Eukaryotic heat shock proteins as molecular links in innate and adaptive immune responses: Hsp60-mediated activation of cytotoxic T cells
Solveig H. Moré,
Minka Breloer and
Arne von Bonin
Bernhard-Nocht Institute for Tropical Medicine, Bernhard-Nochtstr. 74, 20359 Hamburg, Germany
Correspondence to:
A. von Bonin
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Abstract
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Heat shock proteins (HSP) like Hsp60, Hsp70 and gp96 act directly on antigen-presenting cells (APC), e.g. by inducing the secretion of cytokines. Here we analyzed the impact of Hsp60 on the antigen-specific activation of CD8+ T cells in a TCR transgenic system. Hsp60 induced low amounts of IFN-
in the absence of antigenic peptide; however, the release of IFN-
is increased by a factor of 310 following the addition of Hsp60 to purified populations of OT-1 [ovalbumin (OVA)257264/H2-Kb-restricted] T cells and antigen-pulsed peritoneal exudate cells (PEC) as APC. This effect is strictly correlated with the PEC ability to produce IL-12. In contrast, antigen-specific IL-2 secretion and T cell proliferation was not changed in the presence of Hsp60. Hsp60-containing OT-1 T cell cultures produced IFN-
even when the number of antigenic MHC class I complexes was too low to be stimulatory and could not be detected with specific mAb. Hsp60, thus, acts as a catalyzing molecule to initiate both innate and adaptive immune responses, and its presence (e.g. during an infection with cellular destruction) has direct consequences for the activation of otherwise `ignorant' antigen-specific T cells.
Keywords: antigen-presenting cells, danger signal, heat shock protein, Hsp60, peritoneal exudate cells
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Introduction
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The main function ascribed to heat shock proteins (HSP), not only restricted to situations of cellular stress, is to act as chaperones of nascent or aberrantly folded proteins (1). From the immunological point of view, HSP have obtained increasing interest since it could be shown that HSP like Hsp70 (24) and gp96 (5,6), purified from tumor and virus-infected cells (7), are capable of eliciting a protective cytotoxic T lymphocyte (CTL)-mediated immunity even in the absence of CD4+ Th cells (8). This immunogenic potential of HSP is based on antigenic peptides that are associated with Hsp70, Hsp90 and gp96 molecules (9). The uptake of HSPpeptide complexes is mediated by receptors specific for the individual HSP family members that are expressed on the cell surface of antigen-presenting cells (APC) (1014).
Recent studies suggested that autologous HSP preparations are delivering signals to the immune system irrespective of HSP-bound peptides. We have shown that gp96 and Hsp70 preparations derived from syngenic murine tissue are able to activate long-term established antigen-specific CTL clones in vitro and spleen cells in vivo in the absence of antigenic peptides (15). Moreover, mouse and human monocytes were found to mount proinflammatory responses when incubated with recombinant Hsp60, Hsp70 or gp96 molecules (1619). Hsp60 and Hsp70 signaling is mediated by CD14 (17,20), and in the case of Hsp60 the toll-like receptor-4 plays an important role (16).
So far, however, little information exists how HSP influence T cells during a developing immune response. In ex vivo purified T cells expressing a TCR for a MHC class II-associated antigen, Hsp60 enhanced and accelerated the activation of naive T cells (21). Since the T cell-dependent effects of HSP like Hsp70 and gp96 have been analyzed in great detail for cytotoxic T cells, we decided also to investigate a possible T cell activating function for Hsp60 in a MHC class I-restricted system. Here, we investigated the effects of recombinant Hsp60 on cytotoxic T cells derived from TCR transgenic OT-1 mice. We show that the addition of Hsp60 specifically enhances the IFN-
response in OT-1-derived T cells. In contrast to IFN-
secretion, the presence of Hsp60 did not affect the IL-2 production and proliferation of corresponding T cell cultures. Hsp60, which could originate from infected dying cells in vivo, thus, potentially enhances the activation of T cells by specifically inducing the proinflammatory cytokine IFN-
and might help to prime the immune response to control infectious agents optimally.
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Methods
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Mice, cells and antibodies
Eight- to 10-week-old OT-1 TCR transgenic mice [specific for ovalbumin (OVA)257264/H2-Kb] were maintained in the animal facilities at the BNI. All cells were cultured in RPMI 1640 medium supplemented with 10% FCS, HEPES and L-glutamine. To induce peritoneal macrophages, mice were injected with 500 µl pristane (Sigma, Deisenhofen, Germany) i.p. Peritoneal exudate cells (PEC) were harvested 56 days later by rinsing the peritoneum with ice-cold medium. Only freshly prepared PEC were used in the experiments described. FACS analysis of isolated PEC showed a >95% staining for the macrophage surface marker Mac-1. T cells from spleens of OT-1 TCR transgenic mice were enriched via adherence to nylon wool. The resulting cells were typically >70% (<4% B cells) for OT-1-TCR/CD8 double-positive cells. Quantification of cytokines was performed by ELISA, IL-8 antibodies were obtained from R & D Systems (Weisbaden, Germany), all other mAb from PharMingen (Heidelberg, Germany). NO, quantified by the accumulation of nitrite as a stable end-product, was determined by the Griess reaction (22). Briefly, 100 µl Griess reagent (1% sulfanilamide, 0.1% N-1-naphtylene-diamine-dihydrochloride, 2.5% H3PO4; Sigma) was added to 100 µl supernatant and absorbance at 560 nM was measured.
Hsp60
Recombinant human Hsp60 (ESP-740, lot 008408) and Hsp70 (SPP-755, lot 003423) were obtained from Stressgen (Victoria, BC, Canada). Quantification of contaminating endotoxins revealed a concentration of lipopolysaccharide (LPS) in the range of 515 EU/mg Hsp60 (Limulus amebocyte lysate kit, QCL-1000; Biowhittaker, Walkersville, MD).
T cell assays
Purified naive T cells (5x104/well) from OT-1 TCR transgenic mice were incubated for the indicated time periods with syngenic pristane-induced PEC or bone marrow-derived dendritic cells (DC) (5x104/well) that had or had not been pulsed with OVA257264 or OVA252269 peptide (2 h, 37°C) in the presence of native Hsp60 or Hsp60 which had been boiled for 15 min at 95°C. For cytokine quantification, supernatant from the individual cultures was collected and analyzed for IFN-
or IL-2 content respectively. Remaining cultures were labeled with [3H]thymidine for an additional 18 h to quantitate proliferation.
Activation of PEC and DC
Bone marrow-derived DC were generated as previously described (23). Briefly, bone marrow was collected from tibias and femurs of BALB/c mice and resuspended in complete RPMI 1640 medium. Cells (2x106) were placed in round plastic Petri dishes (no tissue culture surface) in 10 ml complete RPMI 1640 containing 20 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF; Biomol, Hamburg, Germany). On day 3, 10 ml RPMI 1640 was added containing 20 ng/ml GM-CSF. On day 6 and 8, 10 ml of the culture supernatants were exchanged with 10 ml fresh medium (20 ng/ml GM-CSF). On day 8, 10 µg/ml Hsp60 or 100 ng/ml LPS was added to the cultures. Control DC received no additional stimulus. On the following day, supernatants were collected and analyzed for cytokines using cytokine-specific ELISA. Cultures treated with heat-inactivated Hsp60 or LPS served as controls. Polymyxin B (100 U/ml; Sigma) was added in some experiments as an internal LPS control. Freshly isolated PEC were treated comparably for cytokine analysis.
FACS analysis
For FACS staining, Fc receptors of cell cultures were blocked for 20 min at 4°C with 100 µg of purified human Ig (Cohn II; Sigma), and subsequently stained with 1:50 dilutions of labeled anti-mouse B7-2, B220 and Mac-1-specific mAb (PharMingen) or with 50 µl of 25-D1.16 supernatant (a kind gift of J. Yewdell, NIH, Bethesda, MD) for 45 min at 4°C. Samples were washed, fixed and analyzed in a FACScan (Becton Dickinson, Heidelberg, Germany).
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Results
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Hsp60 induces antigen-specific IFN-
in OT-1 T cells in a dose-dependent fashion
Since it has been shown that Hsp60 leads to the activation of professional APC, we asked whether freshly purified T cells are also influenced by Hsp60 in their response to antigen. To this end, we employed the mouse strain OT-1 which expresses a transgenic TCR specific for the OVA257264 peptide in the context of H2-Kb. When purified ex vivo OT-1 T cells expressed low amounts of CD25, CD69 and CD44 on the cell surface, suggesting that they belong to the naive T cell population (data not shown). As shown in Fig. 1
, titrated amounts of Hsp60 lead to an increased release of IFN-
in the culture supernatant of purified OT-1 T cells and PEC pulsed with suboptimal amounts of OVA257264. This effect is antigen/peptide-specific since PEC pulsed with an unrelated peptide derived from the LCMV glycoprotein showed no increased IFN-
production (not shown). The detectable IFN-
in the supernatants is produced by the purified T cell population, since cultures containing APC from IFN-
gene-targeted knockout mice show comparable elevated levels of IFN-
in the presence of Hsp60 (M. Breloer et al., unpublished data).

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Fig. 1. The effects of HSP60 are dose dependent. OVA257264 (5 µg/ml)-pulsed C57BL/6 PEC (5x104/well) were incubated with 5x104 OVA257264-specific TCR transgenic OT-1 T cells in the absence or presence of titrated amounts of Hsp60 (x-axis) for 24 h. Shown is the amount of IFN- (A) and IL-2 (B) in the supernatants (y-axis) determined with specific ELISAs. Results are presented as means ± SD of triplicates.
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In contrast to the high induction of IFN-
by Hsp60, the presence of Hsp60 did not change the amounts of detectable IL-2 (Fig. 1B
) or the antigen-dependent proliferation significantly (data not shown). In fact, in some experiments we even observed a reduced secretion of IL-2 in Hsp60-containing cultures. These results indicate that recombinant Hsp60 specifically influences the activation of antigen-specific OT-1 T-cells, leading to increased amounts of IFN-
.
Suboptimal, non-stimulatory MHCpeptide complexes are recognized by OT-1 T cells following the addition of Hsp60
In the first set of experiments shown in Fig. 1
, the peptide was loaded into the MHC class I molecules at concentrations which allowed an activation of ex vivo purified OT-1 T cells. In a second approach, we used an extended version of the OVA257264 peptide, OVA252269 (`OVA-long'), which cannot bind to H2-Kb molecules from the outside (Fig. 2 A and B
) and needs to be processed for H2-Kb-mediated presentation to OT-1 T cells (24). RMA cells, a thymoma line which is not able to process `OVA-long', but is a potent APC for OVA257264, did not activate OT-1 T cells even at 10 times higher peptide concentrations, showing the requirement for further processing (data not shown). As shown in Fig. 2(A)
, however, PEC do not present the extended `OVA-long' peptide at sufficient quantities on the cell surface to be detected by the 25-D1.16 mAb (25). Moreover, Hsp60 did not enhance the binding of the 25-D1.16 mAb, indicating that Hsp60 has no or only a marginal influence on the processing of the `OVA-long' peptides.
Even at high concentrations of `OVA-long', OT-1 T cells were not stimulated (Fig. 2B
). Whereas IFN-
could be detected after 24 h in cultures containing OVA257264 peptide, co-incubation of `OVA-long' pulsed PEC with OT-1 T cells did not result in the activation of the OT-1 T cells. Addition of Hsp60 to cultures containing no antigen induced little amounts of IFN-
(Fig. 2B
, `no peptide'). Interestingly, addition of Hsp60 to cultures containing the `OVA-long' peptide in non-stimulatory concentrations induced IFN-
at early time points (24 h following stimulation), reaching concentrations of IFN-
that are comparable to the OVA257264-induced amounts of IFN-
in OT-1 T cells. These results indicate that T cells which are `ignorant' for low numbers of antigenMHC complexes can be activated to release specifically IFN-
when Hsp60 is present in the close microenvironment. Since the expression of co-stimulatory molecules like members of the B7 family is crucial for an optimal activation of T cells, we analyzed whether Hsp60 influences the expression of B7 molecules. As shown in Fig. 2(C)
, Hsp60 specifically increased the level of B7-2 molecules, whereas the expression levels of B7.1, MHC class (not shown) and MHC class II were less affected.
Since the Hsp60 protein may be contaminated with low amounts of LPS we had to rule out that LPS is the `co-stimulating' agent in the T cell stimulation assay. The addition of 100 ng/ml Escherichia coli-derived LPS, a >100-fold excess of the LPS present in the employed doses of Hsp60, induced a similar increase in antigen-specific IFN-
secretion in OT-1 T cells. While the Hsp60-mediated increase in IFN-
secretion was completely abrogated by boiling the Hsp60 preparations (Fig. 2B
), in contrast, heating of LPS prior to the addition to the OT-1 T cell cultures only weakly reduced the induced IFN-
. These findings show that contaminating LPS in the HSP preparation is not responsible for the observed IFN-
production in OT-1 T cells.
IL-12 plays a central role in Hsp60-mediated IFN-
induction
Recent data revealed that Hsp60 is able to stimulate murine and human APC to release cytokines (16,20). HSP-immunization bypasses CD4 help (5), therefore, a direct activation of professional APC with HSP may directly contribute to the generation of cytotoxic T cells in vivo. As shown in Fig. 3(A)
, professional APC like PEC and bone marrow-derived DC secreted IL-1, IL-6, IL-8, IL-12 and tumor necrosis factor (TNF)-
as well as NO when Hsp60 was added to the cultures, whereas the murine B cell line A20 was not activated (results not shown). Since it is known that IL-12 is an important cytokine for the induction of IFN-
in T and NK cells, we used PEC from IL-12/ mice in an independent experiment. As shown in Fig. 3(B)
, PEC from IL-12/ mice almost completely failed to increase the amount of detectable IFN-
in Hsp60-containing cultures. When stimulated with Hsp60 and LPS, PEC from IL-12/ and C57BL/6 wild-type mice released comparable amounts of, for example, TNF-
and IL-6, indicating that the principle cytokine signaling pathways in IL-12/ PEC are intact (data not shown). We take these results to conclude that the presence of IL-12 is mandatory for the Hsp60-mediated release of IFN-
.
The cytokine-inducing effect of Hsp60, in contrast to LPS-induced effects, was heat labile, since Hsp60 preparations which had been boiled prior to addition to the cultures did not induce cytokines (data not shown). Moreover, Polymyxin B, an antibiotic that binds soluble LPS, had no effect on the stimulation of professional APC with Hsp60 (Fig. 3C
), again arguing against contaminating endotoxins being responsible for the observed induction of cytokines.
Hsp60-induced IFN-
is secreted by T cells and not by NK cells
One major source of IFN-
in an ongoing immune response can be NK or NKT cells (26). The addition of LPS and HSP leads to the induction of IL-12 in professional APC (18), which is an activating cytokine for the secretion of IFN-
in NK cells. To analyze whether Hsp60 induces IFN-
in the NK cell population, we depleted the NK cells in vivo using
-asialo-GM, a potent reagent to eliminate NK1.1+ cells. As shown in Fig. 4(A)
, in vivo treatment of mice with
-asialo-GM resulted in a significant reduction of DX5+ cells (DX5 being a marker of NK and NKT cells (27) in FACS analysis. NK cell-depleted cultures, however, responded to the addition of Hsp60 equally well as normal purified T cells with regard to the IFN-
detectable in the supernatant of purified OT-1 T cells (Fig. 4B
). Taken together, these results show that the high amount of IFN-
produced in cell cultures containing PEC and OT-1 T cells is at least mainly produced by the TCR transgenic T cell population and not by contaminating NK or NKT cells.
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Discussion
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In addition to delivering peptides into the MHC class I-processing pathway, HSP themselves, irrespective of bound peptides, directly act on APC by inducing a release of inflammatory cytokines or by changing the pattern of molecules expressed on the cell surface (16,18,19). This activation of professional APC by HSP, at least in the case of Hsp60, is mediated by evolutionary conserved proteins like CD14 and toll-like receptors, and has immediate consequences for the cellular immune response. The release of chemokines like IL-8 from the APC recruits T cells to lymphoid organs and the infiltrating T cells are then completely activated by recognizing processed peptide fragments of bacterial or viral pathogens in a proinflammatory environment. HSP, thus, would act as initiators of an adaptive T cell response. In fact, in accordance with previous data, we show that Hsp60 acts directly on APC by inducing an inflammatory panel of cytokines in PEC. Moreover, we describe for the first time, at least to our knowledge, that also DC are activated by Hsp60 to secrete a comparable set of proinflammatory cytokines and, moreover, specifically up-regulated their B7-2 molecules, whereas the expression of other surface molecules, like MHC class II, was not significantly changed.
HSP are intracellular proteins and their functions as signaling molecules in the immune system are not directly obvious. However, one can envisage situations, e.g. necrotic cell death, in which intracellular proteins are delivered from inside the cell, acting as danger signals to the immune system (28) and are able to influence cellular responses in the close environment. Regarding the Hsp60-induced effects, parallels with bacterial-derived LPS are obvious. In fact, many of the read-out parameters could be mimicked very closely even by small amounts of LPS. Nevertheless, the performed controls, e.g. boiling of LPS (and Hsp60) as well as the usage of the LPS-specific inhibitor Polymyxin B, largely rule out that endotoxins are responsible for the observed effects.
One could speculate that the release of HSP and especially the release of Hsp60 from necrotic cells subsequently changes the antigen-presenting qualities of macrophages which in turn are able to induce a proinflammatory immune response in naive T cells. Interestingly, the specific enhancement of IFN-
is especially pronounced in ex vivo purified OT-1 T cells and only to much lesser extent in effector cells which had been re-stimulated in vitro (M. Breloer, unpublished observations). Thus, our data support this assumption by showing that Hsp60 creates a microenvironment in which the activation of primary T cells is significantly facilitated by the specific up-regulation of B7-2 molecules and the induction of proinflammatory cytokines. Moreover, this HSP-induced microenvironment leads to the recognition of otherwise `invisible' antigens as demonstrated with the `OVA-long' peptide in our experimental system. We cannot rule out that minor amounts of `OVA-long' are still binding from outside to MHC class I molecules, and the Hsp60-mediated activation of APC and T cells leads to the recognition of these few epitopes. It has been shown in the literature that peptides longer than 810 amino acids can bind to MHC class I molecules (29) and that very few antigenic MHC class I complexes are sufficient to activate cytotoxic T cells in special situations (30). Nevertheless, the few epitopes generated with the help of the `OVA-long' peptide in our experimental system are not sufficient to stimulate the T cells, but in the presence of Hsp60 they become visible and induce IFN-
in OT-1 T cells. These findings may have direct implications for the activation of, for example, potential autoreactive T cells which are tolerant to their antigens in an uninfected, healthy organism, but can in the course of an infection (where tissue damage occurs) be activated by the deliberation of HSP.
Hence, Hsp60 obviously plays a dual role in the course of T cell activation: Hsp60 stimulates cytokine secretion in PEC and DC, and furthermore Hsp60 enhances the activation of cytotoxic T cells through these activated macrophages. Hsp60, thus, acts as a molecular link between the innate immune system, e.g. employing toll-like receptors on APC, and the adaptive immune system, e.g. enhancing and accelerating the stimulation of cytotoxic T cells.
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Acknowledgments
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The authors especially wish to thank Dr Jon Yewdell (Laboratory of Immunology, NIH) for giving us the 25-D1.16 mAb, Svenja Ehrlich for expert technical assistance, and Dr Bernhard Fleischer for support, encouragement and critically reading the manuscript. The work contains major parts of S. H. M.'s PhD thesis. S. H. M. is funded by the Studienstiftung des deutschen Volkes. M. B. is funded by the Fritz Bender Stiftung, München, Germany.
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Abbreviations
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APC antigen-presenting cell |
CTL cytotoxic T lymphocyte |
DC dendritic cell |
HSP heat shock protein |
LPS lipopolysaccharide |
OVA ovalbumin |
PEC peritoneal exudate cell |
TNF tumor necrosis factor |
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Notes
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Transmitting editor: S. H. E. Kaufmann
Received 6 March 2001,
accepted 30 May 2001.
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