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PHAGOCYTES
Exogenous heat shock protein 70 binds macrophage lipid raft microdomain and
stimulates phagocytosis, processing, and MHC-II presentation of antigens
Ruibo Wang, Joseph T. Kovalchin, Peggy Muhlenkamp, and Rajiv Y. Chandawarkar
The extracellular presence of endotoxinfree heat shock protein 70 (HSP70) enhances the rate and capacity of macrophage-mediated phagocytosis at 6 times
the basal rate. It is protein-specific, doseand time-dependent and involves the internalization of inert microspheres, Grampositive and -negative bacteria and fungi.
Structurally, exogenous HSP70 binds the
macrophage plasma membrane, specifically on its lipid raft-microdomain. Disruption of lipid rafts, HSP70-LR interaction,
or denaturing HSP70 abrogates the HSPmediated increase in phagocytosis. Further, HSP70-mediated phagocytosis directly enhances the processing and
presentation of internalized antigens via
the endocytic MHC class-II pathway to
CD4ⴙ T lymphocytes. Modulating the
HSP70-LR interaction presents an opportunity to intervene at the level of hostpathogen interface: a therapeutic tool for
emerging infections, especially when conventional treatment with antibiotics is in-
effective (antibiotic resistance) or unavailable (rapidly spreading, endemic). These
results identify a new role for HSP70, a
highly conserved molecule in stimulating
phagocytosis: a primordial macrophage
function, thereby influencing both innate
and adaptive immune responses. (Blood.
2006;107:1636-1642)
© 2006 by The American Society of Hematology
Introduction
Phagocytosis is a primal protective cellular function that characterizes the innate immune response to microbial invasion.1-4 It is a
complex phenomenon implicating several components of the
plasma membrane, including pattern recognition receptors (PPRs),
cytoskeletal elements, and lipid rafts (LRs).5 Antigen-presenting
cells (APCs) act as sentinels of the host that initiate and execute the
phagocytic response.2,6-9 These cells are activated in response to
certain well-defined substances that provide stimulatory signals
that characterize injury or infection. Typically, these APCs, including macrophages, respond either to the presence of pathogens via
PRRs or to metabolic, physical, or chemical stress, trauma, or other
agents that mediate necrotic-cell lysis and death.
Cell lysis releases a vast number of nonmicrobial, host-derived,
intracellular molecules into the environment, some of which are
potent activators of the immune system. Of these, heat shock
proteins (HSPs) are abundant intracellular molecules, readily
released by cell lysis following injury or infection10-12 where they
exhibit broad immunoactive properties.13 As shown by Basu et al,10
lysate from 1 g tissue was shown to contain 200 ␮g HSP70.
Injury-causing lysis of as little as 0.5 g tissue could potentially
release 100 ␮g/mL concentration of HSP70 into the extracellular
environment where they come into direct contact with cells of the
immune system such as macrophages and dendritic cells. HSPs
have been shown to play a major role in macrophage activation,
preparing the host for defense.10,14,15 Immunologically, HSPs bind
several macrophage-surface receptors and up-regulate key antigenspecific and nonspecific functions, including tumor rejection,
cytokine release, and up-regulation of costimulatory molecules.16-21
Within the confines of the cell, HSPs are chaperones and facilitate
protein synthesis and breakdown.22 They are expressed in all cells
in all forms of life and in a variety of intracellular locations: in the
cytosol (HSP70 and HSP90), nuclei, endoplasmic reticulum (gp96),
and mitochondria. In addition to their ubiquity, the HSPs constitute
the single most abundant group of proteins inside cells. They are
expressed in vast quantities under normal unstressed conditions,
and their expression can be powerfully induced to much higher
levels as a result of heat shock or other forms of stress, including
exposure to toxins, oxidative stress, glucose deprivation, and so
forth, leading up to cell lysis, which releases large quantities of
HSPs into the extracellular milieu. Approximately 10 families of
HSPs are known, and each family consists of 1 to 5 closely
related proteins. Since their discovery, an increasing array of
functions such as folding and unfolding of proteins, degradation of
proteins, assembly of multisubunit complexes, thermotolerance,
buffering of expression of mutations, and others have been
attributed to HSPs.22-24
Our study examines whether HSPs provide stimulatory signals
to the macrophage and promote phagocytosis of particulate
antigen, its processing, and presentation. Here, we show that
the ligation of endotoxin-free HSP70 to the LR microdomain on
the macrophage-cell surface. Functionally LRs have been
implicated in cellular processes by their ability to facilitate
protein-protein interaction, raft-mediated endocytosis, signal
transduction and to play an important role in the biogenesis
of the phagosomes.25-31 The HSP70-LR interaction promotes
phagocytosis of particulate antigen and subsequent antigen processing and presentation to CD4⫹ T lymphocytes in a MHC-II
restricted manner.
From the Division of Plastic Surgery, Department of Surgery, Center for
Immunotherapy, University of Connecticut School of Medicine, Farmington, CT.
Connecticut Health Center MC 1601, 263 Farmington Ave, Farmington CT
06030-1601; e-mail: [email protected].
Submitted June 29, 2005; accepted October 12, 2005. Prepublished online as
Blood First Edition Paper, November 1, 2005; DOI 10.1182/blood-200506-2559.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Rajiv Y. Chandawarkar, Division of Plastic Surgery, University of
© 2006 by The American Society of Hematology
1636
BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
Taken together, these findings identify HSP70 as a new
mammalian agent that can activate a primitive macrophage function: that of phagocytosis. The increase in antigen uptake in
response to binding of the HSP70 to the LR microdomain,
described here for the first time, defines the new role for HSP70, a
highly conserved molecule in influencing a primordial macrophage
function. The clinical applications of these findings range from
developing new anti-infective therapies, to their value in tissue
repair and host defense.
Materials and methods
Cell culture
All cell lines were obtained from American Type Culture Collection
(ATCC, Manassas, VA). RAW 264.7, RAW 309, and J774A.1 were
maintained in DMEM (Gibco, Invitrogen, Grand Island, NY) with 10%
heat inactivated FCS and RAW264.7 NO⫺ was maintained in RPMI
(ATCC) with 10% heat-inactivated FCS at 37°C, 5% CO2.
Mice
I-Ad–restricted DO11.10 TCR-␣␤–transgenic and C57/BL6J mice (6-8
weeks old) were purchased from The Jackson Laboratory (Bar Harbor,
ME). Mice were housed in the vivarium of the Center of Laboratory Animal
Care in University of Connecticut Health Center.
Reagents
All chemicals, unless otherwise specified were purchased from Sigma (St
Louis, MO), and all sterile noncharged plastic ware was from Corning
(Corning, NY). Alexafluor488-labeled Saccharomyces cerevisiae and Escherichia coli were purchased from Molecular Probes (Eugene, OR); fluorescent and unlabeled polystyrene microspheres (size, 3 ␮m) were purchased
from Sigma. Anti-HSP70 antibodies were obtained from Stressgen (San
Diego, CA) respectively.
HSP70-INDUCED PHAGOCYTOSIS
1637
490 nm falls within the absorbance range of trypan blue (475-675 nm), the
fluorescence of the sample only represents the intracellular fluorescence.
External or surface-bound fluorescence is quenched. Using Student t test,
the results were analyzed, and P values were calculated.
Purification of lipid rafts
Lipid rafts were purified as DRMs (detergent resistant membranes) using
nonionic detergents following sucrose gradient centrifugation. The cells
used for lipid raft purification were treated with or without 30 mM MCD for
30 minutes at 37°C, after being washed with cold PBS and incubated with
HSP70-HRP complexes (HRP conjugation kit; Alpha Diagnostic, San
Antonio, TX) for 30 minutes on ice. After incubation, cells were washed 3
times with PBS and lysed with 2 mL MBS (150 mM NaCl, 20 mM MES,
pH 6.5, 500 mM PMSF, and 5 mM iodoacetamide) containing 0.5% Ttriton
X-100 or 1% Brij98 for 30 minutes on ice or 7 minutes at 37°C,
respectively. The cells were mixed with an equal volume of 90% sucrose in
MEB and placed at the bottom of the centrifuge tube. The sample was
overlaid with 5.5 mL 30% sucrose and 4.5 mL 5% sucrose in MBS and
centrifuged at 100 000g for 16 hours (SW28; Beckman Instruments, Palo
Alto, CA). Fractions of 1 mL each were collected from the bottom of the
tube, and each fraction was analyzed by SDS-PAGE and immunoblotting.
Proliferation of CD4ⴙ T cells and IFN-␥ release
Unlabeled S cerevisiae (80 ␮g) was incubated with excess chicken ovary
albumin (OVA) at 37°C for 1 hour and washed repeatedly to eliminate free
OVA protein. S cerevisiae–OVA complexes (Ova-coated yeast) were
administered to macrophages treated with or without HSP70. Macrophages
were then washed and irradiated at 120 Gy (12 000 rad) and assessed for
viability by trypan blue exclusion. Subsequently, they were cocultured with
CD4⫹ T lymphocytes that were purified from spleens of DO11.10 mice and
labeled with CSFE. Purification of CD4⫹ T lymphocytes was performed
using the magnetic-activated cell sorting (MACS) purification columns and
␣CD4 microbeads (Miltenyi Biotech, Auburn, CA). Following the 48-hour
incubation, proliferation of CD4⫹ cells was determined with fluorescenceactivated cell sorting (FACS), and IFN-␥ release in the supernatant was
tested by enzyme-linked immunosorbent assay (ELISA; Pierce,
Rockford, IL).
Heat shock proteins
Heat shock proteins (HSPs) (HSP70, HSP90, and GP96) were purified from
murine livers as described earlier.32,33 HSPs were prepared as a complex
with endogenous peptide, except when using adenosine triphosphate
(ATP)–treated HSP70 that was used to assess the role of peptides in
phagocytosis. ATP-treated HSP70 removes all the peptides associated with
HSP70 itself, whereas the purification of HSP70 using adenosine diphosphate (ADP) does not.34 Purity was established by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis
and immunoblotting, and HSPs were quantified using Bradford analysis.
LPS content was measured by the Limulus Amebocyte Lysate (LAL) assay
(LAL kit QCL-1000; Biowhittaker, Walkersville, MD).
Phagocytosis assay in vitro
RAW 264.7, RAW264.7 (NO⫺), and J774A.1 cells were cultured in DMEM
or RPMI (RAW NO⫺) with 10% FCS, harvested, and washed. Cells
(3 ⫻ 105/well) were incubated in serum-free EMEM in a 24-well plate for 1
hour at 37°C. Alexafluor488-labeled particles (inert polystyrene microspheres, or yeast, S cerevisiae, or Gram-negative bacteria, E coli, 40
particles/macrophage) were added and coincubated in a dark environment
for 60 minutes in serum-free EMEM with or without HSPs, LPS, or control
proteins. At the end of the 60-minute incubation, the plate was covered in a
dark container on ice. Specific measures were taken to exclude the
fluorescence that resulted from particles that were outside of the cell or
sticking to the surface of the cell. This was done by using trypan blue that
quenches all the fluorescence outside of the cell but does not quench the
internalized particles. Specifically, trypan blue (0.8 mg/mL) was added for
60 seconds, and plates were analyzed immediately using FluorImager SI
(Molecular Dynamics/Amersham, Sunnyvale, CA). Because the emission
of Alexafluor488 is approximately 519 nm at excitation approximately,
Results
HSPs enhance macrophage-mediated antigen uptake
Murine macrophage lines (RAW264.7, J774.A1, or RAW264
NO⫺) were treated with either one of the HSPs (HSP70, HSP90, or
gp96) (100 ␮g/mL) or with non-HSP control proteins and coincubated with either Alexafluor488-labeled inert polystyrene microspheres, yeast (S cerevisiae [Sc]), or Gram-negative bacteria (E
coli [Ec]) (40 particles/macrophage). A phagocytosis assay was
performed using specific techniques to exclude external cellsurface binding of particles and measuring the actual fluorescence
from internalized material alone (see “Materials and methods”).
Macrophages treated with any of the 3 HSPs consistently showed
an increase in uptake of microbial or nonmicrobial materials as
compared with those treated with control proteins or with buffer
(Figure 1A). The increase in uptake ranged from 2 to 6 times the
basal rate and included the internalization of a variety of
materials tested.
We focused on HSP70, because of our knowledge of its
molecular functions and its influence on various aspects of tissue
protection.35-45 Specificity of HSP70-mediated antigen uptake was
tested by treating macrophages with either HSP70 (100 ␮g/mL) or
with equimolar amounts of HSP controls, including ␤ galactosidase; phosphorylase B; and mouse serum albumin (molecular
weights of these proteins correspond to those of the HSPs tested);
bovine serum albumin (BSA; 100 ␮g/mL); concavalin A
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1638
WANG et al
Figure 1. Macrophage-mediated phagocytosis is modulated by HSPs. The
ability of macrophage cell lines (as indicated) to phagocytose either Alexafluorlabeled inert microspheres or S cerevisiae or E coli in the absence or presence of
each of the HSPs indicated (HSP70, HSP90, or gp96) or serum albumin (MSA) was
tested in vitro. HSP-mediated phagocytosis was quantified by measuring the
internalized fluorescence measured by FluorImager SI, and relative mean fluorescence intensity (RMFI) was compared between the treatment groups (as indicated).
All the treatment groups were compared with medium alone using the Student t test,
and significance was denoted by P ⬍ .01. The error bar represents 1 SD. The results
shown are a cumulative analysis of 3 experiments, 3 wells/group. *P ⬍ .05 when
compared to medium.
BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
(6 ␮g/mL); and complete and incomplete Freund adjuvant
(10 ␮L/well) (potent stimulators of APC-function). None of the
control proteins used enhanced uptake as compared with treatment
with HSP70. Further, reagents commonly used in HSP purification,
including buffers, ADP (3 mM); and ATP (3 mM); were ineffective
(Figure 2A).
Increasing the doses of HSP70 (range, 10-100 ␮g/mL) revealed
that doses less than 20 ␮g/mL were unable to stimulate uptake, and
the peak effect of HSPs occurred at 40 ␮g/mL (Figure 2B) and
reached a plateau through 100 ␮g/mL. Doses up to 200 ␮g/mL did
not result in any further increase in the quantity of phagocytosis.
Physiologically, this dose is well within the range of the concentration of HSP70 that is observed from lysis of 1 g tissue lysis. As
shown by Basu et al,10 lysate from 1 g tissue was shown to contain
200 ␮g HSP70. In our experiments we used HSP70 in the dose of
100 ␮g/mL that would correspond to 0.5 g lysed tissue. Injurycausing lysis of as little as 0.5 g tissue could potentially mimic the
conditions we use in vitro wherein macrophages are treated with
100 ␮g/mL concentration of HSP70. Viability of cells treated was
confirmed by using trypan blue exclusion to ensure that the HSP
treatment was not cytotoxic. Time titration was performed by
treating macrophages with HSPs or controls for increasing time
periods, ranging from 10 to 90 minutes. In the HSP-treated group,
antigen uptake reached a plateau within 45 minutes. Time points
beyond 90 minutes were not tested. Representative microphotographs of macrophages visualized at ⫻ 10 magnification using
H&E stain at 15, 30, and 60 minutes showed morphologic changes
indicative of ongoing uptake, which ceased at 60 minutes
(Figure 2C).
To address whether HSP70 somehow coated the yeast particles,
thereby acting like an “opsonizing agent,” the following experiment were performed. First, macrophages were pretreated with
Figure 2. HSP70-mediated phagocytosis is specific, titratable, actin dependent, and independent of protein synthesis and HSP-peptides. (A) Macrophages were
treated with HSP70 or non-HSP controls (as indicated) to examine their specific effects on phagocytosis of Alexafluor-labeled yeast (S cerevisiae) at the concentration of 40
particles/macrophage. *P ⬍ .05 when compared to medium. (B) Macrophages were treated with HSP70 (doses indicated) and subsequently tested for their ability to
phagocytose Alexafluor-labeled yeast (S cerevisiae) at the concentration of 40 particles/macrophage. (C) Representative microphotographs (⫻ 10 magnification) of
macrophages (treated for minutes as indicated). At 15 and 30 minutes the macrophages show ongoing phagocytic activity (elongated cells with cytoskeletal alterations),
whereas at 60 minutes the cells are round and appear quiescent. Images were visualized under a Nikon Optiphot microscope (Nikon, Melville, NY) equipped with objective
lenses ranging from 10 ⫻/2.5 to 40 ⫻/16.0. Images were captured with a Kodak DC 120-zoom camera (Kodak, Rochester, NY) and processed with Adobe Photoshop 6.0
software (Adobe Systems, San Jose, CA). (D) Macrophages were pretreated with HSP70 (100 ␮g/mL), as indicated, and washed free of residual HSP70. Macrophages were
then administered yeast and tested in a phagocytosis assay. For comparison, macrophages were with HSP70 and administered the yeast at the same time. (E) HSP70-coated
yeast was prepared by coincubating Alexafluor-labeled S cerevisiae (Sc) with HSP70 and washed until free of unbound HSP70. Experimental groups (as indicated) were tested
for their ability to enhance phagocytosis. (F) Macrophages were treated with HSP70 (100 ␮g/mL) under (1) conditions that prevent actin-mediated cytoskeletal changes (low
temperature and cytochalasin D as indicated) or (2) in the presence or absence of cycloheximide (as indicated) and subjected to a phagocytosis assay or (3)
phagocytosis-enhancing effects of ATP-treated HSP70 (peptide free) was compared with that of ADP-purified HSP70 (with peptides). The error bar represents 1 SD. The
results shown are a cumulative analysis of 3 experiments, 3 wells/group.
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
HSP70 and washed until no free HSPs remained in the wash. Then
they were administered Alexafluor-labeled yeast (S cerevisiae).
Pretreating the macrophages with HSP prior to administering the
yeast enhanced the uptake of yeast to the same extent as when both
(HSP and yeast) were administered simultaneously (Figure 2D).
Second, yeast particles were coincubated with HSP70
(100 ␮g/80 ␮g yeast, 37°C, 60 minutes) to create a HSP-coated
yeast complex. Unbound HSP70 was removed by washing with
PBS. Presence of the HSP-coated yeast complex was confirmed by
SDS-PAGE and immunoblotting with HSP70 antibodies. These
complexes were then coincubated with macrophages with the
following experimental groups: group I, HSP70-coated yeast
(HSP-Sc*); group II, yeast (Sc*) ⫹ HSP70; group III, HSP70treated macrophages ⫹ HSP70-coated yeast (HSP-Sc*). As shown
in Figure 2E, macrophages that were treated with HSP70-coated
yeast (group I) did not demonstrate increased uptake of yeast as
compared with group II in which both the HSP70 and yeast were
administered simultaneously, indicating that HSPs do not act as
opsonizing agents.
Macrophages treated with HSP70 at conditions that inhibit actin
polymerization,46 that is, at either 4°C instead of 37°C or in the
presence of cytochalasin-D (4 ␮M), showed inhibition of the
effects of HSP70 (Figure 2F), indicating that HSP70-mediated
internalization was via actin-dependent, cytoskeletal rearrangement: essential characteristics of phagocytosis.
Macrophages treated with HSP70 either in the presence or
absence of cycloheximide (1 mM; ribosomal protein synthesis
inhibitor)47 showed similar increase in HSP-mediated phagocytic
uptake as did macrophages treated with HSP70 alone (Figure 2F),
indicating that synthesis of new intracellular proteins is not
essential for HSP-mediated phagocytosis. Trypan blue exclusion
was undertaken to confirm that the cells were viable through the
cycloheximide treatment.
Finally, macrophages treated with either ATP-purified HSP70
(ATP removes peptide from the HSP70)34 or with ADP-purified
HSP70 (peptides remain bound to the HSP70) demonstrated the
same degree of HSP-mediated phagocytosis (Figure 2F), indicating
that the HSP70-mediated effect was independent of the presence of
chaperoned peptides.
HSP70-mediated phagocytosis is not due to contamination
by endotoxin
HSP70 used in all assays was purified using LPS-free sterilepackaged plastic ware, baked glass ware (420°F for 4 hours) and
using endotoxin-free culture grade reagents to minimize inadvertent LPS contamination. Further, LPS levels and activity in the
purified HSPs were quantified and confirmed to be less than 1
EU/mg protein. Next, HSPs and LPS were compared for their
influence on phagocytosis in the presence or absence of serum.
These conditions were specifically chosen because LPS-binding
protein (LBP), an essential mediator for LPS function, is not
present in serum-free conditions.48 As a control, macrophages were
treated with LPS in the presence or absence of serum, and their
ability to elicit TNF-␣ release was compared (Figure 3A). Having
established that serum-free conditions rendered LPS ineffective at
stimulating macrophages, the same conditions (serum-free) was used to
test the ability of HSP70 to stimulate macrophages. Briefly, macrophages were treated with HSP70 (100 ␮g /mL) or LPS (different doses
ranging from 25 ng to 1 ␮g/mL) in serum-free conditions for 1 hour, and
a phagocytosis assay was performed. HSP70 was able to enhance
phagocytosis equally whether in the presence or absence of serum,
whereas LPS was unable to enhance phagocytosis in serum-free
HSP70-INDUCED PHAGOCYTOSIS
1639
Figure 3. HSP70-mediated phagocytosis is independent of LPS. (A) Macrophages were treated with LPS in serum-free conditions and resultant TNF-␣
production was measured. (B) Macrophages maintained in serum-free conditions
were treated with either HSP70 or of LPS (as indicated), or heat-denatured HSP70
and a phagocytosis assay was performed. The error bar is 1 SD. The results shown
are a cumulative analysis of 3 experiments, 3 wells/group.
conditions (Figure 3B). Because HSPs do not act via the LBP, their
activity is not affected by the absence of serum.
Last, because heat denatures HSPs but not LPS, HSP70
preparations were heated at 100°C for 20 minutes and tested for
phagocytosis. Heat-denatured HSP70 samples lost the ability to
enhance phagocytosis as compared with intact HSPs (Figure 3B),
indicating that the HSP70-mediated phagocytosis was due to intact
HSP70 alone and not due to any contaminating LPS. Taken
together, these measures indicate that contaminating LPS (if any) is
not responsible for HSP-mediated phagocytosis.
Exogenous HSP70 binds the lipid raft microdomain
of the macrophage plasma membrane
Briefly, RAW264.7 macrophages were administered exogenous
HRP-labeled HSP70 (100 ␮g/mL) or were left untreated for 60
minutes and washed with PBS 3 times to remove free HSPs.
Subsequently, they were lysed in MES buffer and processed for
purification of lipid rafts (see “Materials and methods”). One of 2
different types of detergents was used: Brij98 or Triton X-100 to
fractionate the material to isolate the fraction of DRMs. This
fraction was further purified into light and heavy components using
a sucrose gradient. These fractions obtained (light, LR; heavy,
non-LR) were then analyzed using immunoblotting to detect HRP
activity of HSP70-HRP complexes or tested for the presence of an
LR-associated ganglioside GM1 by using HRP–cholera toxin B.
Similarly, these fractions were tested for the amount of cholesterol
using (Biovision Research Products, Palo Alto, CA). LR fractions
were purified from HSP70-treated macrophages using Brij98
(Figure 4A) or Triton X (Figure 4B) methods showed that both,
exogenously administered HSP70 and GM1, colocalized on the
LR-microdomain of the macrophage plasma-membrane binding
(Figure 4A). Similarly, cholesterol levels of the fractions that
bound exogenous HSP70 were higher. Collectively, these results
indicated that HSP70 bound to the same fractions that were
enriched in LR-associated molecules, including GM1 and cholesterol and copurified with the LR-microdomains as purified by using
2 separate detergents.
Further, macrophages were treated with exogenous HRPlabeled HSP70 (100 ␮g/mL) in the presence of MCD (30 ␮M),
which is known to disrupt the LR-integrity.26 Subsequently,
macrophages were processed for purification of LRs by using
Triton X-100. As shown in Figure 4B, there was a significant
reduction in the binding of exogenously administered HSP70 when
macrophages were treated with MCD. Similar reduction in the
presence of GM1 and cholesterol further supported the evidence
that this reduction in HSP70 binding was due to the disruption of
the LR. To ensure that the viability of cells was not affected by
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1640
WANG et al
BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
Figure 4. HSP70-macrophage interaction occurs on the lipid raft microdomain of macrophage plasma membranes. (A) Macrophages treated with exogenous
HRP-labeled HSP70 (100 ␮g/mL, at 4°C) were washed, lysed with MBS buffer with 1% Brij98. Further fractionation using a sucrose gradient into lipid rafts (LR) (light fractions)
or non-LR (heavy fractions) microdomains was undertaken. These fractions were tested by using detecting HRP activity of HRP-HSP70 complexes (as indicated); the
presence of GM1 by using HRP–cholera toxin-B (as indicated); and assayed for the amount of cholesterol. (B) Similar to the conditions in panel A macrophages were treated
with HSP70-HRP, and the LR-fractions were purified using Triton X-100 (as indicated). These fractions were tested for the presence of HSP70 and GM1 and were assayed for
the amount of cholesterol. Further, the ability of macrophages to bind HSP70 was tested in the presence of LR-disrupting drug MCD. Macrophages were treated
methyl-␤-cyclodextrin (MCD; 30 mM), washed with cold PBS, and then incubated with exogenous HRP-labeled HSP70 (100 ␮g/mL) for 30 minutes on ice. Subsequently,
macrophages were processed for purification of LRs by using Triton X-100 and tested for the presence of HSP70 and GM1 and were assayed quantitatively for the amount of
cholesterol. (C) The influence of the LR-integrity on HSP70-mediated phagocytosis was tested by treating macrophages with nystatin or MCD (both drugs disrupt LRs), and a
phagocytosis assay was performed. Macrophages treated with FCS served as controls to assess the effects of LR-disrupting drugs on opsonic phagocytosis. The results
shown are a cumulative analysis of 3 experiments, 3 wells/group. (D) RAW264.7 macrophages were treated with HSP70 (100 ␮g/mL) in the presence of varying doses of MCD
(as indicated). Error bars indicate one standard deviation.
treatment with MCD, we performed a trypan blue exclusion that
revealed a 95% viability in both MCD-treated or untreated cells.
Next, macrophages were treated with medium alone, HSP70, or
fetal calf serum (FCS) in the presence of either nystatin or MCD
(30␮M) (both agents are known to disrupt the LR integrity26; see
Figure 4B). Subsequently, the cells were administered Alexafluorlabeled yeast (40 particles/macrophage), and the amount of yeast
internalized was quantified as described in “Materials and methods.” As a control, macrophages treated with serum were also
included in the assay to test the effects the LR-disrupting drugs on
FcR␥-mediated antigen uptake. As shown in Figure 4C, HSP70mediated phagocytosis was inhibited in the presence of LR-disrupting
drugs, whereas opsonic phagocytosis via the FcR␥ was not.
Further, macrophages were treated with HSP70 (100 ␮g/mL) in
the presence of varying doses of MCD (ranging from 0 to 30 mM).
As seen in Figure 4D, the inhibitory effect that MCD has on the
macrophage response to HSP70 is titratable depending on the dose
of MCD used.
the antigenic epitope but not to the whole ovalbumin protein.
Resultant proliferation of the CSFE-labeled CD4⫹ was measured
by FACS as an indirect measure of the amount of ovalbumin
protein processed and its peptide epitope presented in context of
MCH-II. To quantify the effector function of the CD4⫹ cells, the
production of IFN-␥ by the CD4⫹ T cells was measured using
ELISA. As shown in Figure 5B HSP70-treated macrophages cause
an increase in CD4⫹ proliferation to 20% as compared with those
treated with medium alone (1.7%). Further, cytochalasin-D treatment abrogated the proliferation caused by HSP70 treatment,
indicating the net result was phagocytosis dependent. The net
increase in CD4⫹ cells in response to HSP70 treatment was
concordant with the increase in their ability to produce IFN-␥ as
compared with treatment with ova-coated yeast alone or HSP70
given alone (Figure 5C). Taken together, these results indicated that
HSP70 mediated a sharp rise in the uptake of the yeast coated with
Increased HSP70-mediated phagocytosis enhances MHC-II
antigen processing and presentation
Alexafluor-labeled S cerevisiae were coated with whole ovalbumin
protein 1 mg/mL by coincubation at 37°C for 20 minutes and
washed until free ovalbumin protein was removed (washes were
tested by gel electrophoresis using SDS-PAGE and immunoblotting). The association itself was confirmed by SDS-PAGE analysis
and immunoblotting using antibody to ovalbumin (Sigma). The
ovalbumin protein-coated–yeast complexes (Ova-coated yeast)
were administered to 4 ⫻ 105 macrophages (40 yeast/macrophage)
that were treated with either HSP70 (100 ␮g/mL) or control
proteins in the presence or absence of cytochalasin-D to confirm
that the process is phagocytosis dependent (Figure 5A). After 2
hours, the macrophages were washed, irradiated to prevent macrophage-proliferation, and cocultured with CSFE-labeled 2 ⫻ 105
CD4⫹ T cells purified from spleens of DO11.10 mice (I-Ad–
restricted DO11.10 TCR-␣␤–transgenic mice). The DO11.10 strain
is transgenic against a MHC-II epitope of ovalbumin protein, and
CD4⫹ T cells from these animals proliferate on being exposed to
Figure 5. Increased HSP70-mediated phagocytosis enhances antigen presentation. (A) Yeast (S cerevisiae) was coated with ovalbumin (the whole protein) by
coincubation at 37°C for 20 minutes. The complexes (Ova-coated yeast) were
administered to macrophages treated with HSP70 (100 ␮g/mL) or control proteins in
the presence or absence of cytochalasin-D. After 2 hours, the macrophages were
washed, irradiated (to prevent macrophage-proliferation), and cocultured with CSFElabeled CD4⫹ T cells purified from spleens of DO11.10 mice (I-Ad–restricted DO11.10
TCR-␣␤–transgenic). (The DO11.10 strain is transgenic against a specific MHC-II
epitope of ovalbumin protein.) Resultant CD4⫹ proliferation was measured using
FACS as an indicator of the amount of the ovalbumin peptide presented in context of
MCH-II antigen presentation. (B) Concurrent production of interferon ␥ (IFN␥) by the
CD4⫹ T cells was quantified using ELISA as a measure of their effector function. The
data shown represents 1 of 3 independent experiments.
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BLOOD, 15 FEBRUARY 2006 䡠 VOLUME 107, NUMBER 4
HSP70-INDUCED PHAGOCYTOSIS
ovalbumin protein antigen, enhanced its processing to generate the
MHC-II–restricted antigenic epitope, and presented it effectively to
generate a CD4⫹ T-cell response.
Discussion
The interaction of extracellular HSP70, a highly conserved intracellular molecule with the macrophage, stimulates them to phagocytose at 6 times the basal rate. Soon after HSP70 treatment,
macrophages rapidly internalize a variety of particulate materials,
including Gram-positive and -negative bacteria (Staphylococcus
aureus, E coli), fungi (S cerevisiae, Candida albicans), and inert
particles (polystyrene microspheres).
Further, HSP70-macrophage interaction occurs on the LR
microdomain of the macrophage-cell surface. Our study identifies
an important role of LRs in the HSP70-macrophage interaction and
its downstream effects. Structurally, it appears that the exogenous
HSP70 binds the macrophage on the lipid raft microdomain of the
plasma membrane. Whether this binding is on a specific LR-bound
receptor or whether it binds certain structural components of the
LR is not currently known and is being investigated. By the same
token, it is possible that the HSP70 is taken up by a different
macrophage receptor and binds the inner leaflet of the LR. This
possibility was not tested. Our study defines a functional role of
lipid rafts. Although their physical existence has been questioned
by some,49 our results define the influence of LRs on HSP
phagocytosis. Disruption of the LRs partially abrogates the HSPmediated effects on phagocytosis. Lipid rafts could provide a
platform for the interaction between the HSP70 and the phagocytic
receptors. Although our results indicate that the interaction with
HSP70 occurs on the lipid raft, the nature of further signaling to
involve the phagocytic receptors is not clear. Our preliminary work
in defining the signaling pathway indicates that it is dependent on
endocytosis (data not shown). Whether the entire HSP70-LR
complex is endocytosed and if so, how this complex actually
activates the phagocytic receptors needs further study. The fact that
cycloheximide is unable to block HSP-mediated phagocytosis
indicates that new protein synthesis is not required, partly explaining the quick onset of HSP-mediated phagocytosis. Disruption of
the LRs abrogates the HSP-mediated phagocytosis, suggesting that
LRs provide a platform for HSP70 and subsequent activation of the
phagocytic receptors. Although several studies have shown that the
endogenous HSP70 binds the LR,26,50 this provides further evidence of exogenous HSP70 binding the LR. Whether LRs play any
other role in the HSP70-mediated phagocytic uptake (other than
providing a structural platform for the HSP70 binding and facilitating further activation) needs further scrutiny. Functionally, LRs
have been implicated in several cellular processes, including their
ability to facilitate protein-protein interaction, raft-mediated
endocytosis, signal transduction, and the biogenesis of the phagosome.27-29,31 Our current work did not study the signaling pathway
involved; however, some of our findings provide vital clues to its
1641
characteristics. The quick onset of phagocytosis, within minutes of
HSP70 treatment, and the nondependence on synthesis of new
proteins (cycloheximide independent) suggest that HSP70mediated enhancement of phagocytosis occurs via a short signaling
pathway, possibly membrane bound and does not involve a gene
up-regulation.
These results provide evidence that HSP70, a phylogenetically
conserved molecule, plays an important role in the innate host
response to pathogens. Physiologically, necrotic-cell lysis from
injury or infection is known to release HSPs into the extracellular
compartment where they activate APC-mediated defenses.10 It has
been shown that the local concentration of HSP70 is in the range of
200 ␮g/g tissue lysed.10 In the context of earlier data, as little as 100
␮g/mL HSP70 (which would correspond to 0.5 g tissue lysate)
released extracellularly either from infection or injury could
potentially elicit macrophage phagocytosis. It is important to note
that the HSP70 is able to stimulate phagocytosis whether it carries
peptides on it or not. Both ADP- and ATP-purified HSP70 are
equally capable of stimulating phagocytosis. Because the nonspecific responses to HSPs are independent of peptides chaperoned by
HSPs, they act similarly to bacterial lipopolysaccharides (LPSs).
As seen in our results, our study has taken special precautions to
exclude the effect of LPS.51,52
Our results reveal that extracellular HSPs are recognized by LR
microdomain of the macrophage-triggering phagocytosis, a primal
mechanism of self-defense. The significance of these findings is far
reaching. This report indicates that the presence of extracellular
HSP70, basically an intracellular molecule whose presence outside
of the cell is unnatural, a result of tissue breakdown from infection
or injury.10 It appears from our findings that a component of the LR
microdomain recognizes the extracellularly released HSP70 to
represent cell lysis and responds to it by up-regulating phagocytic
antigen uptake. Modulating the HSP70-LR interaction presents an
opportunity to intervene at the level of host-pathogen interface, a
therapeutic tool for emerging infections, especially where conventional treatment with antibiotics is ineffective (antibiotic resistance) or unavailable (rapidly spreading, endemic). Sequentially,
HSP70-mediated phagocytosis leads to increased antigenic processing and presentation to CD4⫹ T lymphocytes, which proliferate and
release IFN-␥. In this context, HSP70-LR interaction plays an
important role, not only clearing invading agents but also processing and presenting their antigens to host immune system. Although
prokaryotic HSPs have been shown to be a potential source of
microbial peptide antigens during phagocytic processing of bacteria during infection,53 our results indicate that antigen presentation
is increased when macrophages are treated or pretreated with
HSP70 and not otherwise. The wide range of microbial and
nonmicrobial agents phagocytosed in response to HSP70-LR
interaction as well as its remarkably short time of onset presents the
opportunity to develop a rapidly deployable therapeutic intervention. Potentially, it could not only facilitate early elimination of the
invading agent but also activate the immune system into a state of
heightened preparedness.
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2006 107: 1636-1642
doi:10.1182/blood-2005-06-2559 originally published online
November 1, 2005
Exogenous heat shock protein 70 binds macrophage lipid raft
microdomain and stimulates phagocytosis, processing, and MHC-II
presentation of antigens
Ruibo Wang, Joseph T. Kovalchin, Peggy Muhlenkamp and Rajiv Y. Chandawarkar
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