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Induction by Heat-Shock Protein 27 (Hsp27) Suggests Hsp27 Is Primarily an Antiinflammatory Stimulus1
Department of Surgery, University of Massachusetts Medical School, Worcester, MA 01655
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-15, IL-12) cytokines, hsp27, a small hsp,
has been primarily identified as a substrate of mitogen-activated
protein kinase-activated protein kinase-2 involved in the p38 signaling
pathway and activated during monocyte IL-10 production. Hsp27 can also
act as an endogenous protein circulating in the serum of breast cancer
patients and a protein whose induction correlates to protection from
LPS shock. However, the cytokine-stimulating properties of hsp27 have
been unexplored. In this study, exogenous hsp27 is demonstrated for the
first time as a potent activator of human monocyte IL-10 production,
but only a modest inducer of TNF-. Although exogenous hsp27
stimulation activated all three monocyte mitogen-activated protein
kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun
N-terminal kinase, and p38), only p38 activation was sustained and
required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and
p38 activation were required for induction of TNF- when using the
p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27’s transient
activation of the c-Jun N-terminal kinase pathway, which can
down-regulate IL-10, may contribute to its potent IL-10 induction.
Hsp27’s ERK 1/2 activation was also less sustained than activation by
stimuli like LPS, possibly contributing to its modest TNF-
induction. The failure of either PD98059 or anti-TNF- Ab
to substantially inhibit IL-10 induction implied that hsp27 induces
IL-10 via activation of p38 signaling independently of TNF-
activation and may be predominantly an antiinflammatory monokine
stimulus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-8, IL-1, and PGE2 (10, 11, 12). These
data suggest that some large autologous hsp may stimulate
antiinflammatory cytokine activity. This antiinflammatory function of
hsp is controversial, however, because hsp60 has also been shown to
induce TNF- in a human monocyte cell line and TNF-, as well as
IL-15 and IL-12, in murine bone marrow-derived macrophage (7, 13).
Hsp27, an important member of the small hsp family, has been
investigated primarily for its role as a circulating protein marker of
increased malignancy in breast cancer (14). Hsp27 has been
shown to down-regulate reactive oxygen intermediate (ROI) production,
thereby protecting from TNF--mediated apoptosis (15).
The glutamine induction of rat hsp25, the analogue of human hsp27, has
been shown to correlate with protection from lethal endotoxin shock
(16). Human monocytes from patients with systemic
inflammatory response syndrome have significantly elevated hsp27
expression (17). These data suggest that exogenous hsp27
may also have some antiinflammatory or immune modulatory capacities on
monocytes. IL-10 can also down-regulate ROI activity in monocytes and
macrophages, but, unlike the large hsp, hsp27 has not been previously
shown to exogenously induce production of either pro- or
antiinflammatory cytokines (18, 19). However, hsp27 is a
substrate for mitogen-activated protein kinase (MAPK)-activated protein
kinase-2 (MAPKAPK-2), an important member of the p38 MAPK cascade that
is both activated by cytokine treatment and critical in monocyte
production of cytokines (10, 20, 21, 22). Recently, the
activation (phosphorylation) of p38 MAPK and its substrate, MAPKAPK-2,
has been shown to be crucial to LPS induction of IL-10 in human
monocytes, further suggesting that hsp27 could play an antiinflammatory
role in monocytes (10). Circulating hsp27 is present in
the serum of cancer patients and, in some cases, induces in vivo hsp27
Ab production, suggesting that hsp27 can stimulate as an exogenous
protein (23, 24). Phosphorylated hsp27 has also been
identified as being associated with cell membranes of
lamellipodia in migrating cells, suggesting a possible hsp27
surface expression (25). Although hsp27 phosphorylation
after MAPKAPK-2 activation is necessary for LPS induction of monocyte
IL-10, the effect of exogenous hsp27 on increasing production of IL-10
or any monokine is unexplored. Administration of IL-10 has been shown
to suppress lethal endotoxemia and reduce serum TNF- levels
(26). Because of its antiinflammatory properties, IL-10
has been suggested as a possible therapeutic agent for inflammatory
conditions, such as rheumatoid arthritis and inflammatory bowel disease
(26). Consequently, any monocyte-modulating activity of
hsp27 in increasing IL-10 levels without concomitantly highly inducing
proinflammatory monokines such as TNF- could also have therapeutic
implications.
In this study, hsp27 has been assessed for a novel ability to induce
IL-10 and/or TNF- in human monocytes when added exogenously. We
demonstrate that human hsp27 is a potent inducer of IL-10 in human
monocytes, but only a modest inducer of TNF-. We have also explored
the preferential involvement of different MAPK pathways during
hsp27-induced monocyte IL-10 production to determine whether hsp27
induced IL-10 via the same p38, MAPKAPK-2, hsp27 pathway as LPS
stimulation. Although modest levels of TNF- were induced by hsp27,
only a small portion of the monocyte hsp27 induction of IL-10 was due
to its prior induction of TNF-, a known enhancer of IL-10 in
monocytes (10, 27). Hsp27 independently induced high
levels of monocyte IL-10, while concomitantly stimulating only minimal
levels of TNF-. Hsp27 induction of monocyte IL-10 is totally
dependent on the activation of the p38 MAPK pathway and, unlike
monocyte TNF- production, independent of the ERK pathway activation,
further supporting a TNF--independent IL-10 induction by hsp27.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FBS was purchased from Sigma (St. Louis, MO). Culture media and
other supplements were purchased from Irvine Scientific (Santa Ana,
CA). Muramyl dipeptide (MDP) was generously provided by CIBA-GEIGY
(Basel, Switzerland). Staphylococcal enterotoxin B (SEB) and zymosan A
were purchased from Sigma, and polymyxin B was purchased from
Calbiochem (La Jolla, CA). The mAbs, My4 (CD14) FITC and IgG2b FITC
were purchased from Coulter (Hialeah, FL). Recombinant human hsp27 was
purchased from Stressgen Biotechnologies (Victoria, Canada). Polyclonal
Ab against hsp27 was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA), mAb against hsp27 from Stressgen Biotechnologies, and mAb
against TNF- from Endogen (Woburn, MA). SB203580 and PD98059 were
purchased from Calbiochem (San Diego, CA). Human rTNF- was kindly
provided by The National Institute for Biological Standards and Control
(Potters Bar, U.K.). Phosphoplus p38 MAPK, p44/42 (ERK 1/2) MAPK, and
stress-activated protein kinase (SAPK)/c-Jun N-terminal
kinase (JNK) kits were purchased from New England Biolabs
(Beverly, MA). Anti-phosphoserine mAb was purchased from
Calbiochem. MAPKAPK-2 IP-kinase assay kit was purchased from
Upstate Biotechnology (Lake Placid, NY). 32P and
ECL reagents were purchased from NEN Life Science Products
(Boston, MA).
Separation of monocytes and stimulations
PBMC were first isolated from venous blood of healthy volunteers
by Ficoll-Hypaque density centrifugation. Monocytes were separated from
PBMC by selective adherence to microexudate-coated plastic surfaces, as
described (28). Adherent monocytes (>95% purity, as
checked by flow-cytometric analysis) were collected by treatment
with 10 mM EDTA, suspended in IMDM medium, supplemented with 10% FBS,
50 U/ml penicillin G, 50 µg/ml streptomycin, 50 µg/ml gentamicin,
2.5 µg/ml fungizone, 4 mM L-glutamine, 1 mM sodium
pyruvate, and 1% MEM nonessential amino acids. Endotoxin
contamination was less than 12 pg/ml in the culture medium and FBS.
Polymyxin B was added (20 U/ml) to all the washing and culture media to
block the effect of any contaminating LPS. In some experiments,
polymyxin B was used at a higher concentration (200 U/ml) in monocyte
culture. Monocytes were cultured (1 x 106
cells/ml) for 16–18 h in the presence or absence of 20 µg/ml of MDP
+ SEB (0.5 µg/ml) or human hsp27 (2 µg/ml). Culture supernatants
were harvested and stored at -80°C until they were tested for IL-10
or TNF-. In some experiments, monocytes were also stimulated with
zymosan A (50 µg/ml), a potent inducer of monocyte IL-10 and TNF-
as an additional positive control. In some experiments, monocytes were
stimulated with rTNF- (2.5 ng/ml) alone or in combination with hsp27
(2 µg/ml). In selected experiments, hsp27 was first incubated with
-hsp27 polyclonal Ab (20 µg/ml) for 3 h before its addition
to monocyte culture or -TNF- mAb (10 µg/ml) was added, together
with hsp27, to monocyte culture. In some experiments, monocytes were
first treated with SB203580 (10 µM), or PD98059 (10 µM), or the
DMSO control (solvent used for dissolving both the reagents) for 2
h before addition of hsp27 to the culture.
RNase protection assay
A total of 2 x 106 monocytes was
stimulated in the presence or absence of MDP (20 µg/ml) + SEB (0.5
µg/ml) or hsp27 (2 µg/ml) for 8–9 h. Total cytoplasmic RNA was
isolated using Tri-reagent (Molecular Research Center, Cincinnati, OH),
according to manufacturer’s instructions. Antisense probes were
labeled with [32P]UTP (NEN Life Science
Products) using the Riboquant in vitro transcription labeling kit
(PharMingen, San Diego, CA), according to manufacturer’s instructions.
A mixture of probes, Riboquant hCK-1 (PharMingen), was used to
facilitate the simultaneous quantification of several RNA species. The
antisense probes generated using this probe set include the controls,
GAPDH and L32 and the human cytokine IL-10 and some other human
cytokines, IL-5, IL-4, IL-14, IL-15, IL-9, IL-2, IL-13, and IFN-
.
The RNase protection assays were performed using the Riboquant RNase
protection assay kit (PharMingen), according to manufacturer’s
instructions. In brief, molar excesses of labeled probes were incubated
with RNA derived from cells in hybridization buffer supplied by the
manufacturer for 16–48 h at 56°C. Hybridized samples were then
digested with 5 U of RNase A/T1 mixture for 45 min at 30°C.
Subsequent to digestion, the protected fragments were separated from
digested probe by electrophoresis on an 8 molar urea, 5%
polyacrylamide Tris borate EDTA gel. The gels were then dried, exposed
directly to film, and developed. The band intensities were quantitated
using the National Institutes of Health image software. IL-10 mRNA
levels were adjusted according to L32 and GAPDH levels (used as loading
controls).
Immunoblotting for assessment of activation (phosphorylation) of ERK 1/2, SAPK/JNK, and p38 MAPK, as well as of endogenous hsp27
Monocytes (1.5 x 106 cells) were cultured in serum-free medium for 2 h and then stimulated with hsp27 (2 µg/ml) for different time periods (1 min to 3 h). Western blot analysis was performed, essentially as described previously (29). Briefly, cells were lysed using a buffer consisting of 1% Nonidet P-40, 50 mM HEPES (pH 7.2), 100 mM NaCl, 2 mM EDTA, 1 mM pyrophosphate, 2 mM Na3VO4, 10 mM NaF, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Postnuclear supernatants were harvested after centrifugation of the lysate for 15 min at 14,000 x g at 4°C. Equal amounts of postnuclear lysates were boiled for 5 min in the presence of SDS sample buffer (reducing) and subjected to SDS-12% PAGE and then transferred to nitrocellulose membrane (Millipore, Bedford, MA) in transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% (v/v) methanol). Membranes were first rinsed in TTBS (TBS with 0.1% Tween-20) and then blocked for 1 h at room temperature in TTBS-5% w/v nonfat dry milk. The membrane was then incubated overnight at 4°C with antiphospho-p38 MAPK Ab (rabbit polyclonal; New England Biolabs) (1/1000 dilution in TTBS-1% BSA). Ab-Ag complexes were detected with the aid of HRP-conjugated anti-rabbit secondary Ab (1/2000 dilution) (New England Biolabs), followed by detection of the bands with ECL reagent (NEN Life Science Products). The same membranes were used for detection of several other proteins, such as phospho-ERK 1/2 (p44/42), phospho-SAPK/JNK, total p38 MAPK, total ERK 1/2, and total SAPK/JNK by sequential stripping of Abs, by incubation of the membrane for 30 min at 50°C in a specific buffer (2% SDS, 100 mM 2-ME, 62.5 mM Tris-HCl, pH 6.7), and then reprobing the blot with respective Ab (all Abs; New England Biolabs) using the procedure as mentioned above for the assessment of phospho-p38 MAPK.
Activation of endogenous hsp27 was assessed, as described above, for activation of the MAPKs. In brief, monocytes were activated with recombinant hsp27 (2 µg/ml) for different time periods (5–120 min). Cells were washed twice at 4°C to remove recombinant hsp27 from the culture and then lysed as above. Equal amounts of postnuclear lysates were then subjected to SDS-15% PAGE and immunoblotted with anti-phosphoserine mAb (1/500 dilution) for the detection of phospho-hsp27. Recombinant hsp27 was used as a positive control. Presence of equivalent amounts of hsp27 in the cell lysates was confirmed by stripping the blot and reprobing with anti-hsp27 mAb.
MAPKAPK-2 assay
Monocytes (1.5 x 106) were cultured
in serum-free medium for 2 h and then stimulated with MDP (20
µg/ml) + SEB (0.5 µg/ml), hsp27 (2 µg/ml), or UV (as positive
control) for 30 min. Postnuclear lysates were prepared as described
above. Protein (A + G) (20 µl of beads/sample) (Santa Cruz
Biotechnology) was first washed twice with ice-cold PBS and then the
MAPKAPK-2 assay was performed as described, using a specific kit
(Upstate Biotechnology) (10). In brief, washed protein (A
+ G) was incubated with anti-MAPKAPK-2 sheep polyclonal Ab for
1 h at 4°C. In some experiments, protein (A + G) was incubated
with sheep IgG for the Ab control. Ab-bound protein (A + G) was then
washed twice with ice-cold PBS, followed by incubation with the
postnuclear lysate sample for 2 h at 4°C in ice-cold RIPA buffer
(50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 1 mM
Na3VO4, 0.1% 2-ME, 1%
Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate,
0.1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 50 mM NaF)
with thorough mixing. The protein (A + G)-enzyme immune complex was
washed once with ice-cold RIPA buffer containing 0.5 M NaCl, and then
twice with ice-cold RIPA buffer and once with kinase assay buffer (20
mM MOPS, pH 7.2, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM
Na3VO4, 1 mM DTT). The
beads were resuspended in 10 µl of kinase assay buffer, followed by
addition of 10 µl of 1 mM hsp27 peptide sequence KKLNRTSVA (used as
substrate). Reactions were initiated by the addition of 10 µl of
[-32P]ATP (10 µCi/assay) diluted in
magnesium/ATP mixture (75 mM magnesium chloride and 500 mM ATP in
kinase assay buffer). The reaction was allowed to proceed for 30 min at
30°C before termination. This was achieved by spotting the assay
mixture onto squares of p81 paper and then placing them in 0.75%
orthophosphoric acid. The squares were washed three times in the acid
and once in acetone before scintillation counting.
Flow cytometric analysis
Cell phenotype verification in our monocyte populations was conducted using anti-CD14 mAb. IgG2b FITC was used as the isotype control. Fluorescent measurements were done on the Coulter Epics XL flow cytometer. Briefly, 5 x 105 cells were incubated with conjugated mAb or with the appropriate isotypic control for 1 h at dilutions suggested by the manufacturer. Samples were washed twice with PBS and resuspended in 500 µl PBS for fluorescent analysis.
IL-10 and TNF- ELISA
IL-10 and TNF- levels in the culture supernatants were
determined by specific ELISA kit (Endogen), according to the
instructions of the manufacturer. The sensitivity of the assay was 5
pg/ml.
Statistical analysis
Results are expressed as mean ± SEM. Statistical significance was calculated by the Student’s t test (paired) using the StatView program. Statistical significance was accepted for p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate pro- or antiinflammatory monokine induction by
hsp27, we treated human monocytes with recombinant human hsp27 and
assessed the culture supernatants for IL-10 and TNF- levels. One
recurring problem with using recombinant proteins to induce monokines
is that even picogram amounts of contaminating LPS can augment other
stimuli, confusing LPS independent and dependent monokine induction by
exogenous recombinant proteins. A combination of SEB plus MDP, rather
than LPS, was used as a positive control for induction of monokines.
MDP, or MDP + SEB, induce significant quantities of IL-10 and TNF-
in monocyte/macrophage, even in the presence of polymyxin B (28, 30). Consequently, MDP + SEB was used as a control stimuli and
20 U/ml of polymyxin B was included in all media to detect monokine
production induced exclusively by hsp27. Hsp27 induced significantly
(p = 0.0009) higher amounts of IL-10 as
compared with adherence-stimulated, untreated monocytes (10-fold
higher) or even SEB + MDP-stimulated monocytes (3- to 4-fold higher)
(Fig. 1
A). Even increasing the
added polymyxin B to 200 U/ml did not diminish monocyte IL-10 induction
by recombinant hsp27. The hsp27 induction of IL-10 protein was maximal
(
10-fold increase) at 16–18 h. MDP + SEB-induced monocyte IL-10
levels continued to slightly increase up to 40 h of culture, but
still remained 3-fold less than hsp27-induced IL-10 levels. Hsp27
increased both the kinetics of monocyte IL-10 induction as well as the
absolute levels, suggesting hsp27 induction was direct and not working
through induction of a second monokine. Combination of hsp27 with SEB +
MDP did not significantly increase IL-10 induction over hsp27 alone
(3400–3700 pg/ml), suggesting maximal monocyte IL-10 levels were
induced by hsp27. In a separate set of experiments, zymosan A, a
stimuli of IL-10, reported equal in potency to LPS, induced
x845 pg IL-10 vs x4272 pg by hsp27
(30). Hsp27-induced monocyte IL-10 production was dose
dependent, with 1–5 µg/ml being the optimum concentration (Fig. 1B). Finally, to further demonstrate the specificity of
hsp27 direct induction of monocyte IL-10, hsp27 was treated with
specific anti-hsp27 Ab for 3 h before its addition to the
monocyte cultures. Anti-hsp27 treatment, but not addition of irrelevant
Ab, could abolish the hsp27 induction of monocyte IL-10 (Fig. 1C). These findings suggest that hsp27 itself potently
induces monocyte IL-10.
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Adherence alone induces some IL-10 in human monocytes in the
absence of any other stimulants (28). Consequently, hsp27
might only be augmenting IL-10 protein translation of
adherence-stimulated, already transcribed IL-10 mRNA rather than
increasing hsp27 mRNA levels. To explore this possibility, we assessed
monocyte IL-10 mRNA expression with the RNase protection assays (Fig. 2). Hsp27 induced almost 7.2-fold
increases in mRNA levels, as compared with only adherence-stimulated
monocytes. Hsp27-induced IL-10 mRNA levels were 3.2-fold higher than
the control, MDP + SEB-induced IL-10 mRNA levels, again demonstrating
the potency of hsp27 as an IL-10 inducer.
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, while
maximally stimulating IL-10 independently of TNF-
Recently, hsp60 has been shown to induce 750 pg/ml TNF- in
Mono Mac-6, a human monocyte cell line (7). Moreover,
TNF- is a potent augmentor of IL-10 production in human monocytes
(10, 27). TNF- induction occurs before IL-10 induction
in human monocytes after LPS stimulation (31). Thus,
exogenously added hsp27 could first induce high levels of monocyte
TNF-, which only subsequently autocrine stimulated the monocytes to
produce IL-10. Maximal LPS induction of monocyte IL-10 has been
reported to require prior induction of endogenous TNF- (27, 32). Because of the rapidity of monocyte TNF- induction, it
was possible that hsp27 induction of monocyte IL-10 similarly was
dependent on TNF-, despite the observed increase in IL-10 induction
kinetics. We, therefore, assessed hsp27-induced TNF- production in
human monocytes. Hsp27 significantly (p =
0.0003) induced TNF- levels in human monocytes (Fig. 3A). However, in contrast to
hsp27’s exaggerated monocyte IL-10-inducing potential (10-fold vs
3-fold, as compared with monocyte IL-10-inducing potential of SEB +
MDP), hsp27 and SEB + MDP induced almost identical levels of monocyte
TNF- (Fig. 3A). In addition, the combination of SEB + MDP
+ hsp27 further significantly (p = 0.002)
increased monocyte TNF- production from 483 ± 74 for hsp27
alone to 1737 ± 267 pg/ml. These data are in contrast to the
failure of the same combination (SEB + MDP + hsp27) to increase
monocyte IL-10 production over maximal IL-10 production (3400 pg/ml)
induced by hsp27 alone.
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Ab, along with
hsp27, to the monocyte culture to delineate any critical role of
endogenously produced TNF- levels during hsp27-induced monocyte
IL-10 production. As can be seen in Fig. 3
B,
anti-TNF- Abs did partially (40%) inhibit hsp27-induced
IL-10 production. This anti-TNF- Ab completely inhibited
TNF--induced IL-10 production. To further test a requirement for
TNF- in the hsp27 induction of IL-10, we compared the monocyte IL-10
induction by 2500 pg of TNF alone or in combination with hsp27 to hsp27
alone. As previously reported, TNF- alone induced only minimal
monocyte IL-10 levels (10 ; Fig. 3C). As
illustrated in Fig. 3C, addition of 2500 pg/ml of exogenous
TNF-, along with 2 µg/ml hsp27, failed to augment monocyte IL-10
induction over hsp27 alone (3098 ± 660 vs 3021 ± 729 pg).
This 2500 pg of TNF- is five times the levels of TNF- induced by
hsp27 (Fig. 3A). Even addition of 10,000 pg/ml of TNF-
failed to augment the hsp27 induction of IL-10 (data not shown). As a
further examination of hsp27 potency in inducing IL-10, but its minimal
TNF- induction, we compared zymosan A induction of these two
monokines with hsp27 induction. As illustrated in Table I, zymosan A induced high levels of
monocyte TNF- (12,738 ± 3,747), but more modest levels of
IL-10 (845 ± 320), while hsp27 had the opposite pattern, inducing
exaggerated levels of IL-10 (4272 ± 217 pg/ml) vs modest TNF-
levels (301 ± 190 pg/ml). In total, these data suggest that hsp27
is an unusually potent stimuli for monocyte IL-10, but only a modest
stimuli for monocyte TNF-, and that TNF- induction is not
necessary for maximal hsp27 induction of monocyte IL-10.
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LPS-induced monokine production involves the activation of three
different MAPK, p38, p44/42 (ERK 1/2), and p46/54 (SAPK/JNK-1/2).
However, LPS induction of monocyte IL-10 requires only activation of
the p38 pathway, while TNF- induction requires both ERK and p38
(10, 22, 33). Therefore, hsp27 induction of IL-10 could
involve only the p38 pathway or both the ERK and p38 pathway if TNF-
production is required. In addition, endogenous hsp27 is itself a
substrate in the required p38 activation pathway by which LPS induces
IL-10. Consequently, the hsp27 preferential induction of monocyte IL-10
might represent just adding more hsp27 as a substrate, not increasing
the overall activation of the MAPK activation pathways. To assess
whether there was activation (phosphorylation) of these MAPKs after
hsp27 addition, human monocytes were stimulated at different time
points, and activated p38, ERK 1/2, and JNK 1/2 measured using the
respective Abs against their phosphorylated forms. As can be seen in
Fig. 4, hsp27 activated all three MAPKs
pathways, but to different degrees. Phosphorylation of ERK 1/2, JNK
1/2, as well as p38 MAPK was clearly increased at 20 min after addition
of hsp27. Maximal stimulation was observed at 40 min after hsp27
addition for all three MAPKs. However, activation of p38 MAPK persisted
up to 180 min when P-p44 ERK, as well as P-p54 and P-p46 JNK were
clearly declining (Fig. 4). In addition, hsp27 induction of P-p54 JNK
was minimal compared with its activation of p38 and ERK. Activation of
MAPKAPK-2 (a substrate of p38 MAPK) has been shown as necessary to LPS
induction of IL-10 in human monocytes (10). Therefore, we
also assessed the activation of MAPKAPK-2 during hsp27-induced
activation and IL-10 production of human monocytes by in vitro kinase
assay, using a sequence of hsp27 (KKLNRTSVA) as the substrate. As can
be seen in Fig. 5A, the
immunoprecipitate (using -MAPKAPK-2 sheep polyclonal Ab; control
sheep IgG had no MAPKAPK-2 activity) from hsp27-activated monocyte
lysate had significantly increased MAPKAPK-2 activity vs that from
adherence-stimulated untreated monocyte lysate, suggesting that
exogenously added hsp27 could activate (phosphorylate) human monocyte
endogenous hsp27. To test this possibility, we stimulated human
monocytes at different time points with recombinant hsp27 and measured
activated (phosphorylated at serine residues) endogenous hsp27 in the
cell lysates by Western blotting, using an Ab against phosphoserine.
The presence of equivalent amounts of hsp27 in the cell lysates was
tested by reprobing the stripped blot with anti-hsp27 Ab. As can be
seen in Fig. 5B, exogenous hsp27 addition could activate
monocyte endogenous hsp27. Moreover, just as hsp27 induced prolonged
p38 activation (Fig. 4), endogenous monocyte hsp27 remained activated
for prolonged periods (up to 2 h tested) after addition of
exogenous recombinant hsp27. These data suggest that hsp27 is a
potent inducer of IL-10 in human monocytes because it
differentially activates the MAPK pathways that play critical roles in
inducing monokine production. The next sets of experiments
examined which of the different MAPKs had critical roles in
hsp27-induced IL-10 and TNF- production by monocytes.
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Although the p38 MAPK pathway is critical in LPS induction of
IL-10, the differential involvement of MAPK pathways in cytokine
production has been shown to be stimulus dependent and might contribute
to hsp27 preferential induction of IL-10 (10, 21, 33, 34).
To assess any essential role of differential MAPK activation for
induction of monocyte IL-10 or TNF- by hsp27, we added different
MAPK inhibitors to the monocyte culture before addition of hsp27.
SB203580 was used to block the effect of p38 MAPK, whereas PD98059 was
used to inhibit the effect of MEK 1/2 (the enzyme responsible for
activation of ERK 1/2) (10, 33). SB203580 could
significantly (p = 0.002) block hsp27-induced
IL-10 production (Fig. 6). Monocyte IL-10
production was inhibited 80% by SB203580, which also blocked 90%
of the TNF- activity induced by hsp27, indicating a potential
critical role of p38 MAPK pathway during induction of both monocyte
IL-10 and TNF- production by hsp27. However, even in the presence of
SB203580, hsp27 induced a small amount of IL-10, which was still
significantly (p = 0.002) increased over that
of adherence only-stimulated monocytes (Fig. 6). In addition, the
SB203580 treatment had no effect on the hsp27 induction of ERK
activation in these monocytes, indicating that the inhibition of
hsp27-induced monocyte IL-10 was not a result of general loss of
monocyte viability, nor did the DMSO control affect hsp27-induced
monocyte IL-10 or TNF- production (data not shown). In contrast to
the inhibitory effects of SB203580, PD98059 had no inhibitory effect on
hsp27-induced monocyte IL-10 production, but blocked 68% of the
TNF- induced by hsp27 (Fig. 6). These data suggest that activation
of the ERK 1/2 pathway is not required for induction of monocyte IL-10
by hsp27, but that both the ERK 1/2 and p38 pathways are involved in
hsp27 induction of monocyte TNF-. These data further support that
monocyte TNF- production induced by hsp27 is not required for
maximal IL-10 production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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activation of the p38 MAPK pathway
and is seen as a circulating protein that can eventually induce Abs in
breast cancer and cystic fibrosis patients with arthritis (23, 36). Increased hsp27 expression has also been suggested as
protecting from TNF--mediated apoptosis (15). Induction
of hsp27 has been reported to result in protection from lethal
endotoxin shock and to be highly induced in monocytes from patients
with systemic inflammatory shock syndrome, supporting a possible
antiinflammatory role for hsp27 (16, 17). Hsp60, one of
the large hsp, can induce monocyte TNF-, and TNF- induction
augments monocyte IL-10 levels (7, 32). These
data, combined with those emphasizing the pivotal role of MAPKAPK-2,
the kinase that phosphorylates hsp27, led us to investigate exogenous
hsp27 as a stimuli for monocyte IL-10 or TNF-.
Hsp27 addition to primary human monocyte cultures induced exaggerated
IL-10 production (3400 pg/ml). In contrast, LPS-stimulated IL-10
levels reported for primary human monocytes range from 500-1500 pg/ml,
similar to our SEB + MDP- and zymosan A-induced monocyte IL-10 levels
of 800–1100 pg/ml (30, 31, 41). However, LPS
simultaneously stimulates the same human monocytes to produce from
6,000 to 15,000 pg of TNF- (30, 31, 41). In our
polymyxin B-containing system, zymosan A similarly induced high levels
of TNF- (13,000 pg/ml), but only 800–900 pg of IL-10. Hsp27
induced 300–500 pg/ml of monocyte TNF-, somewhat similar to the
reported 750 pg/ml induced by human hsp60 in the human Mono Mac-6 cell
line (7). Hsp27’s exaggerated stimulation of human
monocyte IL-10 vs its modest induction of TNF- is further
illustrated by our data showing that combining SEB + MDP + hsp27
induced no further increase in IL-10 levels, but augmented monocyte
TNF- levels more than 3-fold. Similarly, TNF- addition did not
further increase hsp27-induced IL-10 levels. These data imply that
hsp27 alone induced maximal monocyte IL-10, but only minimal TNF-
levels, and that hsp27 may preferentially activate human monocyte IL-10
production. Increased production of IL-10 by APC has been suggested to
polarize the immune responses to Th2 (42). The paradigm
that bacterial hsp may pivotally act as stimulating danger signals
whose immune recognition is highly conserved, while autologous Hsp
could have preferential immune regulatory activity has been previously
postulated (6, 7, 43, 44). Our data showing recombinant
human hsp27 to preferentially induce antiinflammatory IL-10 rather than
proinflammatory TNF- in human monocytes are consistent with this
paradigm.
The mechanism(s) behind hsp27 preferential induction of monocyte IL-10
may involve preferential activation of the MAPK p38 pathway. Previous
reports demonstrated that the p38 but not the ERK pathway was required
for LPS induction of IL-10 (10). However, LPS induction of
maximal monocyte IL-10 appears also to be pivotally dependent on its
induction of TNF- and/or IL-1 (10, 32). LPS-stimulated
maximal IL-10 production by monocytes is consequently delayed, peaking
at 24 h after LPS stimuli and continuing for 96 h
(27). In contrast, hsp27-induced monocyte IL-10 levels
were maximal at 18 h, and anti-TNF- mAb only inhibited 40%
of hsp27-induced IL-10 levels in our experiments vs a reported 70%
inhibition of LPS-induced IL-10 (32). These data suggest
that exogenous human hsp27 directly stimulates human monocytes to
produce IL-10. Inhibition of the p38 MAPK pathway with SB203580 reduced
our hsp27-induced monocyte IL-10 levels by 80%, while the ERK
inhibitor, PD98059, which inhibited TNF- production almost 70%, had
no effect on maximal IL-10 production. The significant level of
hsp27-induced IL-10 generated even in the face of MAPKAPK-2 inhibition
by SB203580 might result from other pathways of hsp27 phosphorylation
since activated PKC-
has been also demonstrated to directly
phosphorylate hsp27 (20). Alternatively, the SB203580 may
only be capable of inhibiting 80% of MAPKAPK-2 activity because of
prior adherence-induced activation. The exaggerated monocyte IL-10
levels induced by hsp27 could result from the persistence of
phosphorylated p38 (P-p38) in the hsp27-induced monocytes. Similar to
LPS-induced human monocyte activation data, levels of phosphorylated
ERK p42/p44, JNK p46/p54, and p38 peaked at 40-min post-hsp27
stimulation, then both ERK p42/p44 and JNK p46/p54 declined
(33). In contrast to the published data for LPS induction
of human monocyte MAPK pathways, hsp27-induced P-p38 persisted even at
180 min (33). Since P-p38 activation of MAPKAPK-2 is
required for monocyte IL-10 induction, this increased persistence of
P-p38 may partially explain both the higher levels of MAPKAPK-2
induced by hsp27 (2-fold) vs SEB + MDP (1.3-fold) and hsp27’s
exaggerated induction of IL-10. Phosphorylation of endogenous hsp27 was
also prolonged in hsp27-induced monocytes. Alternatively or
additionally, the rapid disappearance of JNK p46/p54 after hsp27
stimulation might be allowing continued monocyte IL-10 production. A
critical down-regulatory role for activation of the JNK pathway has
been implicated for IL-10 production, since JNK
1-/- Th cells produced
exaggerated IL-10 in response to CD3 induction (45). Our
data suggested that JNK was only minimally activated by hsp27. The
importance of this minimal JNK pathway activation in hsp27 induction of
exaggerated monocyte IL-10 needs to be further explored, utilizing JNK
inhibitors. However, JNK inhibitors are not yet commercially
available.
The ability of exogenous hsp27 to differentially stimulate MAPK pathway activation and distinctive cytokine production might imply that it is binding to a monocyte surface receptor rather than nonspecifically internalized by pinocytosis. The different stimulatory capacity of autologous vs microbial hsp, despite their sequence homology, along with their surface binding of peptides, has led several investigators to propose a putative hsp receptor (7, 8, 35). The receptor for advanced glycation end products (RAGE) has been proposed as a likely candidate for an hsp receptor (8). The RAGE levels on monocytes are up-regulated in diabetics (46), a disease in which large hsp have been shown to have immunoregulatory activity (47) and RAGE activation induces MAPK signaling pathways (48). Most of the hypotheses developed and the immune regulation investigations performed involved the large hsp. As far as we could determine, our study represents the first described data of cytokine-inducing activities for exogenous hsp27 and is consistent with differential monocyte activation through an hsp27 receptor.
In summary, we have demonstrated a novel role for exogenous human hsp27
in preferentially inducing human monocytes to produce IL-10 by
activating the p38 MAPK, MAPKAPK-2, and endogenous hsp27 in a prolonged
fashion. Exogenous hsp27 was able to induce monocyte IL-10 mRNA
increases as well as protein increases. Increased IL-10 induction may
be responsible for the reported ability of hsp27 to down-regulate
TNF--mediated apoptosis and ROI induction (3, 15).
Whether our demonstration of hsp27-mediated antiinflammatory function
is limited to human monocytes or is a unique property of autologous
hsp27 vs large hsp remains to be investigated. The ability of hsp27 to
induce a predominant in vitro antiinflammatory monokine profile could
imply that some autologous small hsp serve as antiinflammatory agents
in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carol L. Miller-Graziano, Department of Surgery, University of Massachusetts Medical Center, 55 Lake Avenue North, Room S3-716, Worcester, MA 01655.
3 Abbreviations used in this paper: hsp, heat shock protein; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase-2; MDP, muramyl dipeptide; RAGE, receptor for advanced glycation end products; ROI, reactive oxygen intermediate; SAPK, stress-activated protein kinase; SEB, staphylococcal enterotoxin B.
Received for publication July 16, 1999. Accepted for publication July 7, 2000.
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