Exaggerated Human Monocyte IL-10 Concomitant to Minimal TNF- α Induction by Heat-Shock Protein 27 (Hsp27) Suggests Hsp27 Is Primarily an Antiinflammatory This information is current as Stimulus of September 24, 2021. Asit K. De, Karen M. Kodys, Berhan S. Yeh and Carol Miller-Graziano J Immunol 2000; 165:3951-3958; ; doi: 10.4049/jimmunol.165.7.3951 Downloaded from http://www.jimmunol.org/content/165/7/3951

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Exaggerated Human Monocyte IL-10 Concomitant to Minimal TNF-␣ Induction by Heat-Shock Protein 27 (Hsp27) Suggests Hsp27 Is Primarily an Antiinflammatory Stimulus1

Asit K. De, Karen M. Kodys, Berhan S. Yeh, and Carol Miller-Graziano2

Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-␣, 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 Downloaded from 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 http://www.jimmunol.org/ 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. The Journal of Immunology, 2000, 165: 3951–3958.

ystemic inflammatory responses, as well as exaggerated also been shown to induce TNF-␣ in a human monocyte cell line local inflammatory cytokine production, have been impli- and TNF-␣, as well as IL-15 and IL-12, in murine bone marrow- S cated in mediating multiple organ failure and rheumatoid derived macrophage (7, 13). arthritis (1, 2). During shock inflammatory stress, heat shock pro- Hsp27, an important member of the small hsp family, has been teins (hsp),3 which are stress response proteins found in all species, investigated primarily for its role as a circulating protein marker of by guest on September 24, 2021 are up-regulated (3–5). These hsp are thought to play a pivotal role increased malignancy in breast cancer (14). Hsp27 has been shown in protecting cells during stress and inflammatory responses (3–6). to down-regulate reactive oxygen intermediate (ROI) production, Recently, the large hsp have also been suggested as danger signals thereby protecting from TNF-␣-mediated apoptosis (15). The glu- that first activate monokine production, then stimulate and/or reg- tamine induction of rat hsp25, the analogue of human hsp27, has ulate the magnitude of the immune response (7, 8). Immunization been shown to correlate with protection from lethal endotoxin of mice with hsp65 protects against pristane-induced arthritis by shock (16). Human monocytes from patients with systemic inflam- inducing IL-10- and IL-4-producing CD4 T cells (9). Both IL-4 matory response syndrome have significantly elevated hsp27 ex- and IL-10 are potent down-regulators of monocyte production of pression (17). These data suggest that exogenous hsp27 may also proinflammatory mediators, such as TNF-␣, IL-8, IL-1, and PGE 2 have some antiinflammatory or immune modulatory capacities on (10–12). These data suggest that some large autologous hsp may monocytes. IL-10 can also down-regulate ROI activity in mono- stimulate antiinflammatory cytokine activity. This antiinflamma- cytes and macrophages, but, unlike the large hsp, hsp27 has not tory function of hsp is controversial, however, because hsp60 has been previously shown to exogenously induce production of either pro- or antiinflammatory cytokines (18, 19). However, hsp27 is a Department of Surgery, University of Massachusetts Medical School, Worcester, substrate for mitogen-activated protein kinase (MAPK)-activated MA 01655 protein kinase-2 (MAPKAPK-2), an important member of the p38 Received for publication July 16, 1999. Accepted for publication July 7, 2000. MAPK cascade that is both activated by cytokine treatment and The costs of publication of this article were defrayed in part by the payment of page critical in monocyte production of cytokines (10, 20–22). Re- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cently, the activation (phosphorylation) of p38 MAPK and its sub- 1 This work was supported by Public Health Service Grant GM36214-13. Its contents strate, MAPKAPK-2, has been shown to be crucial to LPS induc- are solely the responsibility of the authors and do not necessarily represent the official tion of IL-10 in human monocytes, further suggesting that hsp27 views of the National Institutes of Health. could play an antiinflammatory role in monocytes (10). Circulating 2 Address correspondence and reprint requests to Dr. Carol L. Miller-Graziano, De- hsp27 is present in the serum of cancer patients and, in some cases, partment of Surgery, University of Massachusetts Medical Center, 55 Lake Avenue North, Room S3-716, Worcester, MA 01655. induces in vivo hsp27 Ab production, suggesting that hsp27 can 3 Abbreviations used in this paper: hsp, heat shock protein; ERK, extracellular signal- stimulate as an exogenous protein (23, 24). Phosphorylated hsp27 related kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein ki- has also been identified as being associated with cell membranes nase; MAPKAPK-2, MAPK-activated protein kinase-2; MDP, muramyl dipeptide; RAGE, receptor for advanced glycation end products; ROI, reactive oxygen interme- of lamellipodia in migrating cells, suggesting a possible hsp27 diate; SAPK, stress-activated protein kinase; SEB, staphylococcal enterotoxin B. surface expression (25). Although hsp27 phosphorylation after

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 3952 Hsp27 INDUCES IL-10 IN HUMAN MONOCYTES

MAPKAPK-2 activation is necessary for LPS induction of mono- monocytes were also stimulated with zymosan A (50 ␮g/ml), a potent inducer cyte IL-10, the effect of exogenous hsp27 on increasing production of monocyte IL-10 and TNF-␣ as an additional positive control. In some ex- ␣ of IL-10 or any monokine is unexplored. Administration of IL-10 periments, monocytes were stimulated with rTNF- (2.5 ng/ml) alone or in combination with hsp27 (2 ␮g/ml). In selected experiments, hsp27 was first has been shown to suppress lethal endotoxemia and reduce serum incubated with ␣-hsp27 polyclonal Ab (20 ␮g/ml) for 3 h before its addition TNF-␣ levels (26). Because of its antiinflammatory properties, to monocyte culture or ␣-TNF-␣ mAb (10 ␮g/ml) was added, together with IL-10 has been suggested as a possible therapeutic agent for in- hsp27, to monocyte culture. In some experiments, monocytes were first treated flammatory conditions, such as rheumatoid arthritis and inflam- with SB203580 (10 ␮M), or PD98059 (10 ␮M), or the DMSO control (solvent used for dissolving both the reagents) for 2 h before addition of hsp27 to the matory bowel disease (26). Consequently, any monocyte-modu- culture. lating activity of hsp27 in increasing IL-10 levels without concomitantly highly inducing proinflammatory monokines such RNase protection assay as TNF-␣ could also have therapeutic implications. A total of 2 ϫ 106 monocytes was stimulated in the presence or absence of In this study, hsp27 has been assessed for a novel ability to MDP (20 ␮g/ml) ϩ SEB (0.5 ␮g/ml) or hsp27 (2 ␮g/ml) for 8–9 h. Total induce IL-10 and/or TNF-␣ in human monocytes when added ex- cytoplasmic RNA was isolated using Tri-reagent (Molecular Research ogenously. We demonstrate that human hsp27 is a potent inducer Center, Cincinnati, OH), according to manufacturer’s instructions. Anti- 32 of IL-10 in human monocytes, but only a modest inducer of sense probes were labeled with [ P]UTP (NEN Life Science Products) ␣ using the Riboquant in vitro transcription labeling kit (PharMingen, San TNF- . We have also explored the preferential involvement of Diego, CA), according to manufacturer’s instructions. A mixture of probes, different MAPK pathways during hsp27-induced monocyte IL-10 Riboquant hCK-1 (PharMingen), was used to facilitate the simultaneous production to determine whether hsp27 induced IL-10 via the same quantification of several RNA species. The antisense probes generated us- p38, MAPKAPK-2, hsp27 pathway as LPS stimulation. Although ing this probe set include the controls, GAPDH and L32 and the human Downloaded from modest levels of TNF-␣ were induced by hsp27, only a small 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 portion of the monocyte hsp27 induction of IL-10 was due to its using the Riboquant RNase protection assay kit (PharMingen), according prior induction of TNF-␣, a known enhancer of IL-10 in mono- to manufacturer’s instructions. In brief, molar excesses of labeled probes cytes (10, 27). Hsp27 independently induced high levels of mono- were incubated with RNA derived from cells in hybridization buffer sup- cyte IL-10, while concomitantly stimulating only minimal levels of plied by the manufacturer for 16–48 h at 56°C. Hybridized samples were then digested with5UofRNase A/T1 mixture for 45 min at 30°C. Sub-

␣ http://www.jimmunol.org/ TNF- . Hsp27 induction of monocyte IL-10 is totally dependent sequent to digestion, the protected fragments were separated from digested on the activation of the p38 MAPK pathway and, unlike monocyte probe by electrophoresis on an 8 molar urea, 5% polyacrylamide Tris bo- TNF-␣ production, independent of the ERK pathway activation, rate EDTA gel. The gels were then dried, exposed directly to film, and further supporting a TNF-␣-independent IL-10 induction by developed. The band intensities were quantitated using the National Insti- hsp27. tutes of Health image software. IL-10 mRNA levels were adjusted accord- ing to L32 and GAPDH levels (used as loading controls).

Materials and Methods Immunoblotting for assessment of activation (phosphorylation) Reagents of ERK 1/2, SAPK/JNK, and p38 MAPK, as well as of FBS was purchased from Sigma (St. Louis, MO). Culture media and other endogenous hsp27 supplements were purchased from Irvine Scientific (Santa Ana, CA). Mu- by guest on September 24, 2021 Monocytes (1.5 ϫ 106 cells) were cultured in serum-free medium for 2 h ramyl dipeptide (MDP) was generously provided by CIBA-GEIGY (Basel, and then stimulated with hsp27 (2 ␮g/ml) for different time periods (1 min Switzerland). Staphylococcal enterotoxin B (SEB) and zymosan A were to 3 h). Western blot analysis was performed, essentially as described pre- purchased from Sigma, and polymyxin B was purchased from Calbiochem viously (29). Briefly, cells were lysed using a buffer consisting of 1% (La Jolla, CA). The mAbs, My4 (CD14) FITC and IgG2b FITC were Nonidet P-40, 50 mM HEPES (pH 7.2), 100 mM NaCl, 2 mM EDTA, purchased from Coulter (Hialeah, FL). Recombinant human hsp27 was 1 mM pyrophosphate, 2 mM Na VO , 10 mM NaF, 1 mM PMSF, 10 purchased from Stressgen Biotechnologies (Victoria, Canada). Polyclonal 3 4 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin. Postnuclear supernatants were Ab against hsp27 was purchased from Santa Cruz Biotechnology (Santa harvested after centrifugation of the lysate for 15 min at 14,000 ϫ g at 4°C. Cruz, CA), mAb against hsp27 from Stressgen Biotechnologies, and mAb Equal amounts of postnuclear lysates were boiled for 5 min in the presence against TNF-␣ from Endogen (Woburn, MA). SB203580 and PD98059 of SDS sample buffer (reducing) and subjected to SDS-12% PAGE and were purchased from Calbiochem (San Diego, CA). Human rTNF-␣ was then transferred to nitrocellulose membrane (Millipore, Bedford, MA) in kindly provided by The National Institute for Biological Standards and transfer buffer (25 mM Tris, 192 mM glycine, pH 8.3, 20% (v/v) metha- Control (Potters Bar, U.K.). Phosphoplus p38 MAPK, p44/42 (ERK 1/2) nol). Membranes were first rinsed in TTBS (TBS with 0.1% Tween-20) MAPK, and stress-activated protein kinase (SAPK)/c-Jun N-terminal and then blocked for1hatroom temperature in TTBS-5% w/v nonfat dry kinase (JNK) kits were purchased from New England Biolabs (Beverly, milk. The membrane was then incubated overnight at 4°C with antiphos- MA). Anti-phosphoserine mAb was purchased from Calbiochem. pho-p38 MAPK Ab (rabbit polyclonal; New England Biolabs) (1/1000 MAPKAPK-2 IP-kinase assay kit was purchased from Upstate Biotech- dilution in TTBS-1% BSA). Ab-Ag complexes were detected with the aid nology (Lake Placid, NY). 32P and ECL reagents were purchased from of HRP-conjugated anti-rabbit secondary Ab (1/2000 dilution) (New En- NEN Life Science Products (Boston, MA). gland Biolabs), followed by detection of the bands with ECL reagent (NEN Separation of monocytes and stimulations Life Science Products). The same membranes were used for detection of several other proteins, such as phospho-ERK 1/2 (p44/42), phospho- PBMC were first isolated from venous blood of healthy volunteers by SAPK/JNK, total p38 MAPK, total ERK 1/2, and total SAPK/JNK by Ficoll-Hypaque density centrifugation. Monocytes were separated from sequential stripping of Abs, by incubation of the membrane for 30 min at PBMC by selective adherence to microexudate-coated plastic surfaces, as 50°C in a specific buffer (2% SDS, 100 mM 2-ME, 62.5 mM Tris-HCl, described (28). Adherent monocytes (Ͼ95% purity, as checked by flow- pH 6.7), and then reprobing the blot with respective Ab (all Abs; New cytometric analysis) were collected by treatment with 10 mM EDTA, England Biolabs) using the procedure as mentioned above for the as- suspended in IMDM medium, supplemented with 10% FBS, 50 U/ml sessment of phospho-p38 MAPK. penicillin G, 50 ␮g/ml streptomycin, 50 ␮g/ml gentamicin, 2.5 ␮g/ml Activation of endogenous hsp27 was assessed, as described above, for fungizone, 4 mM L-glutamine, 1 mM sodium pyruvate, and 1% MEM activation of the MAPKs. In brief, monocytes were activated with recom- nonessential amino acids. Endotoxin contamination was less than 12 pg/ml in binant hsp27 (2 ␮g/ml) for different time periods (5–120 min). Cells were the culture medium and FBS. Polymyxin B was added (20 U/ml) to all the washed twice at 4°C to remove recombinant hsp27 from the culture and washing and culture media to block the effect of any contaminating LPS. In then lysed as above. Equal amounts of postnuclear lysates were then sub- some experiments, polymyxin B was used at a higher concentration (200 jected to SDS-15% PAGE and immunoblotted with anti-phosphoserine U/ml) in monocyte culture. Monocytes were cultured (1 ϫ 106 cells/ml) for mAb (1/500 dilution) for the detection of phospho-hsp27. Recombinant 16–18 h in the presence or absence of 20 ␮g/ml of MDP ϩ SEB (0.5 ␮g/ml) hsp27 was used as a positive control. Presence of equivalent amounts of or human hsp27 (2 ␮g/ml). Culture supernatants were harvested and stored at hsp27 in the cell lysates was confirmed by stripping the blot and reprobing Ϫ80°C until they were tested for IL-10 or TNF-␣. In some experiments, with anti-hsp27 mAb. The Journal of Immunology 3953

MAPKAPK-2 assay Monocytes (1.5 ϫ 106) were cultured in serum-free medium for2hand then stimulated with MDP (20 ␮g/ml) ϩ SEB (0.5 ␮g/ml), hsp27 (2 ␮g/ ml), or UV (as positive control) for 30 min. Postnuclear lysates were pre- pared as described above. Protein (A ϩ G) (20 ␮l 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 ϩ G) was in- cubated with anti-MAPKAPK-2 sheep polyclonal Ab for1hat4°C. In some experiments, protein (A ϩ G) was incubated with sheep IgG for the Ab control. Ab-bound protein (A ϩ 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 so- dium pyrophosphate, 10 mM sodium glycerophosphate, 0.1 mM PMSF, 1 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, and 50 mM NaF) with thorough mix- ing. The protein (A ϩ 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 -glycerol phosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM DTT). The beads were resuspended in 10 ␮l of kinase assay buffer, followed by ad- ␮

dition of 10 l of 1 mM hsp27 peptide sequence KKLNRTSVA (used as Downloaded from substrate). Reactions were initiated by the addition of 10 ␮lof[␥-32P]ATP (10 ␮Ci/assay) diluted in magnesium/ATP mixture (75 mM magnesium chloride and 500 mM ATP in kinase assay buffer). The reaction was al- lowed 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. http://www.jimmunol.org/ Flow cytometric analysis Cell phenotype verification in our monocyte populations was conducted using anti-CD14 mAb. IgG2b FITC was used as the isotype control. Flu- orescent measurements were done on the Coulter Epics XL flow cytometer. Briefly, 5 ϫ 105 cells were incubated with conjugated mAb or with the appropriate isotypic control for1hatdilutions suggested by the manufac- ␮ turer. Samples were washed twice with PBS and resuspended in 500 l FIGURE 1. Hsp27 induces IL-10 in human monocytes independently. PBS for fluorescent analysis. A, Human monocytes were cultured (1 ϫ 106 cells/ml) for 16–18 h in the ␣ presence or absence of MDP (20 ␮g/ml) ϩ SEB (0.5 ␮g/ml) or recombi- IL-10 and TNF- ELISA by guest on September 24, 2021 nant human hsp27 (2 ␮g/ml). IL-10 levels in the culture supernatants were IL-10 and TNF-␣ levels in the culture supernatants were determined by tested by ELISA. Data are expressed as mean Ϯ SEM. Representative of ϭ ءء ϭ ء -specific ELISA kit (Endogen), according to the instructions of the manu facturer. The sensitivity of the assay was 5 pg/ml. seven experiments. , p 0.0001 and , p 0.0009, as compared with only adherence-stimulated monocyte IL-10 levels. B, Human monocytes Statistical analysis were cultured as above in the presence of different concentrations of hsp27 Ϯ and then tested for IL-10 levels in the culture supernatants. Representative Results are expressed as mean SEM. Statistical significance was calcu- ␮ lated by the Student’s t test (paired) using the StatView program. Statistical of three experiments. C, Hsp27 was incubated with anti-hsp27 (20 g/ml, significance was accepted for p Ͻ 0.05. final concentration) for 3 h before its addition to the monocyte culture. p ϭ 0.016 as compared with only ,ءءء .Representative of three experiments Results hsp27-induced monocyte IL-10 levels. Hsp27 induces high levels of IL-10 in human monocytes independent of LPS augmentation The hsp27 induction of IL-10 protein was maximal (Ϸ10-fold in- To investigate pro- or antiinflammatory monokine induction by crease) at 16–18 h. MDP ϩ SEB-induced monocyte IL-10 levels hsp27, we treated human monocytes with recombinant human continued to slightly increase up to 40 h of culture, but still re- hsp27 and assessed the culture supernatants for IL-10 and TNF-␣ mained 3-fold less than hsp27-induced IL-10 levels. Hsp27 in- levels. One recurring problem with using recombinant proteins to creased both the kinetics of monocyte IL-10 induction as well as induce monokines is that even picogram amounts of contaminating the absolute levels, suggesting hsp27 induction was direct and not LPS can augment other stimuli, confusing LPS independent and working through induction of a second monokine. Combination of dependent monokine induction by exogenous recombinant pro- hsp27 with SEB ϩ MDP did not significantly increase IL-10 in- teins. A combination of SEB plus MDP, rather than LPS, was used duction over hsp27 alone (Ϸ3400–3700 pg/ml), suggesting max- as a positive control for induction of monokines. MDP, or MDP ϩ imal monocyte IL-10 levels were induced by hsp27. In a separate SEB, induce significant quantities of IL-10 and TNF-␣ in mono- set of experiments, zymosan A, a stimuli of IL-10, reported equal cyte/macrophage, even in the presence of polymyxin B (28, 30). in potency to LPS, induced ϫ845 pg IL-10 vs ϫ4272 pg by hsp27 Consequently, MDP ϩ SEB was used as a control stimuli and 20 (30). Hsp27-induced monocyte IL-10 production was dose depen- U/ml of polymyxin B was included in all media to detect mono- dent, with 1–5 ␮g/ml being the optimum concentration (Fig. 1B). kine production induced exclusively by hsp27. Hsp27 induced sig- Finally, to further demonstrate the specificity of hsp27 direct in- nificantly ( p ϭ 0.0009) higher amounts of IL-10 as compared with duction of monocyte IL-10, hsp27 was treated with specific anti- adherence-stimulated, untreated monocytes (10-fold higher) or hsp27 Ab for 3 h before its addition to the monocyte cultures. even SEB ϩ MDP-stimulated monocytes (3- to 4-fold higher) Anti-hsp27 treatment, but not addition of irrelevant Ab, could (Fig. 1A). Even increasing the added polymyxin B to 200 U/ml did abolish the hsp27 induction of monocyte IL-10 (Fig. 1C). These not diminish monocyte IL-10 induction by recombinant hsp27. findings suggest that hsp27 itself potently induces monocyte IL-10. 3954 Hsp27 INDUCES IL-10 IN HUMAN MONOCYTES

Hsp27 induces monocyte IL-10 at the mRNA levels 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-stimu- lated, 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 as- says (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 ϩ SEB-induced IL-10 mRNA levels, again demon- strating the potency of hsp27 as an IL-10 inducer.

Hsp27 induces only modest levels of monocyte TNF-␣, while maximally stimulating IL-10 independently of TNF-␣ Recently, hsp60 has been shown to induce Ϸ750 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 Downloaded from (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 re- ported to require prior induction of endogenous TNF-␣ (27, 32). Because of the rapidity of monocyte TNF-␣ induction, it was pos- http://www.jimmunol.org/ sible that hsp27 induction of monocyte IL-10 similarly was de- pendent on TNF-␣, despite the observed increase in IL-10 induc- tion kinetics. We, therefore, assessed hsp27-induced TNF-␣ production in human monocytes. Hsp27 significantly ( p ϭ 0.0003) induced TNF-␣ levels in human monocytes (Fig. 3A). However, in contrast to hsp27’s exaggerated monocyte IL-10-inducing poten- tial (10-fold vs 3-fold, as compared with monocyte IL-10-inducing ϩ ϩ FIGURE 3. Hsp27 induces TNF-␣ in human monocytes, but hsp27-in- potential of SEB MDP), hsp27 and SEB MDP induced almost ␣ by guest on September 24, 2021 ␣ duced monocyte IL-10 production can be independent of TNF- . A, Hu- identical levels of monocyte TNF- (Fig. 3A). In addition, the ϫ 6 ϩ ϩ ϭ man monocytes were cultured (1 10 cells/ml) for 16–18 h in the combination of SEB MDP hsp27 further significantly ( p presence or absence of MDP (20 ␮g/ml) ϩ SEB (0.5 ␮g/ml) or hsp27 ␣ Ϯ 0.002) increased monocyte TNF- production from 483 74 for (2 ␮g/ml). TNF-␣ levels in the culture supernatants were tested by ELISA. hsp27 alone to 1737 Ϯ 267 pg/ml. These data are in contrast to the Data are expressed as mean Ϯ SEM. Representative of seven experiments. -p ϭ 0.0003, as compared with only adherence ,ءء p ϭ 0.009 and ,ء -failure of the same combination (SEB ϩ MDP ϩ hsp27) to in stimulated monocyte TNF-␣ levels. B, Human monocytes were cultured as above in the presence of hsp27 alone or in combination with anti-TNF-␣ Ab (10 ␮g/ml) and then tested for IL-10 levels in the culture supernatants. p ϭ 0.03 as compared with hsp27- ,ءءء .Representative of five experiments induced IL-10 levels. C, Human monocytes were cultured as above in the presence of hsp27, TNF-␣ (2.5 ng/ml), or their combination and then tested for IL-10 levels in the culture supernatants. Representative of three experiments.

crease monocyte IL-10 production over maximal IL-10 production (Ϸ3400 pg/ml) induced by hsp27 alone. In the next experiments, we added anti-TNF-␣ Ab, along with hsp27, to the monocyte culture to delineate any critical role of endogenously produced TNF-␣ levels during hsp27-induced FIGURE 2. Hsp27 increases IL-10 mRNA in human monocytes. A total monocyte IL-10 production. As can be seen in Fig. 3B, anti-TNF-␣ of 2 ϫ 106 monocytes was stimulated in the presence or absence of MDP Abs did partially (Ϸ40%) inhibit hsp27-induced IL-10 production. (20 ␮g/ml) ϩ SEB (0.5 ␮g/ml) or hsp27 (2 ␮g/ml) for 8–9 h and then total This anti-TNF-␣ Ab completely inhibited TNF-␣-induced IL-10 cytoplasmic RNA was isolated. Multiprobe RNase protection assay was production. To further test a requirement for TNF-␣ in the hsp27 performed to measure the mRNA levels for IL-10 and also L32 and induction of IL-10, we compared the monocyte IL-10 induction by GAPDH (loading controls). Equivalent amounts of RNA were treated with 2500 pg of TNF alone or in combination with hsp27 to hsp27 [32P]UTP-labeled Riboquant hck-1 probe mixture and then digested with alone. As previously reported, TNF-␣ alone induced only minimal RNase A/T1 mixture. The protected fragments were then analyzed by elec- trophoresis on an 8 molar urea 5% polyacrylamide Tris borate EDTA gel, monocyte IL-10 levels (10; Fig. 3C). As illustrated in Fig. 3C, followed by drying of the gel and autoradiography. The gel was exposed addition of 2500 pg/ml of exogenous TNF-␣, along with 2 ␮g/ml for 6 h (for IL-10 bands, upper panel) and 1 h (for L32 and GAPDH bands, hsp27, failed to augment monocyte IL-10 induction over hsp27 lower panel). Representative of three experiments. alone (3098 Ϯ 660 vs 3021 Ϯ 729 pg). This 2500 pg of TNF-␣ is The Journal of Immunology 3955

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-␣ induc- tion, 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 Ϯ 3,747), but more mod- est levels of IL-10 (845 Ϯ 320), while hsp27 had the opposite pattern, inducing exaggerated levels of IL-10 (4272 Ϯ 217 pg/ml) vs modest TNF-␣ levels (301 Ϯ 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.

Exogenous hsp27 induction of monocyte IL-10 is dependent on sustained p38 activation and can activate endogenous hsp27 LPS-induced monokine production involves the activation of three different MAPK, p38, p44/42 (ERK 1/2), and p46/54 (SAPK/JNK- Downloaded from 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, endog- enous hsp27 is itself a substrate in the required p38 activation http://www.jimmunol.org/ pathway by which LPS induces IL-10. Consequently, the hsp27 preferential induction of monocyte IL-10 might represent just add- ing 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, FIGURE 4. Activation (phosphorylation) of different MAPK pathways human monocytes were stimulated at different time points, and in human monocytes by hsp27. A total of 1.5 ϫ 106 monocytes was cul- activated p38, ERK 1/2, and JNK 1/2 measured using the respec- tured for2hinserum-free medium, followed by stimulation with hsp27 tive Abs against their phosphorylated forms. As can be seen in Fig. (2 ␮g/ml) for different time periods (1–180 min). Cells were lysed as de- 4, hsp27 activated all three MAPKs pathways, but to different de- tailed in Materials and Methods. Equal amounts of postnuclear lysates by guest on September 24, 2021 grees. Phosphorylation of ERK 1/2, JNK 1/2, as well as p38 were immunoblotted (SDS-12% PAGE, followed by transfer to nitrocel- lulose membrane) with anti-phospho-p38 MAPK Ab. The same mem- MAPK was clearly increased at 20 min after addition of hsp27. branes were used for detection of other MAPK (both phosphorylated and Maximal stimulation was observed at 40 min after hsp27 addition total) by sequential stripping of the membranes, followed by reprobing the for all three MAPKs. However, activation of p38 MAPK persisted blots with respective Ab. Representative of three experiments. 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 activate (phosphorylate) human monocyte endogenous hsp27. To ERK. Activation of MAPKAPK-2 (a substrate of p38 MAPK) has test this possibility, we stimulated human monocytes at different been shown as necessary to LPS induction of IL-10 in human time points with recombinant hsp27 and measured activated (phos- monocytes (10). Therefore, we also assessed the activation of phorylated at serine residues) endogenous hsp27 in the cell lysates MAPKAPK-2 during hsp27-induced activation and IL-10 produc- by Western blotting, using an Ab against phosphoserine. The pres- tion of human monocytes by in vitro kinase assay, using a se- ence of equivalent amounts of hsp27 in the cell lysates was tested quence of hsp27 (KKLNRTSVA) as the substrate. As can be seen by reprobing the stripped blot with anti-hsp27 Ab. As can be seen in Fig. 5A, the immunoprecipitate (using ␣-MAPKAPK-2 sheep in Fig. 5B, exogenous hsp27 addition could activate monocyte en- polyclonal Ab; control sheep IgG had no MAPKAPK-2 activity) dogenous hsp27. Moreover, just as hsp27 induced prolonged p38 from hsp27-activated monocyte lysate had significantly increased activation (Fig. 4), endogenous monocyte hsp27 remained acti- MAPKAPK-2 activity vs that from adherence-stimulated untreated vated for prolonged periods (up to 2 h tested) after addition of monocyte lysate, suggesting that exogenously added hsp27 could exogenous recombinant hsp27. These data suggest that hsp27 is a potent inducer of IL-10 in human monocytes because it differ- entially activates the MAPK pathways that play critical roles in inducing monokine production. The next sets of experiments Table I. Contrasting levels of monocytea IL-10 or TNF-␣ induction by hsp27 and zymosan A examined which of the different MAPKs had critical roles in hsp27-induced IL-10 and TNF-␣ production by monocytes.

Unstimulated Hsp27 Zymosan A Critical role of p38 MAPK, but not of ERK 1/2 during IL-10 (pg/ml)b 302 Ϯ 163 4,272 Ϯ 217 845 Ϯ 320 hsp27-induced monocyte IL-10 production TNF␣ (pg/ml)b 48 Ϯ 1.5 301 Ϯ 190 12,738 Ϯ 3,747 Although the p38 MAPK pathway is critical in LPS induction of a Monocytes were cultured (n ϭ 3) in medium alone (unstimulated), hsp27 IL-10, the differential involvement of MAPK pathways in cytokine (2 ␮g/ml), or zymosan A (50 ␮g/ml) for 16–18 h and the culture supernatants were simultaneously tested for IL-10 and TNF-␣. production has been shown to be stimulus dependent and might b Tested by ELISA and expressed as mean Ϯ SEM. contribute to hsp27 preferential induction of IL-10 (10, 21, 33, 34). 3956 Hsp27 INDUCES IL-10 IN HUMAN MONOCYTES Downloaded from http://www.jimmunol.org/

FIGURE 6. SB203580, but not PD98059, inhibits hsp27-induced monocyte IL-10 production. Monocytes (1 ϫ 106 cells/ml) were treated with SB203580 (10 ␮M) or PD98059 (10 ␮M) for 2 h before addition of FIGURE 5. Hsp27 induced MAPKAPK-2 activity and activates endog- hsp27 (2 ␮g/ml) to the monocyte culture. Monocytes were then cultured for enous hsp27 in human monocytes. A, A total of 1.5 ϫ 106 monocytes were 16–18 h and tested for IL-10 or TNF-␣ levels in the culture supernatants. cultured in serum-free medium for 2 h and then stimulated with MDP (20 Data are expressed as mean Ϯ SEM. Representative of seven experiments by guest on September 24, 2021 p ϭ ,ء .␮g/ml) ϩ SEB (0.5 ␮g/ml), hsp27 (2 ␮g/ml), or UV (as positive control) for IL-10 production and of five experiments for TNF-␣ production for 30 min. Cells were then lysed and the postnuclear lysates were used for 0.002 as compared with hsp27-induced IL-10 levels; #, p ϭ 0.002 as com- -p ϭ 0.04 as com ,ءء ;assessment of MAPKAPK-2 activity by immunoprecipitation of the en- pared with only adherence-stimulated IL-10 levels zyme by anti-MAPKAPK-2 Ab, followed by in vitro kinase assay using pared with hsp27-induced TNF-␣ levels. hsp27 peptide sequence (KKLNRTSVA) as the substrate. Incorporation of [␣-32P]ATP into the substrate was assessed by scintillation counting and ءء ϭ ء expressed as cpm. Representative of three experiments. , p 0.04 and , lated monocytes (Fig. 6). In addition, the SB203580 treatment had ϭ p 0.03, as compared with only adherence-stimulated monocyte MAP- no effect on the hsp27 induction of ERK activation in these mono- KAPK-2 activity. B, Monocyte cell lysates (after stimulation with hsp27 cytes, indicating that the inhibition of hsp27-induced monocyte for 5–120 min) were subjected to SDS-15% PAGE, followed by immuno- blotting with anti-phosphoserine Ab. Recombinant hsp27 loaded in the IL-10 was not a result of general loss of monocyte viability, nor right-most lane served as positive control. Bands (phospho-hsp27) corre- did the DMSO control affect hsp27-induced monocyte IL-10 or sponding to 27 kDa molecular mass are shown (upper panel). The blot was TNF-␣ production (data not shown). In contrast to the inhibitory then stripped and reprobed with anti-hsp27 Ab to show the presence of effects of SB203580, PD98059 had no inhibitory effect on hsp27- equivalent amounts of total hsp27 (both nonphospho and phospho) in each induced monocyte IL-10 production, but blocked 68% of the lane (lower panel). TNF-␣ induced by hsp27 (Fig. 6). These data suggest that activa- tion of the ERK 1/2 pathway is not required for induction of mono- cyte IL-10 by hsp27, but that both the ERK 1/2 and p38 pathways To assess any essential role of differential MAPK activation for are involved in hsp27 induction of monocyte TNF-␣. These data induction of monocyte IL-10 or TNF-␣ by hsp27, we added dif- further support that monocyte TNF-␣ production induced by hsp27 ferent MAPK inhibitors to the monocyte culture before addition of is not required for maximal IL-10 production. hsp27. SB203580 was used to block the effect of p38 MAPK, whereas PD98059 was used to inhibit the effect of MEK 1/2 (the Discussion enzyme responsible for activation of ERK 1/2) (10, 33). SB203580 This is the first demonstration of a novel role for exogenous hsp27 could significantly ( p ϭ 0.002) block hsp27-induced IL-10 pro- in the preferential induction of monocyte IL-10 production. Hsp duction (Fig. 6). Monocyte IL-10 production was inhibited Ϸ80% interactions in the immune response have been a focus of many by SB203580, which also blocked 90% of the TNF-␣ activity in- recent investigations (6, 13). Primarily, the large hsp (6, 7) have duced by hsp27, indicating a potential critical role of p38 MAPK been examined for their stimulation or regulation of T cells and pathway during induction of both monocyte IL-10 and TNF-␣ pro- their augmentation of Ag presentation (6, 9, 13, 35). However, one duction by hsp27. However, even in the presence of SB203580, of the small hsp, hsp27, has been implicated as interacting with hsp27 induced a small amount of IL-10, which was still signifi- granzyme A in mediating granzyme A-induced cell death and in cantly ( p ϭ 0.002) increased over that of adherence only-stimu- cystic fibrosis arthritis, as well as glaucoma (36, 37). Although The Journal of Immunology 3957 hsp27 is capable of acting as a classic thermoprotective protein, its monocytes to produce IL-10. Inhibition of the p38 MAPK pathway thermoprotective and protein chaperone activities are independent with SB203580 reduced our hsp27-induced monocyte IL-10 levels of its phosphorylation (38). Hsp27 can play other physiologic roles by 80%, while the ERK inhibitor, PD98059, which inhibited when phosphorylated (15, 20, 39). There are a number of sugges- TNF-␣ production almost 70%, had no effect on maximal IL-10 tions that hsp27 might be exogenously active on monocytes. production. The significant level of hsp27-induced IL-10 generated Hsp27 is both more constitutively expressed and more cytokine even in the face of MAPKAPK-2 inhibition by SB203580 might inducible in monocytes than in T cells (40). Hsp27 is phosphory- result from other pathways of hsp27 phosphorylation since acti- lated in monocytes as a substrate of MAPKAPK-2 after LPS or vated PKC-␦ has been also demonstrated to directly phosphorylate TNF-␣ activation of the p38 MAPK pathway and is seen as a hsp27 (20). Alternatively, the SB203580 may only be capable of circulating protein that can eventually induce Abs in breast cancer inhibiting 80% of MAPKAPK-2 activity because of prior adher- and cystic fibrosis patients with arthritis (23, 36). Increased hsp27 ence-induced activation. The exaggerated monocyte IL-10 levels expression has also been suggested as protecting from TNF-␣- induced by hsp27 could result from the persistence of phosphor- mediated apoptosis (15). Induction of hsp27 has been reported to ylated p38 (P-p38) in the hsp27-induced monocytes. Similar to result in protection from lethal endotoxin shock and to be highly LPS-induced human monocyte activation data, levels of phosphor- induced in monocytes from patients with systemic inflammatory ylated ERK p42/p44, JNK p46/p54, and p38 peaked at 40-min shock syndrome, supporting a possible antiinflammatory role for post-hsp27 stimulation, then both ERK p42/p44 and JNK p46/p54 hsp27 (16, 17). Hsp60, one of the large hsp, can induce monocyte declined (33). In contrast to the published data for LPS induction of ␣ ␣ TNF- , and TNF- induction augments monocyte IL-10 levels human monocyte MAPK pathways, hsp27-induced P-p38 persisted (7, 32). These data, combined with those emphasizing the pivotal even at 180 min (33). Since P-p38 activation of MAPKAPK-2 is Downloaded from role of MAPKAPK-2, the kinase that phosphorylates hsp27, led us required for monocyte IL-10 induction, this increased persistence of to investigate exogenous hsp27 as a stimuli for monocyte IL-10 or P-p38 may partially explain both the higher levels of MAPKAPK-2 ␣ TNF- . induced by hsp27 (2-fold) vs SEB ϩ MDP (1.3-fold) and hsp27’s Hsp27 addition to primary human monocyte cultures induced exaggerated induction of IL-10. Phosphorylation of endogenous Ϸ exaggerated IL-10 production ( 3400 pg/ml). In contrast, LPS- hsp27 was also prolonged in hsp27-induced monocytes. Alterna- stimulated IL-10 levels reported for primary human monocytes tively or additionally, the rapid disappearance of JNK p46/p54 http://www.jimmunol.org/ range from 500-1500 pg/ml, similar to our SEB ϩ MDP- and after hsp27 stimulation might be allowing continued monocyte zymosan A-induced monocyte IL-10 levels of Ϸ800–1100 pg/ml IL-10 production. A critical down-regulatory role for activation of (30, 31, 41). However, LPS simultaneously stimulates the same the JNK pathway has been implicated for IL-10 production, since human monocytes to produce from 6,000 to 15,000 pg of TNF-␣ Ϫ Ϫ JNK 1 / Th cells produced exaggerated IL-10 in response to (30, 31, 41). In our polymyxin B-containing system, zymosan A CD3 induction (45). Our data suggested that JNK was only min- similarly induced high levels of TNF-␣ (Ϸ13,000 pg/ml), but only imally activated by hsp27. The importance of this minimal JNK Ϸ800–900 pg of IL-10. Hsp27 induced Ϸ300–500 pg/ml of pathway activation in hsp27 induction of exaggerated monocyte monocyte TNF-␣, somewhat similar to the reported 750 pg/ml

IL-10 needs to be further explored, utilizing JNK inhibitors. How- by guest on September 24, 2021 induced by human hsp60 in the human Mono Mac-6 cell line (7). ever, JNK inhibitors are not yet commercially available. Hsp27’s exaggerated stimulation of human monocyte IL-10 vs its The ability of exogenous hsp27 to differentially stimulate modest induction of TNF-␣ is further illustrated by our data show- MAPK pathway activation and distinctive cytokine production ing that combining SEB ϩ MDP ϩ hsp27 induced no further in- crease in IL-10 levels, but augmented monocyte TNF-␣ levels might imply that it is binding to a monocyte surface receptor rather more than 3-fold. Similarly, TNF-␣ addition did not further in- than nonspecifically internalized by pinocytosis. The different crease hsp27-induced IL-10 levels. These data imply that hsp27 stimulatory capacity of autologous vs microbial hsp, despite their alone induced maximal monocyte IL-10, but only minimal TNF-␣ sequence homology, along with their surface binding of peptides, levels, and that hsp27 may preferentially activate human monocyte has led several investigators to propose a putative hsp receptor IL-10 production. Increased production of IL-10 by APC has been (7, 8, 35). The receptor for advanced glycation end products suggested to polarize the immune responses to Th2 (42). The par- (RAGE) has been proposed as a likely candidate for an hsp recep- adigm that bacterial hsp may pivotally act as stimulating danger tor (8). The RAGE levels on monocytes are up-regulated in dia- signals whose immune recognition is highly conserved, while au- betics (46), a disease in which large hsp have been shown to have tologous Hsp could have preferential immune regulatory activity immunoregulatory activity (47) and RAGE activation induces has been previously postulated (6, 7, 43, 44). Our data showing MAPK signaling pathways (48). Most of the hypotheses developed recombinant human hsp27 to preferentially induce antiinflamma- and the immune regulation investigations performed involved the tory IL-10 rather than proinflammatory TNF-␣ in human mono- large hsp. As far as we could determine, our study represents the cytes are consistent with this paradigm. first described data of cytokine-inducing activities for exogenous The mechanism(s) behind hsp27 preferential induction of mono- hsp27 and is consistent with differential monocyte activation through cyte IL-10 may involve preferential activation of the MAPK p38 an hsp27 receptor. pathway. Previous reports demonstrated that the p38 but not the In summary, we have demonstrated a novel role for exogenous ERK pathway was required for LPS induction of IL-10 (10). How- human hsp27 in preferentially inducing human monocytes to pro- ever, LPS induction of maximal monocyte IL-10 appears also to be duce IL-10 by activating the p38 MAPK, MAPKAPK-2, and en- pivotally dependent on its induction of TNF-␣ and/or IL-1 (10, dogenous hsp27 in a prolonged fashion. Exogenous hsp27 was 32). LPS-stimulated maximal IL-10 production by monocytes is able to induce monocyte IL-10 mRNA increases as well as protein consequently delayed, peaking at 24 h after LPS stimuli and con- increases. Increased IL-10 induction may be responsible for the tinuing for 96 h (27). In contrast, hsp27-induced monocyte IL-10 reported ability of hsp27 to down-regulate TNF-␣-mediated apo- levels were maximal at 18 h, and anti-TNF-␣ mAb only inhibited ptosis and ROI induction (3, 15). Whether our demonstration of 40% of hsp27-induced IL-10 levels in our experiments vs a re- hsp27-mediated antiinflammatory function is limited to human ported 70% inhibition of LPS-induced IL-10 (32). These data sug- monocytes or is a unique property of autologous hsp27 vs large gest that exogenous human hsp27 directly stimulates human hsp remains to be investigated. The ability of hsp27 to induce a 3958 Hsp27 INDUCES IL-10 IN HUMAN MONOCYTES predominant in vitro antiinflammatory monokine profile could im- 23. 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