [Full Text] [PDF]
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A. Biragyn, R. Schiavo, P. Olkhanud, K. Sumitomo, A. King, M. McCain, F. E. Indig, G. Almanzar, and D. Baatar Tumor-Associated Embryonic Antigen-Expressing Vaccines that Target CCR6 Elicit Potent CD8+ T Cell-Mediated Protective and Therapeutic Antitumor Immunity J. Immunol., July 15, 2007; 179(2): 1381 - 1388. [Abstract] [Full Text] [PDF]
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 K. Babaahmady, W. Oehlmann, M. Singh, and T. Lehner Inhibition of Human Immunodeficiency Virus Type 1 Infection of Human CD4+ T Cells by Microbial HSP70 and the Peptide Epitope 407-426 J. Virol., April 1, 2007; 81(7): 3354 - 3360. [Abstract] [Full Text] [PDF]
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 A. Shamaei-Tousi, J. P. Halcox, and B. Henderson Stressing the obvious? Cell stress and cell stress proteins in cardiovascular disease Cardiovasc Res, April 1, 2007; 74(1): 19 - 28. [Abstract] [Full Text] [PDF]
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 Z. Ye and Y.-H. Gan Flagellin Contamination of Recombinant Heat Shock Protein 70 Is Responsible for Its Activity on T Cells J. Biol. Chem., February 16, 2007; 282(7): 4479 - 4484. [Abstract] [Full Text] [PDF]
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J. Pido-Lopez, T. Whittall, Y. Wang, L. A. Bergmeier, K. Babaahmady, M. Singh, and T. Lehner Stimulation of Cell Surface CCR5 and CD40 Molecules by Their Ligands or by HSP70 Up-Regulates APOBEC3G Expression in CD4+ T Cells and Dendritic Cells J. Immunol., February 1, 2007; 178(3): 1671 - 1679. [Abstract] [Full Text] [PDF]
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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]
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 R. A. Floto, P. A. MacAry, J. M. Boname, T. S. Mien, B. Kampmann, J. R. Hair, O. S. Huey, E. N. G. Houben, J. Pieters, C. Day, et al. Dendritic Cell Stimulation by Mycobacterial Hsp70 Is Mediated Through CCR5. Science, October 20, 2006; 314(5798): 454 - 458. [Abstract] [Full Text] [PDF]
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X.-Y. Wang, H. Arnouk, X. Chen, L. Kazim, E. A. Repasky, and J. R. Subjeck Extracellular Targeting of Endoplasmic Reticulum Chaperone Glucose-Regulated Protein 170 Enhances Tumor Immunity to a Poorly Immunogenic Melanoma J. Immunol., August 1, 2006; 177(3): 1543 - 1551. [Abstract] [Full Text] [PDF]
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J. B. Flechtner, K. P. Cohane, S. Mehta, P. Slusarewicz, A. K. Leonard, B. H. Barber, D. L. Levey, and S. Andjelic High-Affinity Interactions between Peptides and Heat Shock Protein 70 Augment CD8+ T Lymphocyte Immune Responses J. Immunol., July 15, 2006; 177(2): 1017 - 1027. [Abstract] [Full Text] [PDF]
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 L.A. Bergmeier and T. Lehner Innate and Adaptive Mucosal Immunity in Protection against HIV Infection Advances in Dental Research, April 1, 2006; 19(1): 21 - 28. [Abstract] [Full Text] [PDF]
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J. D. Johnson and M. Fleshner Releasing signals, secretory pathways, and immune function of endogenous extracellular heat shock protein 72 J. Leukoc. Biol., March 1, 2006; 79(3): 425 - 434. [Abstract] [Full Text] [PDF]
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J.-E. Park, J. Facciponte, X. Chen, I. MacDonald, E. A. Repasky, M. H. Manjili, X.-Y. Wang, and J. R. Subjeck Chaperoning Function of Stress Protein grp170, a Member of the hsp70 Superfamily, Is Responsible for its Immunoadjuvant Activity Cancer Res., January 15, 2006; 66(2): 1161 - 1168. [Abstract] [Full Text] [PDF]
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 K. R. Qazi, M. R. Qazi, E. Julian, M. Singh, M. Abedi-Valugerdi, and C. Fernandez Exposure to Mycobacteria Primes the Immune System for Evolutionarily Diverse Heat Shock Proteins Infect. Immun., November 1, 2005; 73(11): 7687 - 7696. [Abstract] [Full Text] [PDF]
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 J. D. Johnson, J. Campisi, C. M. Sharkey, S. L. Kennedy, M. Nickerson, and M. Fleshner Adrenergic receptors mediate stress-induced elevations in extracellular Hsp72 J Appl Physiol, November 1, 2005; 99(5): 1789 - 1795. [Abstract] [Full Text] [PDF]
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 A. Clayton, A. Turkes, H. Navabi, M. D. Mason, and Z. Tabi Induction of heat shock proteins in B-cell exosomes J. Cell Sci., August 15, 2005; 118(16): 3631 - 3638. [Abstract] [Full Text] [PDF]
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R. Morita, T. Uchiyama, and T. Hori Nitric Oxide Inhibits IFN-{alpha} Production of Human Plasmacytoid Dendritic Cells Partly via a Guanosine 3',5'-Cyclic Monophosphate-Dependent Pathway J. Immunol., July 15, 2005; 175(2): 806 - 812. [Abstract] [Full Text] [PDF]
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A. A. R. Tobian, C. V. Harding, and D. H. Canaday Mycobacterium tuberculosis Heat Shock Fusion Protein Enhances Class I MHC Cross-Processing and -Presentation by B Lymphocytes J. Immunol., May 1, 2005; 174(9): 5209 - 5214. [Abstract] [Full Text] [PDF]
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Y. Wang, T. Whittall, E. McGowan, J. Younson, C. Kelly, L. A. Bergmeier, M. Singh, and T. Lehner Identification of Stimulating and Inhibitory Epitopes within the Heat Shock Protein 70 Molecule That Modulate Cytokine Production and Maturation of Dendritic Cells J. Immunol., March 15, 2005; 174(6): 3306 - 3316. [Abstract] [Full Text] [PDF]
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 S.-N. Lin, K. Ayada, Y. Zhao, K. Yokota, R. Takenaka, H. Okada, R. Kan, S. Hayashi, M. Mizuno, Y. Hirai, et al. Helicobacter pylori heat-shock protein 60 induces production of the pro-inflammatory cytokine IL8 in monocytic cells J. Med. Microbiol., March 1, 2005; 54(3): 225 - 233. [Abstract] [Full Text] [PDF]
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A. A. R. Tobian, D. H. Canaday, and C. V. Harding Bacterial Heat Shock Proteins Enhance Class II MHC Antigen Processing and Presentation of Chaperoned Peptides to CD4+ T Cells J. Immunol., October 15, 2004; 173(8): 5130 - 5137. [Abstract] [Full Text] [PDF]
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M.-F. Tsan and B. Gao Endogenous ligands of Toll-like receptors J. Leukoc. Biol., September 1, 2004; 76(3): 514 - 519. [Abstract] [Full Text] [PDF]
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S. Jang, S. Uematsu, S. Akira, and P. Salgame IL-6 and IL-10 Induction from Dendritic Cells in Response to Mycobacterium tuberculosis Is Predominantly Dependent on TLR2-Mediated Recognition J. Immunol., September 1, 2004; 173(5): 3392 - 3397. [Abstract] [Full Text] [PDF]
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M. Nikulina, C. Habich, S. B. Flohe, F. W. Scott, and H. Kolb Wheat Gluten Causes Dendritic Cell Maturation and Chemokine Secretion J. Immunol., August 1, 2004; 173(3): 1925 - 1933. [Abstract] [Full Text] [PDF]
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 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]
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K. Campbell, H. Diao, J. Ji, and L. Soong DNA Immunization with the Gene Encoding P4 Nuclease of Leishmania amazonensis Protects Mice against Cutaneous Leishmaniasis Infect. Immun., November 1, 2003; 71(11): 6270 - 6278. [Abstract] [Full Text] [PDF]
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),
HSP70359–610 (•), or HSP701–358 (
).
Three days after cultures, the supernatants were collected for assays
of RANTES and MIP-1
