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Identification of Stimulating and Inhibitory Epitopes within the Heat Shock Protein 70 Molecule That Modulate Cytokine Production and Maturation of Dendritic Cells ¹

Yufei Wang^*, Trevor Whittall^*, Edward McGowan, Justine Younson, Charles Kelly, Lesley A. Bergmeier^*, Mahavir Singh and Thomas Lehner^2,*

^* Mucosal Immunology Unit, Department of Oral Medicine and Pathology and Department of Oral Immunology, Guy’s, King’s and St. Thomas’ Hospital Medical and Dental Schools, London, United Kingdom; and Lionex Diagnostics and Therapeutics, Braunschweig, Germany

	Abstract

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The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407–426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457–496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457–496 but enhanced by p407–426.

	Introduction

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Heat shock proteins (HSP) ³ are intracellular chaperones, present in most mammalian cells and microorganisms. Microbial HSP (mHSP) are of considerable interest in stimulating production of cytokines (1, 2, 3, 4, 5, 6, 7) and chemokines (8, 9) and in inducing maturation of dendritic cells (DC) (6, 7). By virtue of these functions, they have adjuvant properties when administered with Ags by the systemic (10, 11, 12, 13) or mucosal routes (8). Some but not all of these properties are shared with the homologous mammalian HSP which are intracellular molecules and play an essential role as chaperones of proteins (14). An important property of mHSP70 is their ability to present external proteins to HLA class II and cross-presentation to HLA class I pathways (15, 16). Furthermore, the concept that the HSP groove may bind critical tumor peptides has led to protection in the development of tumors, by immunization with peptide-bound HSP70 and HSP95 purified by ADP chromatography from tumor cells (17, 18).

mHSP70, like human HSP70 (huHSP70), consists of three functionally distinct domains, an N-terminal 44-kDa ATPase portion (aa 1–358), followed by an 18-kDa peptide-binding domain (aa 359–494) and a C-terminal 10-kDa fragment (aa 495–609) (19). Immunological functions of the three different domains in stimulating monocytes and DC have not been fully defined. However, the C-terminal portion (aa 359–610) stimulates production of CC chemokines, IL-12, TNF-, and NO; induces Th1 polarization and maturation of DC; and functions in vivo as an adjuvant (7, 8, 9). The ATPase domain of mHSP70 largely lacks these functions, and removal of the ATPase domain enhances HSP70 stimulation in the production of cytokines, chemokines, and maturation of DC. However, a sequence required for CTL stimulation after immunization with an OVA-mHSP70 fusion protein was identified in the ATPase portion (20), as well as a suppressor epitope stimulating production of IL-10 and TGF-1 (21, 22).

Interaction between HSP70 and APC is receptor mediated. CD14 and TLR4 are implicated in binding HSP60/65 (23, 24) and were also reported as receptors for huHSP70 (25), both mHSP70 and huHSP70 bind CD40 but use different sites of the molecule (9, 26). Because a major costimulatory pathway of interaction is the one between CD40 on APC and CD40L on T cells (27, 28, 29), the finding that mHSP70 binds and stimulates CD40⁺ cells to produce chemokines and cytokines may be important in the interphase between innate and adaptive immunity (30). The significance of HSP70 binding and activating the CD40-costimulatory pathway has now been confirmed in vivo (31, 32). DC play a central role in the immunological repertoire, with modulating functions according to the state of maturation of the DC (33). Ag processing by immature DC and Ag presentation by mature DC, with potent immune responses, is the hallmark of DC. The concept has been suggested that mature DC are involved in immunogenicity and immature or steady state DC in tolerance (33).

The aim of this study was first to identify an epitope within mHSP70 that is responsible for the stimulating functions of HSP70, in terms of cytokine and chemokine production and maturation of DC. During the process of using overlapping peptides, we found an epitope within the HSP70 molecule that inhibits cytokine and chemokine production and maturation of DC elicited by HSP70. Here we show that the cytokine- and chemokine-stimulating functions and maturation of DC reside in the peptide-binding portion of HSP70 (aa 359–494). Stimulation of monocytes or DC in vitro with peptide 407–426 generates IL-12, TNF-, and CCL5 and enhances maturation of DC. In contrast, peptide 457–496 inhibits production of cytokines, chemokines, and maturation of DC stimulated with HSP70 or CD40L. Consistent with these functions, investigation of signaling by the p38 MAP kinase phosphorylation pathway revealed that p407–426 enhanced, whereas p457–496 inhibited, phosphorylation in HSP70- or CD40L-stimulated maturation and IL-12 production by DC.

	Materials and Methods

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Reagents

Soluble CD40L trimer (CD40LT) was kindly donated by Dr. F. Villinger (Atlanta, GA). Human recombinant GM-CSF (Leucomax) was obtained from Sandoz Pharmaceuticals and human rIL-4 was from R&D Systems. The fluorochrome-conjugated mAbs to CCR7, CXCR4, CD14, and CD40 were purchased from Immunotech. The Abs to human CD83 and control murine mAb isotypes were obtained from Serotec and DAKO.

Preparation of mHSP70 and its fragments

HSP70, HSP70_359–610, and HSP70_359–494 were prepared from the Escherichia coli pop strain using the pJLA603 vector (34). Cloned inserts were verified by DNA sequence analysis. HSP70 was expressed with no additional amino acid residues, whereas HSP70_359–610 and HSP70_359–494 were expressed with a C-terminal (His)₆ tag. HSP70 was purified by ion exchange chromatography using Q-Sepharose resin followed by ATP-affinity chromatography. HSP70_359–610 and HSP70_359–494 recombinant polypeptides were prepared by affinity chromatography using Ni²⁺-chelating resin, and the identity of the polypeptides was confirmed by N-terminal sequence analysis (10 cycles each). The HSP preparations were further treated with polymyxin B-coated beads (Sigma-Aldrich) to remove LPS. The LPS content of the HSP preparations was determined by the Limulus amebocyte lysate assay (Sigma-Aldrich), and showed <0.006 U/µg of HSP70 or 5 pg/µg of the HSP preparation.

Determination of substrate-binding activity of HSP70 and fragments

Surface plasmon resonance was used to measure binding of HSP70, HSP70_359–610, and HSP70_359–494 to a peptide that included the sequence NRLLLTG, which is a substrate for HSP70 binding (19). The peptide (NRLLLTGGSPSPVC) was synthesized by standard Fmoc chemistry (as described below) and included a spacer sequence C-terminal to the HSP70-binding motif and a C-terminal Cys for coupling. The peptide was coupled (3000 resonance U) to the surface of flow cell 2 of a CM-5 sensorchip (Biacore International) using the thiol-coupling procedure as described in the manufacturer’s instructions. Flow cell 1 served as a noninteracting reference surface. Binding of fluid phase HSP70, HSP70_359–610, and HSP70_359–494 was measured over a range of concentrations (50–200 nM), using BIAcore X. Buffer was 0.15 M NaCl, 0.005% (v/v) Tween 20 in 0.01 M HEPES, pH 7.4. The flow rate was 5 µl/min, and the injection volume was 20 µl. The surface was regenerated by injection of a pulse (5 µl) of 2 M KSCN.

Preparation of human monocytes and monocyte-derived DC

The human monocytic THP1 cell line was obtained from the Medical Research Council (National Institute for Biological Standards and Control, Porters Bar, U.K.). The nonadherent THP1 cells were maintained on RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, and 100 IU/ml penicillin and 100 µg/ml streptomycin. Human primary monocytes were isolated from PBMC prepared from healthy donors by centrifugation on a Ficoll-Hypaque density gradient (Amersham Biosciences). The CD14⁺ monocytes were enriched by depletion of CD14^– cells using a Monocyte Isolation Kit (MACS; Miltenyi Biotec). The purity of isolated monocytes was consistently >90% when analyzed by flow cytometry with Ab to CD14. Human DC were generated by culturing monocytes with GM-CSF (400 U/ml) and IL-4 (100 U/ml) for 5 days (35). These monocyte-derived DC were generally considered to be immature DC, defined by surface expression of DC markers CD83, CD80, CD86, and CD40 and were CD14^–.

Induction of the CC chemokine (CCL5, RANTES) and the cytokines TNF- and IL-12

Human monocytes (1 x 10⁶/ml) or monocyte-derived DC (2 x 10⁵/ml) were incubated with HSP70, HSP70_359–610 or HSP70_359–494 at concentrations of 0.005–0.5 µM in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. DC were cultured in RPMI 1640, conditioned with 400 U/ml GM-CSF and 100 U/ml IL-4. After 48 h of incubation, the supernatant was used to assay the chemokine CCL5 and the cytokines IL-12 and TNF-.

Investigations of the effects of BAPTA-AM and proteinase K on the production of TNF- and IL-12 by stimulation with HSP70 compared with LPS

To exclude the possibility that any contaminated LPS in the HSP70 or its fragments may have affected the results of these investigations, the intracellular calcium chelator BAPTA-AM and treatment with proteinase K-agarose were conducted. DC cultures were stimulated with 20 µg/ml HSP70, 5 µg/ml HSP70_359–610, 5 µg/ml HSP70_359–494 or 500 ng/ml LPS and then treated with BAPTA-AM at concentrations of 1–100 µM. To study the effect of proteinase K, 100 µg of HSP70, the fragments or LPS was incubated with 100 µg of proteinase K-agarose beads (equivalent to 0.005 enzyme U) in a total volume of 100 µl of PBS buffer for 1 h at 37°C. The proteinase K beads were removed by centrifugation, the digested supernatants were collected, and 3 concentrations of each were used to stimulate DC. After stimulation for 2 days, the DC culture supernatants were collected and assayed for TNF- or IL-12.

Determination of the minimal concentration of LPS that affects the stimulating function of mHSP70 and HSP70_359–494

DC were stimulated with mHSP70 (20 µg/ml) HSP70_359–494 (5 µg/ml) or CD40LT (10 µg/ml) in the presence of increasing concentrations of LPS (10 pg–100 ng/ml). The production of IL-12 was assayed after incubation for 48 h.

Preparation of overlapping 20-mer peptide-binding portion of HSP70 (p359–494), alanine substitution of residues in the identified p407–426-stimulating domain, and preparation of the 40-mer p457–496

A panel of peptides (20-mers overlapping by 10 amino acid residues) that span residues 357–496 of HSP70 were synthesized by standard Fmoc chemistry using a peptide synthesis block (BT7400; Biotech Instruments). Peptides were synthesized on Rink amide MBHA resin and released using 82.5% TFA, 5% anisole, 5% phenol, 2.5% ethane dithiol, 5% H₂O (v/v/w/v/v). Peptides were recovered by precipitation with ether, washed three times with ether, and dried. The peptides were subsequently dissolved in 10 mM acetic acid or 10 mM NH₄HCO₃ and lyophilized. Using the same procedure, a further panel of peptides was prepared in which each amino acid residue within the sequence of the identified stimulating p407–426 was in turn substituted by alanine. Selected peptides were analyzed by mass spectroscopy to confirm the sequence. The inhibitory 40-mer peptide 457–496 was synthesized by Bachem to a purity of 93.7%, determined by high pressure liquid chromatography (M_r 4392.6). The sequences of the two peptides are: p407–426, Q P S V Q I Q V Y Q G E R E I A A H N K; p457–496, I V H V T A K D K G T G K E N T I R I Q E G S G L S K E D I D R M I K D A E A H.

Effect of stimulating monocytes or DC with synthetic peptides derived from HSP70_359–494

To study the effect of the synthetic peptides, human monocytes (1 x 10⁶/ml) or monocyte-derived DC (2 x 10⁵/ml) were incubated with 50 µg/ml of the 13 overlapping 20-mer peptides (overlapping by 10 residues), corresponding to the HSP70_359–496 peptide-binding fragment. The 20-mer peptides were used first singly and then by combining two adjacent 20-mers or using the single 40-mer in concentrations of 50 µg/ml with 10 µg/ml HSP70, 10 µg/ml CD40LT, or 500 ng/ml LPS. These were added to DC or monocytes, and after 3 days of culture TNF-, IL-12, or CCL-5 was assayed in the supernatants. The critical residues within p407–426 were identified by alanine substitution of each of the 20 N-terminal residues of 407–426, and these peptides were then used to stimulate DC to produce TNF- and IL-12.

Stimulation of maturation of DC by the peptides

Monocyte-derived DC were prepared as above in 96-well flat-bottom tissue culture plates. After incubation for 5 days, 50 µl of medium with 4 or 20 µg/ml each of HSP70, HSP_359–610, HSP_359–494, or 0.2 or 2 µg/ml CD40LT or 5 or 50 ng/ml LPS were added. In another experiment, immature DC were treated with 50 µg/ml HSP peptide (407–426 or 457–476/477–496 or p457–496). After 30 min, 10 µl of medium with HSP70, CD40LT, or LPS were added. The cells were incubated for 2 days, before harvesting into 12- x 75-mm tubes, using PBS with 0.02% EDTA to help release of adherent cells. The isolated cells were stained with fluorochrome-conjugated Abs to DC markers or conjugated mouse IgG isotype as a control, for 30 min on ice. After two washings, the cells were fixed in 1% formaldehyde-PBS solution before analysis by flow cytometry. The cells were examined on a Coulter XL-MCL flow cytometer, and the data were analyzed using Win MDI software.

Effect of the HSP70-binding motif NRLLLTG on the production of TNF- or IL-12

To study the effect of peptide bound by HSP70 or HSP70_359–494 on the production of TNF- or IL-12, a synthetic peptide, NRLLLTG, which has high affinity binding for HSP70, was used (19). Human monocytes or DC cultures were stimulated with 10 µg/ml HSP70 or 5 µg/ml HSP70_359–494, in the presence of 5–50 µg/ml NRLLLTG. Two days after culture, the concentration of TNF- or IL-12 was assayed in the supernatants.

ELISA for CCL-5, IL-12, and TNF-

The CC chemokine CCL5 in the culture supernatants was assayed using specific ELISA-paired Ab (R&D Systems). IL-12p40 and TNF- in the culture supernatants were detected using paired specific Abs to IL-12p40 or to TNF- (BD Pharmingen), with sensitivity limits of 8 pg/ml for TNF- and 15 pg/ml for IL-12. The IL-12p40 Ab detects only the p40 molecule and does not detect IL-12 p70. The supernatants were diluted x5 for CCL5 and x2 for IL-12 or TNF- assay. The results were expressed in picograms per milliliter.

Assay of p38 MAP kinase phosphorylation and the effect of corresponding inhibitor

For the phospho-p38 assay, DC, monocytes, or HEK 293 cells (transfected or nontransfected) were treated with HSP70 CD40L or LPS in the presence or absence of p38 inhibitors (SB 203580; Sigma) for 30 min and then lysed in isotonic buffer with 6 M urea and 0.1% Triton X-100. The phospho-p38 content was assayed by ELISA using IC duo-set reagents (R&D Systems), according to the manufacturer’s instructions. Samples were also subjected to SDS-PAGE and Western blotting to demonstrate p38-specific phosphorylation.

Transfection of HEK 293 cells with CD40

Full length human CD40 cDNA in the pcDNA plasmid vector (Invitrogen) was a gift from Dr. B. Seed (36). pCDM8 encoding the E. coli -galactosidase (Lac-Z) was used as a control. The HEK 293 cells were cultured in six-well plates up to 30–50% confluence and transfected using LipofectAmine Plus (Technologies), according to the manufacturer’s protocol. CD40 was detected by flow cytometry using FITC-conjugated CD40 mAb (Serotec). The percentage of CD40⁺ cells was consistently >65% at days 2 and 3 after transfection. The p38 MAP kinase assay was conducted with CD40-transfected and untransfected HEK cells, which were treated for 20 min with 30 µg/ml HSP70, 10 µg/ml peptide-binding domain (359–494), 20 µg/ml CD40LT, or 1 µg/ml LPS. The 20-mer peptide 407 or the 40-mer peptide 457–496 was added to the cultures at a concentration of 50 µg/ml. After incubation, the cells were washed with cold PBS medium, detached from culture plates with 0.5% EDTA, centrifuged at 500 x g, and the cell pellets were lysed with 50 µl of cell lysis buffer before the p38 MAP kinase assay.

Statistical analysis

The data were expressed as mean ± SEM, and the differences between the groups were analyzed by Student’s t test.

	Results

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Stimulation of monocytes and DC by the HSP70 peptide-binding domain (359–494) in comparison with p359–610 and the wild-type HSP70

To define the stimulatory domain within the C-terminal part of HSP70, we prepared the peptide-binding fragment 359–494 (19). Human monocytes were purified from PBMC to >90% purity, and these as well as the monocyte-derived DC were incubated with various concentrations (0.01–0.5 µM) of wild-type mHSP70, the C-terminal portion of mHSP70 (p359–610) or the peptide-binding domain (p359–494). Two days after stimulation, production of TNF- or IL-12p40 in the supernatant was analyzed by ELISA. Resting cultures of monocytes or those treated with BSA produced negligible levels of TNF- (Fig. 1A). The mHSP70 stimulated only low levels of TNF- production (61.6 ± 10.4 pg/ml) in concentrations up to 0.5 µM. The peptide-binding domain (p359–494), however, elicited greatly enhanced dose-dependent increase in the production of TNF-, in comparison with mHSP70, and the response was almost identical with that of the C-terminal portion of HSP70 (p359–610). At a concentration of 0.5 µM, stimulation with the peptide-binding domain (p359–494) generated 341 ± 75.2 pg/ml TNF-, compared with 306 ± 19 pg/ml stimulated by the C-terminal portion (p359–610). This result suggests that the peptide-binding domain (p359–494) is largely responsible for stimulation of TNF- production, that removal of the 10-kDa C-terminal fragment had no effect on HSP70 stimulation of cytokines, and that deletion of the ATPase domain enhances the cytokine-stimulatory activity.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Comparative stimulation of monocytes and DC with 3 HSP70 preparations. Dose-dependent stimulation of monocytes (A) and DC (B and C) to produce TNF- and IL-12p40 by HSP70 and its two fragments; the effects of intracellular calcium chelator BAPTA-AM (D) or treatment with proteinase K (E) on the production of TNF- stimulated by HSP70, the two fragments, or LPS.

Investigation of contamination of the HSP70 preparations with LPS

Any contamination of HSP70 with LPS was studied first by using the intracellular calcium chelator BAPTA-AM to examine the effect on HSP70 and its C-terminal fragments. TNF- production was inhibited in a dose-dependent manner with the calcium chelator when monocytes were stimulated with HSP70 or the two peptide binding fragments but not with LPS (Fig. 1D). Proteinase K was then used, and this had an inhibitory effect on TNF- production by HSP70 and its two C-terminal fragments but did not affect LPS (Fig. 1E). These results are consistent with the chemokine and cytokine functions being generated by HSP70 independent of any LPS contamination.

To exclude further the possibility that contamination of HSP70 with LPS might have been responsible for the stimulating activity, we determined the minimal concentration of LPS which affects the stimulating activity of DC by HSP70. The results show that 10, 100, or 1000 pg/ml LPS does not affect the concentration of IL-12p40 produced by DC stimulated with either HSP70 or HSP70_359–494, and it required 10⁴ pg/ml LPS to approximately double the concentration of IL-12 by the 2 HSP70 preparations (Table I). Similar results were found with CD40LT which required 10⁴ pg/ml LPS to double the IL-12 concentration (Table I). Indeed, 10⁴ pg/ml LPS alone was required to stimulate TNF- production. As the LPS contamination of the HSP70 preparations was 0.001 U/µg of HSP70 (1 U = 10 ng of LPS) the level of contamination was 10 pg, which was 1000 times lower than any IL-12-stimulating activity that was detected with LPS either alone or with HSP70.

To identify minimal cytokine and chemokine stimulatory epitopes, 20-mer peptides overlapping by 10 aa, corresponding to the sequence of peptide-binding domain (aa 357–496), were synthesized. The cytokine stimulatory activity of the peptides was tested on monocyte-derived DC, after incubation with the 13 overlapping peptides for 2 days. Production of IL-12p40 (Fig. 2A) by DC stimulated with the peptides showed that p407–426 was a dominant epitope in stimulating a 10-fold increase in IL-12p40 (275.4 ± 41.2 pg/ml) compared with the unstimulated control (23.1 ± 10.3 pg/ml). The adjacent peptides 387–406 and 417–446 showed slightly raised production of IL-12. A minor epitope was identified at the N-terminal end (aa 357–376) which yielded 107.6 ± 24 pg/ml IL-12p40. The native HSP70 elicited 3-fold greater production of IL-12p40 (886.5 ± 277.8 pg/ml) than p407–426.

View larger version (36K): [in this window] [in a new window]   FIGURE 2. Identification of a mHSP70-stimulating epitope and the critical residues. A and B, Effect of overlapping 20-mer synthetic peptides, derived from the HSP70 peptide-binding domain (aa 359–494), on the production of IL-12p40 (A) and TNF- (B) by monocyte-derived DC; C and D, effect of alanine substitution of each residue within p407–426 on stimulation of production of IL-12 (C) or TNF- (D) by DC.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Identification of a stimulating epitope within peptide 387–426 and inhibiting epitope within peptide 457–496 using DC costimulated with HSP70 and with two adjacent or overlapping 20-mer peptides. B, Stimulating epitope p407–426 at the base of L3, 4 and L4, 5 and the suppressive epitope (p457–496) in the sheet.

To identify the critical residues within the p407–426 sequence, a panel of 20 peptides was synthesized in which each residue in turn was substituted with alanine. These peptides were then used to stimulate monocyte-derived DC to produce IL-12 and TNF-. The N-terminal residues Q407, P408, S409, and V410 appeared to be critical, given that alanine substitution significantly reduced stimulation of IL-12 and TNF- production (Fig. 2, C and D). Alanine substitution of E420 also showed significant loss of activity in stimulation of production of the two cytokines. Stimulation of CCL5 production by the THP1 monocyte cell line showed the same critical residues as those of the two cytokines produced by DC, but in addition Q411A and H424A were critical (data not presented). The stimulatory peptide epitope 407–426 is situated at the base of L3, 4 and L4, 5 of the peptide-binding domain (Fig. 3B).

Identification of inhibitory peptide epitope within the C-terminal portion of HSP70

The results of epitope mapping suggested that the N-terminal (aa 357–396) and the C-terminal (aa 427–496) portions were largely devoid of TNF--stimulating activity and had little effect on IL-12 production (Fig. 2, A and B). This raised the possibility that some of the peptides might inhibit cytokine or chemokine production. Whereas costimulation of DC with HSP70 and p387–426 enhanced production of TNF-, pooling two adjacent 20-mers (aa 457–476 with 477–496) consistently inhibited TNF- production by DC from 187 (±64) to 78 (±41) pg/ml (p = 0.001; Fig. 3A) and monocytes from 257(±77) to 149 (±46) pg/ml (p = 0.01) (not shown). Similar analysis of IL-12 production by monocytes showed inhibition of HSP70-stimulated IL-12 production by p457–496, from 336.1 (±77.8) to 232.6 (±97.3) pg/ml (p = 0.08) and DC from 460.0 (±246) to 269.0 (±182) pg/ml (p = 0.04) (not shown). The inhibitory effect of these 2 adjacent peptides was then demonstrated with monocytes (p = 0.01) and DC (p = 0.001) prepared from 13 normal subjects (Fig. 4A). This inhibition may not be specific to HSP70, in that CD40L-stimulated production of TNF- by DC was also inhibited, although it failed to reach the 5% level of significance (Fig. 4B). However, LPS stimulated TNF- production by DC was not affected (Fig. 4B). CCL5 production by monocytes was similarly inhibited with p457–496 but not p357–396 (not shown). Thus, the two adjacent peptides 457–496 significantly inhibited TNF- and IL-12 production by DC and monocytes, as well as CCL-5 production stimulated by HSP70. The inhibitory effect of p457–496 on TNF- (Fig. 4C) and IL-12 (Fig. 4D) production was confirmed by synthesizing a single 40-mer peptide which also inhibited significantly HSP70 and CD40LT stimulated but not LPS-stimulated cytokine production (Fig. 4D). Single 20-mers (i.e., p457–476 or 467–486) consistently failed to inhibit the cytokines.

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Specificity of the suppressor epitope p457–496. Treatment with the two adjacent peptides (p457–496) in HSP70-stimulated production of TNF- by monocytes (n = 13) and DC (A) (n = 13) and comparison of HSP70, CD40LT and LPS stimulated DC (B); C and D, dose-dependent inhibition of DC by the 40-mer peptide 457–496 of HSP70-, CD40LT-, or LPS-stimulated production of TNF- and IL-12; E, effect of peptide-binding motif NRLLLTG on HSP70 or HSP70359–494 stimulation of DC to produce IL-12; F, binding of HSP70, HSP70359–610, and HSP70359–494 to NRLLLTG was measured by surface plasmon resonance. The superimposed sensograms are duplicates of each polypeptide at a concentration of 100 µM. *, p = 0.01; **, p = 0.002; ***, p = 0.001.

Peptide mapping suggested that the cytokine-stimulating epitope (aa 407–426) forms the base of the peptide-binding groove (Fig. 3B). Only 1 of the 5 critical residues (V410) is shared (V436) with the 10 defined anchor points for peptide binding (19). We have therefore tried to determine whether HSP70 binding by a high affinity peptide motif (aa NRLLLTG) might affect HSP70 or HSP70_359–494 stimulation of monocytes to produce IL-12 and TNF-. Significant changes in IL-12 production by monocytes stimulated with either HSP70 or HSP70_359–494 (Fig. 4E) were not found with a range of concentrations of peptide NRLLLTG. Similar results were found with TNF- and DC (data not presented). We confirmed, by surface plasmon resonance that both HSP70_359–610 and HSP70_359–494 bound to NRLLLTG with affinity comparable with that of HSP70 (K_D 10^–9 M) (Fig. 4F).

Effect of peptide-binding domain (359–494) in comparison with p359–610 and wild-type HSP70 on the maturation of DC

We have demonstrated previously that mHSP70 and the C-terminal fragment of HSP70_359–610 but not the N-terminal fragment (1–358) elicit maturation of human DC (7). Here we have compared maturation elicited by the peptide-binding fragment aa 359–494 with the C-terminal fragment and wild-type HSP70. Peptide 359–494 elicited maturation of DC comparable with those of the other two HSP70 stimulants, when assessed by the proportion of CD83- and CCR7-expressing cells (Table II). All three HSP70 preparations, as well as the CD40LT and LPS significantly up-regulated maturation of the immature DC (p < 0.01), with the exception of CCR7 in LPS-stimulated DC (Table II).

Because p407–426 stimulated immature DC to produce cytokines and chemokines and enhanced HSP70 stimulation, we examined the possibility that this peptide might also have an effect on maturation of DC. The expression of CD83, CCR7, and CXCR4 showed only slight enhancement of maturation of DC treatment with p407–426, compared with unstimulated or p457–496-stimulated DC (with the exception of CXCR4; Table III and Fig. 5). However, maturation of DC by costimulation of HSP70 with the peptide significantly increased the proportion of DC expressing all three maturation markers when compared with stimulation without peptide or with the control p457–496 (p = 0.01–0.0001). To determine whether the enhancement of maturation of DC with p407–426 was HSP70 specific, we costimulated immature DC with CD40LT and the peptide (Table III), and showed significant increase in the three maturation markers compared with stimulation of CD40LT alone or with p457–496 (p < 0.01–0.001). Costimulation of LPS with p407–426 showed variable results with no significant increase in CXCR4, just reaching the 5% level of significance with CD83 and CCR7, compared with stimulation by LPS alone, but higher p values were reached when analyzed against p457–496 (Table III). Thus, the cytokine- and chemokine-stimulating epitope p407–426 enhanced maturation of DC, irrespective of whether HSP70 or CD40L was used, but costimulation with LPS gave inconsistent results.

View larger version (37K): [in this window] [in a new window]   FIGURE 5. Effect of costimulation of DC with HSP70, CD40LT, or LPS and peptides 407–426 or 457–496 on the expression of CD83 and CCR7; heavy line, without peptide; light line, with 407–426; dotted line, 457–496 peptide. Data by flow cytometry showing percent and mean fluorescence intensity.

The possibility that the two adjacent peptides (457–476/477–496) might inhibit maturation of DC when costimulated with HSP70, CD40LT, or LPS was then explored. The results suggest that these peptides inhibit maturation of DC, isolated from normal subjects and evaluated in HSP70 stimulated cells by the expression of CD83 (p = 0.003), CCR7 (p = 0.049), and CXCR4 (p = 0.0002) (Table IVA, Fig. 5). Similar analyses of DC costimulated with CD40LT and the peptides again showed very significant inhibition of maturation of DC for CD83, CCR7, and CXCR4 (p = 0.003–0.00001). Although costimulation of LPS with the peptide showed inhibition, the levels of significance were lower than those with HSP70 or CD40LT (Table IVA and Fig. 5). As with the cytokines, inhibition of maturation was confirmed by costimulation of the single 40-mer (peptide 457–496) with HSP70 or CD40L, but not with LPS (Table IVB).

We have investigated the effect of stimulating and inhibitory peptides on DC maturation and function by evaluating the p38 MAP kinase transduction pathway which is critical in CD40L-CD40 regulation of maturation of DC (26, 37, 38). We present evidence that HSP70, CD40L, and LPS stimulate DC to generate phosphorylated p38 which is inhibited by p457–496 in HSP70- and to a lesser extent CD40L but not LPS-stimulated DC, whereas p407–426 slightly enhanced p38 phosphorylation stimulated by HSP70 (Fig. 6A). We have also demonstrated the effect of these peptides on p38 MAP kinase phosphorylation with monocytes that showed inhibition by costimulation of p457–496 with HSP70 from 2725 ± 1109 to 870 ± 222 pg/ml) or with CD40L from 3162 ± 1902 to 1025 ± 75 pg/ml but not with LPS (data not shown). The effect of these peptides was confirmed by Western blots; costimulation of HSP70 or CD40L with p457–496 inhibited, whereas p407–426 enhanced p38 MAP kinase phosphorylation (Fig. 6B). Costimulation of LPS with the two peptides had no effect.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Effect of peptides 407–426 and 457–496 on p38 MAP kinase phosphorylation stimulated by HSP70, CD40L, or LPS by ELISA of lysed DC. A, By ELISA; B, by Western blotting; C, CD40-transfected HEK 293 cells; D, HEK 293 cells alone.

We then studied the effect of p38 MAP kinase inhibitor (SB203580) on IL-12 production by DC stimulated with HSP70, CD40L, or LPS and demonstrated consistent dose-dependent inhibition of IL-12 production (Fig. 7A). The inhibitor was not toxic to the cells, given that they retained their viability on direct inspection and trypan blue uptake test. The involvement of the p38 MAP kinase phosphorylation pathway in the maturation of DC was then explored, using the p38 inhibitor. Maturation of DC stimulated with HSP70, CD40L, or LPS was inhibited, as shown by the mean fluorescence intensity of DC expressing cell surface HLA-II Ag (Fig. 7B).

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Effect of p38 MAP kinase inhibitor (SB203580) on production of IL-12 by DC after treatment with HSP70 (1 µg/ml), CD40LT (1 µg/ml) or LPS (200 ng/ml) (A) and maturation of DC evaluated by expression of MHC class II 2 days after treatment with HSP70 (20 µg/ml), CD40LT (2 µg/ml), or LPS (500 ng/ml) in the presence and absence of p38 inhibitor (25 µM) (B).

	Discussion

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We attempted then to identify the cytokine- and chemokine-stimulating epitopes by using overlapping 20-mer peptides, derived from the sequence of the 18-kDa peptide binding domain (359–494), to activate human monocytes and DC. This identified p407–426 as a major epitope stimulating TNF-, IL-12, and CCL-5 from monocytes and DC, the two principal human APCs. Alanine substitutions of each of the 20 residues within p407–426 revealed that four residues at the N-terminal end (Q407, P408, S409, V410) and E420 at the C-terminal end significantly inhibited production of IL-12 and TNF- by DC or monocytes. These results suggest that the five residues are critical in stimulating DC and monocytes to produce cytokines. Mapping the 20 amino acids onto the structure of HSP70 (19) showed that they are located at the base of the peptide-binding groove of HSP70 and involve loops 3, 4 and 4, 5 (Fig. 3B).

This raised the question of whether the cytokine- and chemokine-stimulating activities involve the peptide substrate binding site. This was examined by engaging the peptide-binding domain with the high affinity peptide NRLLLTG. Stimulation of monocytes to produce TNF- or IL-12 with HSP70_359–494 that was pretreated with NRLLLTG showed comparable activity to stimulation with HSP70 alone. The results suggest that the stimulating activity of p359–494 is independent of the peptide-binding capacity of HSP70. The cytokine- and chemokine-stimulating epitope (p407–426) resides in the peptide-binding base between loops 3, 4 and 4, 5 of HSP70, whereas ligation with peptide NRLLLTG involves loops 1, 2 and 3, 4. Indeed, only 1 of the 10 anchor residues of HSP70 (V436) binding pNRLLLTG (19) is shared with the 5 critical residues (V410) defined within the stimulating epitope p407–426. These results are consistent with the recent report that a HSP70 peptide-binding mutant prevented peptide binding but retained cytokine and chemokine stimulation of DC (39).

To explore further the function of the 18-kDa peptide-binding domain and the defined p407–426 epitope, we studied maturation of DC stimulated by these peptides. HSP70_359–494 elicited maturation of DC comparable with that stimulated by native HSP70, HSP70_359–610, CD40LT, or LPS. The p407–426 epitope induced significant enhancement of maturation of DC when stimulated with either HSP70 or CD40LT. This was observed with CD83, CCR7, and CXCR4 phenotypic markers of DC. However, immature DC were only slightly affected by stimulation with p407–426. The observation that this peptide enhanced maturation of DC when stimulated not only with HSP70 but also with CD40LT is consistent with the peptide exerting its effect by ligation of the CD40 molecule, shown to be a receptor for HSP70 (9, 26). The mechanism responsible for maturation of DC is not understood. Signaling through CD40 by engagement of the trimerized CD40L is one of the best defined pathways driving DC maturation (40, 41). Other receptors, such as TLR (42) and TNF- (43) receptors may also be involved in the process of DC maturation. TLR2 and TLR4 may interact with human HSP70 and therefore may mediate HSP-stimulated DC maturation (44), although paradoxically HSP70 can be internalized into TLR4^– DC (45).

Contamination of HSP70 with LPS has been raised (46, 47), especially stimulation with huHSP70 which may share with LPS the CD14, TLR2, and TLR4 receptors (24). Removal of LPS from human HSP70 seems to have abrogated activation of human DC, without affecting peptide delivery (46). We addressed this issue by applying four criteria: 1) all three mHSP70 preparations were depleted of LPS by passing them down a Q-Sepharose and then polymyxin B column which left <0.001 enzyme U per µg of the HSP preparation; 2) HSP70 treated with proteinase K largely abrogated the stimulating activity of HSP70, whereas similar treatment of LPS had no effect; 3) treatment with the intracellular calcium-chelating agent BAPTA-AM inhibited the HSP70 calcium-dependent stimulating activity but had no effect on LPS stimulation which is calcium independent (9, 25); 4) the minimal concentration of LPS that stimulated IL-12 production or doubled the HSP70-stimulating activity was 1000 times higher than that contaminating the HSP70 preparations. Quite apart from these findings, synthetic peptides derived from the sequence of HSP70 that are free of any detectable LPS affected cytokine and chemokine production and maturation of DC. Thus, LPS contamination is most unlikely to have been involved in the present investigations.

A putative suppressor epitope became evident when costimulation of DC with HSP70 and the two adjacent 20-mer peptides (457–476 and 477–496) showed inhibition of TNF- production. The peptide specificity was demonstrated by lack of inhibition with any other two adjacent peptides. The HSP70 specificity was also established given that costimulation with LPS and p457–476/477–496 showed negligible inhibition of TNF- production. Comparable inhibition of HSP70-stimulated IL-12 production and CCL5-production by p457–496 was observed with monocytes. The results were then repeated with a single newly synthesized 40-mer peptide 457–496; this elicited similar responses to those of the two adjacent 20-mer peptides. The effect of p457–496 on maturation of DC was then studied; this also showed very significant inhibition of HSP70- or CD40LT-stimulated cell surface expression of CD83, CCR7, and CXCR4, and lesser inhibition was observed with LPS when the two adjacent peptides were used. Similar inhibition was obtained by costimulation with the single 40-mer p457–496 and HSP70 or CD40LT but not with LPS. Thus, costimulation of p457–496 with either HSP70 or CD40L inhibited DC maturation.

The effect of stimulating and inhibitory peptides on DC functions and maturation was then studied by the p38 MAP kinase phosphorylation pathway, which is critical in CD40-CD40L regulation of maturation of DC (26, 37, 38). Indeed, HSP70, CD40L, and LPS stimulate human DC to phosphorylate p38 which is inhibited by p457–496 only in the HSP70- or CD40L- but not the LPS-stimulated DC. In contrast, p407–426 slightly enhanced p38 phosphorylation. Western blot analysis of p38 MAP kinase phosphorylation confirmed the results of ELISA, showing inhibition on costimulation with HSP70 and p457–496 but enhancement with p407–426. Furthermore, the p38 MAP kinase inhibitor (SB203580) showed consistently a dose-dependent inhibition of HSP70-, CD40L- or LPS-dependent IL-12 production.

To demonstrate that the p38 MAP kinase is generated by interaction between HSP70 and CD40, we transfected HEK 293 cells with CD40 and treated these cells and HEK 293 cells alone with the three stimulants. As with the DC, HSP70- or CD40L-stimulated p38 MAP kinase phosphorylation was inhibited by p457–496 and slightly enhanced with p407–426. However, LPS failed to activate p38 and was not affected by either p457–496 or p407–426, because the cells lack CD14 receptors. The HEK-293 cells alone failed to respond to any of the stimulants. The results suggest that the two peptides modulate the CD40-dependent activation of p38 MAP kinase phosphorylation stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation pathway is also involved in maturation of DC stimulated by HSP70, CD40L, or LPS, as demonstrated by inhibition of HLA class II expression by the p38 inhibitor. This is consistent with the p38 MAP kinase data on maturation of DC recently reported (38). Thus, the p38 MAP kinase phosphorylation pathway has now been demonstrated to be involved in the alternative CD40-HSP70 pathway by ELISA of p38, by Western blot of p38 and also by inhibition of IL-12 production and DC maturation using a p38 inhibitor.

The results of this investigation have identified two discontinuous peptide epitopes within the peptide-binding portion of HSP70 (aa 357–494). The stimulating epitope p407–426 is located at the base of the peptide-binding groove of HSP70 and involves loops 3, 4 and 4, 5 (Fig. 3B). However, the inhibitory epitope p457–496 resides within the -pleated sheet. The mechanism whereby 2 opposing functions in cytokine and chemokine production and DC maturation reside within 2 peptides, 30 residues apart, must be elucidated. There are three clear differences between the two functionally opposing epitopes: 1) cytokine- and chemokine-stimulating functions of p407–426 are not dependent on HSP70 or CD40L activation, unlike the inhibitory functions of p457–496, which are HSP70 or CD40L dependent; 2) the stimulating epitope resides within the peptide-binding domain, sharing 1 of the 5 critical anchor residues, whereas the inhibitory epitope is downstream from this site; 3) p38 MAP kinase phosphorylation is inhibited by p457–496 but enhanced by p407–426. These data are consistent with the concept that p457–496 is an inhibitor of HSP70 or CD40L binding of CD40, whereas p407–426 acts as an agonist of CD40.

mHSP70 is one of the most commonly found molecules in Gram-positive and -negative organisms, parasites, and some viruses. The functional significance of this molecule has been greatly enhanced by the finding that CD40 is a prime receptor for mHSP70 (9). The importance of the CD40-CD40L costimulatory pathway has been well established (29) and plays a crucial part in the interphase between innate and adaptive immunity. The finding that mHSP70 may substitute CD40L in the CD40-costimulatory pathway greatly enhances the significance of mHSP70. Indeed, mHSP70 serves as an alternative ligand to CD4⁺CD40L⁺ helper cells in protection of Mycobacterium tuberculosis infection in CD40L knockout mice (31). Coadministration of the tolerogenic LCMV peptide with human HSP70 interacts with CD40 and may reverse tolerance and promote DC to elicit autoimmune diabetes (32). Human ATPase portion of HSP70 also binds CD40 but at a different site (26); it is the peptide binding (C-terminal) part of mHSP70 that binds the CD40 molecule (9, 26). Furthermore, the Th1-polarizing adjuvant effect of mHSP70 has been recently used in prevention of SIV infection in macaques (48) and may prove to be important in the treatment of HIV-infected patients, with low CD4⁺ T cells and CD40L expression, that may be rectified by the alternative mHSP70-CD40 pathway.

	Disclosure

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by European Commission Grants SHP-CT-2003 503240 and QLK2-CT-1999-01321 and by Guy’s and St. Thomas’ Trust.

² Address correspondence and reprint requests to Dr. Thomas Lehner, Mucosal Immunology Unit, Guy’s, King’s and St. Thomas’ Hospital Medical and Dental Schools, Guy’s Tower, Floor 28, London SE1 9RT, U.K. E-mail address: thomas.lehner{at}kcl.ac.uk

³ Abbreviations used in this paper: HSP, heat shock protein; mHSP70, microbial HSP70; huHSP70, human HSP70; CD40LT, soluble CD40L trimer; DC, dendritic cell.

Received for publication September 29, 2004. Accepted for publication December 3, 2004.

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