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Am J Physiol Cell Physiol 286: C739–C744, 2004;
10.1152/ajpcell.00364.2003.
Invited Review
Cytokine function of heat shock proteins
Min-Fu Tsan1,2 and Baochong Gao1
1
Research Service, Veterans Affairs Medical Center, Washington 20422; and 2Department
of Medicine, Georgetown University, Washington, District of Columbia 20007
tumor necrosis factor-␣; lipopolysaccharide; macrophages; innate immune system
HEAT SHOCK PROTEINS (HSPs) are the most phylogenetically
conserved proteins present in all prokaryotes and eukaryotes
(20, 27, 45). Traditionally, HSPs are regarded as intracellular
molecules. With the availability of recombinant bacterial and
human HSPs, there has been an intense interest in the extracellular function of HSPs in recent years. It has been shown
that HSPs are potent activators of the innate immune system,
capable of inducing proinflammatory cytokine production by
the monocyte-macrophage system, and the activation and maturation of dendritic cells (antigen-presenting cells) (5, 17, 21,
38, 52, 81–83, 85). The purpose of this focused review is to
critically evaluate the reported cytokine function of HSPs, with
particular emphasis on the question of whether the reported
cytokine effects are in fact due to HSPs or to the contaminant(s) that are present in the HSP preparations.
HEAT SHOCK PROTEINS
HSPs are expressed both constitutively (cognate proteins)
and under stressful conditions (inducible forms). In addition to
heat shock, a variety of stressful situations including environmental (ultraviolet radiation or heavy metals), pathological
(infections or malignancies), or physiological (growth factors
or cell differentiation) stimuli induce a marked increase in HSP
synthesis, known as the stress response (33, 45). Upon necrotic
cell death, HSPs are leaked into the extracellular compartment
(10). In addition, HSPs can be released extracellularly independent of necrotic cell death in response to a number of
stressful conditions (8, 29, 44). The mechanism and the physAddress for reprint requests and other correspondence: M.-F. Tsan, MidAtlantic Regional Office (10R), Office of Research Oversight, Dept. of
Veterans Affairs, 50 Irving St. NW, Washington, DC 20422 (E-mail: [email protected]).
http://www.ajpcell.org
iological significance of the HSP release are not clear. However, HSPs are present in circulation of normal individuals (57,
87), and their circulating levels are decreased in aging (62) and
increased in a number of pathological conditions such as
hypertension (58), atherosclerosis (87, 89), and after openheart surgery (18).
The primary function of the HSPs appears to serve as
molecular chaperones in which they recognize and bind to
nascent polypeptide chains and partially folded intermediates
of proteins, preventing their aggregation and misfolding, or as
chaperonins that directly mediate protein folding (20, 27, 33,
45). The classification of HSPs is based on their related
function and size (molecular mass). Using the nomenclature
adopted after the Cold Spring Harbor Meeting of 1996 (30),
family names are written in uppercase, e.g., HSP70, whereas
members of a family are conventionally written as Hsps, e.g.,
Hsp70.
Major classes of HSPs include the small HSPs, HSP40, 60,
70, 90, and 110 families (20, 27, 45). In mammalian species,
the HSP60 (chaperonin) family consists of mitochondrial
Hsp60 (mt-Hsp60) and cytosolic Hsp60 (T-complex polypeptide-1) (20, 27, 45). The mt-Hsp60 exists in a dynamic equilibrium among monomers, heptamers, and tetradecamers (20,
41). It dissociates into monomers at low concentrations and
assembles into tetradecamers in the presence of ATP and
mt-Hsp10, the cofactor of mt-Hsp60 (42). The cytosolic Hsp60
forms heterooligomeric ring structures and functions in cytosol
to fold cytoskeletal proteins such as actin and tubulin (46). The
HSP70 family includes the constitutive cytosolic Hsc70 (or
Hsp73), the stress-induced cytosolic Hsp70 (or Hsp72), the
endoplasmic reticulum (ER) Bip (or Grp78), and the mitochondrial mt-Hsp70 (20, 27, 45). The Hsp70 is composed of two
major functional domains. The NH2-terminal, highly conC739
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Tsan, Min-Fu, and Baochong Gao. Cytokine function of heat shock proteins. Am
J Physiol Cell Physiol 286: C739–C744, 2004; 10.1152/ajpcell.00364.2003.—
Extensive work in the last 10 years has suggested that heat shock proteins (HSPs)
may be potent activators of the innate immune system. It has been reported that
Hsp60, Hsp70, Hsp90, and gp96 are capable of inducing the production of
proinflammatory cytokines by the monocyte-macrophage system and the activation
and maturation of dendritic cells (antigen-presenting cells) in a manner similar to
the effects of lipopolysaccharide (LPS) and bacterial lipoprotein, e.g., via CD14/
Toll-like receptor2 (TLR2) and CD14/TLR4 receptor complex-mediated signal
transduction pathways. However, recent evidence suggests that the reported cytokine effects of HSPs may be due to the contaminating LPS and LPS-associated
molecules. The reasons for previous failure to recognize the contaminant(s) as
being responsible for the reported HSP cytokine effects include failure to use highly
purified, low-LPS preparations of HSPs; failure to recognize the heat sensitivity of
LPS; and failure to consider contaminant(s) other than LPS. Thus it is essential that
efforts should be directed to conclusively determine whether the reported HSP
cytokine effects are due to HSPs or to contaminant(s) present in the HSP preparations before further exploring the implication and therapeutic potential of the
putative cytokine function of HSPs.
Invited Review
C740
HSP AND CYTOKINE FUNCTION
HSPS AND CYTOKINE FUNCTION
The above-described molecular chaperone function and presentation of antigens depend on the peptide-binding properties
of HSPs. Recent studies suggest that HSPs may also have
potent cytokine-like function independent of peptide binding.
HSPs such as Hsp60, Hsp70, Hsp90, and gp96 from a
variety of sources, including purified preparations from bacterial (23, 65, 69) and mammalian (4, 10, 52, 68, 71, 83) sources
as well as recombinant bacterial (15, 22, 37, 39, 53, 54, 66, 81,
86, 91) and human (5, 6, 13, 17, 18, 37, 38, 51, 81, 82)
products, have been shown to be potent activators of the innate
immune system (85). These HSP preparations have been
shown to induce the production of proinflammatory cytokines
such as tumor necosis factor (TNF)-␣, interleukin (IL)-1, IL-6,
and IL-12 and the release of nitric oxide (NO) and C-C
chemokines by monocytes, macrophages, and dendritic cells.
They also induce the maturation of dendritic cells as demonstrated by the upregulation of MHC class I and II molecules,
CD86, CD40, etc. (10, 21, 52, 68, 71, 86). The Hsp60 and
Hsp70 preparations purified from bacterial sources or from
recombinant bacterial and human products are capable of
inducing the above effects in concentrations ranging from ⬍1
AJP-Cell Physiol • VOL
␮g/ml to a few micrograms per milliliter, whereas Hsp70,
Hsp90, and gp96 isolated from mouse liver require concentrations that are one to two orders of magnitude higher (e.g.,
10–100 ␮g/ml). These HSP cytokine effects, compared with
their molecular chaperone function, are unique in that they
require no HSP-associated peptides, no ATP hydrolysis, no
cofactors, and no protein complex assembly. A new term,
“chaperokine,” has been coined for HSPs to indicate their dual
functions as molecular chaperones and cytokines (5).
Furthermore, the observed HSP cytokine effects are mediated via the CD14/Toll-like receptor (both TLR2 and TLR4)
complex signal transduction pathways leading to the activation
of nuclear factor-␬B (NF-␬B) and mitogen-activated protein
kinases (MAPKs), i.e., ERKs (p42 and p44 extracellular signal-regulated kinases), JNK (c-Jun NH2-terminal kinase), and
p38 kinase (5, 6, 15, 18, 38, 51, 81–83). The CD14 and TLR
receptor complexes are pattern recognition receptors involved
in the innate immunity for the pathogen recognition and host
defense (3, 47). CD14, the lipopolysaccharide (LPS) receptor,
is a glycophosphatidyl inositol (GPI)-anchored membrane protein lacking transmembrane and intracellular domains (28, 76).
TLRs are type I transmembrane proteins with an extracellular
domain containing a leucine-rich repeat and a cytoplasmic
domain analogous to that of the IL-1 receptor (IL-1R) family
(47, 67). An adapter protein, MyD88 (myeloid differentiation
protein 88), binds to the Toll/IL-1R homology (TIR) motif
through its own TIR motif, whereas a death domain on its
COOH terminus recruits IL-1R-associated kinase (IRAK) to
the complex (48). IRAK is then autophosphorylated and released from the complex to bind TRAF6 (TNF receptorassociated factor 6), which can then activate either the NF-␬B
or the MAPKs (47, 49). Together with CD14 and an accessory
protein MD2, TLR4 initiates signaling cascades in response to
LPS, whereas TLR2 initiates the signal cascades in response to
bacterial lipoprotein, Gram-positive bacteria, yeast, and spirochetes (2, 14, 34, 47, 67).
The reported activation of the innate immune system by
HSPs, as described above, has been hailed as an important new
function of HSPs with broad biological significance. The
induction of proinflammatory cytokines by Hsp60 and Hsp70
may contribute to the pathogenesis of autoimmune diseases
and chronic inflammation (55). Chlamydial Hsp60 frequently
colocalizes with human Hsp60 in macrophages of atherosclerotic plaques (39). Induction of proinflammatory cytokine
release from macrophages by chlamydial Hsp60 would provide
a potential mechanism by which chlamydial infections may
promote atherogenesis and precipitate acute ischemic events
(37, 39). Likewise, the activation and maturation of dendritic
cells by gp96 may be responsible for the gp96-induced tumor
immunity by inducing both the innate and adaptive immune
responses (50). Thus it has been proposed that through their
cytokine function, HSPs may serve as a “danger signal” to
the innate immune system at the site of tissue injury (17, 85)
and that HSPs could be the endogenous ligands for the
TLR2 and TLR4 (51, 82). In fact, HSPs are considered to be
the prototype of endogenous ligands for Toll-like receptors
(12). There is considerable interest to further explore the
implications and therapeutic potential of these HSP cytokine
effects (7, 55, 88).
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served ATPase domain binds ADP and ATP tightly and hydrolyzes ATP, whereas the COOH-terminal domain is required
for polypeptide binding (20, 27). The HSP90 family includes
the cytosolic Hsp90 (␣ and ␤) and the ER form, gp96 (grp94).
Glucose-regulated proteins (grp) such as grp78 and grp94/gp96
are molecular chaperones in the ER that are upregulated in
response to glucose starvation and other stressful stimuli that
disrupt protein folding in the ER (30, 70). For more detailed
description of HSPs, we refer the reader to excellent reviews of
the subject (20, 27, 33, 45, 55, 70, 87).
Bacterial HSPs, particularly Hsp60 and Hsp70, are highly
immunogenic, capable of inducing antibody production and
T-cell activation (92). The antibodies and T cells against
bacterial Hsp60 and Hsp70 also recognize mammalian Hsp60
and Hsp70, respectively, due to cross-reactivity (36). These
anti-Hsp60 and anti-Hsp70 antibodies and T cells injure tissues
and cause inflammatory reactions. Thus Hsp60 and Hsp70
have been implicated in the pathogenesis of a number of
autoimmune diseases and inflammatory conditions such as type
1 diabetes (1, 19), Crohn’s disease (77), atherosclerosis (56,
87), and juvenile chronic arthritis (60, 64).
HSPs also play important roles in antigen presentation,
cross-presentation, and tumor immunity (43, 72, 73). HSPs of
the cytosol, such as Hsp70 and Hsp90, and of the ER, such as
gp96, bind antigenic peptides generated within the cells and are
part of the endogenous pathway of antigen presentation by the
major histocompatibility complex (MHC) class I molecules
(32, 43, 74). Peptides that are chaperoned by HSPs when
released extracellularly are taken up by antigen-presenting
cells via ␣2-macroglobulin receptor (CD91)-mediated endocytosis, resulting in representation by the MHC molecules (9, 43,
75). Vaccination of mice with Hsp70, Hsp90, and gp96 isolated from murine tumor cells elicits immune response sufficient for tumor rejection and suppression of metastatic tumor
progression (73). This tumor immunity results from tumorderived peptides associated with the HSPs, rather than from the
HSPs themselves (80).
Invited Review
C741
HSP AND CYTOKINE FUNCTION
HSPS VS. CONTAMINANTS
Fig. 1. Effect of heat inactivation on endotoxin
activity and tumor necrosis factor (TNF)-␣-inducing
activity of lipopolysaccharide (LPS). A stock solution of LPS at 4 ng/ml was heated in a boiling water
bath for 1 h. A: endotoxin activities of the nonheated LPS and heated LPS were determined using
the Limulus amebocyte lysate assay. B: murine macrophages were treated with LPS or heated LPS at the
indicated concentrations for 4 h. TNF-␣ concentrations in media were then determined. Values represent means ⫾ SD of 3 experiments. *P ⬍ 0.05 vs.
non-heated LPS. [Reprinted from Gao and Tsan (25)
with permission.]
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The reported HSP cytokine effects are similar to those of
LPS and bacterial lipoprotein. Because the recombinant bacterial and human HSPs are produced by Escherichia coli
expressing HSP cDNAs, the final preparations may be contaminated with bacterial products. Likewise, HSP preparations
isolated from bacteria or murine tissues are also frequently
contaminated with LPS. The fact that various HSPs all have
similar effects and share the same CD14/TLR2 and CD14/
TLR4 receptor complexes is of considerable concern.
Ample examples exist in the literature demonstrating how
contaminants can lead to misleading conclusions. For example,
in 1998 with the use of the commercially available LPS
preparation, it was first reported (90) that TLR2 mediated the
LPS-induced activation of NF-␬B and could be the long sought
after LPS signal transducer. There followed a period of uncertainty regarding whether TLR2 or TLR4 was the LPS signal
transducer (35, 59, 61). In 2000, it was then demonstrated that
TLR2 could not mediate cellular response by repurified commercial preparations of LPS (31, 78). In 2002, two lipoproteins
(Lip12 and Lip19) extracted from E. coli LCD25 LPS were
identified to be the major components responsible for TLR2mediated cell activation in the commercial LPS preparations
(40). Thus failure to recognize the presence of lipoproteins in
the commercially available LPS preparation led to the erroneous attribution of lipoprotein signal transducer, TLR2, as the
LPS signal transducer (35, 90).
Investigators are cognizant of the possibility of contamination, particularly LPS, and have attempted to rule out the
possibility of LPS contamination being responsible for the
observed HSP cytokine effects. Most studies have used two
criteria: first, LPS is resistant to heat inactivation (5, 17, 18, 23,
38, 65, 66, 86), and second, LPS effects are inhibitable by
polymyxin B (5, 17, 18, 38, 65, 66, 86). Other less frequently
used criteria include the effect of anti-HSP antibodies (15, 65)
and other LPS inhibitors such as lipid IVa (5), LPS (10, 52), or
lipid A (18) from Rhodopseudomonas spheroides. Because the
observed HSP cytokine effects were heat sensitive, either not
inhibitable or only partially inhibitable by polymyxin B, not
inhibitable by other LPS inhibitors, and/or inhibitable by antiHSP antibodies, it was concluded that the observed HSP
cytokine effects could not have been due to LPS contamination. However, doubts about these criteria have been raised.
Wallin et al. (85) noted that highly purified murine liver Hsp70
had no cytokine effects even at concentrations as high as
200–300 ␮g/ml. On the other hand, a LPS-contaminated prep-
aration at Hsp70 concentrations as low as 50–100 ng/ml caused
cytokine effects that were heat sensitive and were not inhibitable by polymyxin B.
Recent studies in which HSP preparations essentially free of
LPS were used suggest that the previously reported cytokine
function of HSPs may be due to the contaminants. Bausinger et
al. (11) reported that LPS-free recombinant human Hsp70
(rhHsp70) did not induce the activation of dendritic cells. Gao
and Tsan (24, 25) demonstrated that LPS was heat sensitive
(Fig. 1) and that the ability of commercially available rhHsp70
to induce TNF-␣ production was entirely due to the contaminating LPS (24), whereas that of rhHSP60 was due to contamination by LPS as well as LPS-associated molecules (25). Reed
et al. (63) reported that the activation of NF-␬B and the
production of NO by gp96 were due to LPS contamination.
Importantly, all of these investigators demonstrated that these
highly purified, essentially LPS-free HSPs retained their normal molecular chaperone functions or ATPase activity (11, 24,
25, 63). Thus failure of Hsp60, Hsp70, and gp96 to induce
cytokine or NO production by macrophages or to activate
antigen-presenting cells was not due to defective HSPs as a
result of purification.
The fact that LPS is sensitive to heat inactivation (24, 25, 84)
has not been widely appreciated. Most investigators are not
aware that macrophages are extremely sensitive to LPS. LPS at
a concentration of 0.1–0.2 ng/ml is sufficient to maximally
induce TNF-␣ release from murine macrophages (24). However, in most studies LPS was used at concentrations ranging
from 10 to 500 ng/ml to test for heat sensitivity (5, 17, 18, 23,
38, 65, 66, 86). At these concentrations, even if heat treatment
inactivated 99% of the LPS used in the studies, there would
still be sufficient residual LPS to induce TNF-␣ release, giving
the impression that LPS was heat resistant. Thus, unless one
uses an LPS concentration similar to the LPS concentration
present in the HSP preparation, the result could be misleading.
Although LPS may be the most frequent contaminant, nonLPS contaminant(s) capable of inducing proinflammatory cytokines may also contribute to the reported cytokine effects of
HSPs. Gao and Tsan (25) showed that 50% of the TNF-␣inducing activity of the commercially available rhHsp60 (no.
NSP-540; StressGen Biotechnologies, Victoria, BC, Canada)
was due to non-LPS contaminant(s) that was heat sensitive but
not inhibitable by polymyxin B. The presence of non-LPS
contaminant(s) could partially explain previous reports that the
observed cytokine effects of HSPs were either not inhibitable
Invited Review
C742
HSP AND CYTOKINE FUNCTION
AREAS OF UNCERTAINTY
Although recent evidence suggests that the cytokine effects
that have been reported in the last 10 years may be due to the
effects of contaminants such as LPS and LPS-associated molecules (11, 24, 25, 63), a number of uncertainties exist.
It is not clear why anti-Hsp60 antibodies could inhibit
Hsp60-induced proinflammatory cytokine production by macrophages (15, 65). It is possible that HSPs bind LPS and that
binding of anti-HSP antibodies to HSPs interferes with the
interaction of HSP-bound LPS with the CD14/TLR4 receptor
complex, thus inhibiting the effect of LPS. There is supporting
evidence that Hsp70, Hsp90, and gp96 bind LPS (16, 63, 79).
If the reported cytokine effects are conclusively shown to be
due to contaminants, not due to HSPs themselves, then one
important question will be whether HSPs have any effect on
the innate immune system. Habich et al. (26) have described a
macrophage receptor for Hsp60 that is specific and saturable,
suggesting that Hsp60 may have some effect on macrophages
that is different from the reported cytokine effects mediated
through the CD14/TLR receptor complexes. Reed et al. (63)
reported that a highly purified, low-LPS preparation of gp96
was able to elicit a marked increase in ERK phosphorylation
but not the activation of p38 and JNK, an effect distinct from
that of LPS. Thus HSPs may activate macrophage signal
transduction independent of LPS.
In conclusion, extensive investigation in the past 10 years
has suggested that HSPs may be potent activators of the innate
immune system. It has been shown that Hsp60, Hsp70, Hsp90,
and gp96 are capable of inducing the production of proinflammatory cytokines by the monocyte-macrophage system, as well
as the activation and maturation of antigen-presenting cells in
a manner similar to the effects of LPS and bacterial lipoprotein,
e.g., via CD14/TLR2 and CD14/TLR4 receptor complex-mediated signal transduction pathways. However, recent evidence
suggests that the reported cytokine effects of HSPs may be due
to LPS and LPS-associated molecules. Thus it is essential that
efforts should be directed to conclusively determine whether
the reported HSP cytokine effects are due to HSPs or to
contaminant(s) present in the HSP preparations before exploring further the implication and therapeutic potential of the
putative cytokine function of HSPs.
AJP-Cell Physiol • VOL
GRANTS
This work is supported by the Medical Research Service, Office of Research and Development, Department of Veterans Affairs.
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