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The Journal of Immunology, 2000, 165: 2582-2587.
Copyright © 00 by The American Association of Immunologists

Saturation, Competition, and Specificity in Interaction of Heat Shock Proteins (hsp) gp96, hsp90, and hsp70 with CD11b+ Cells1

Robert J. Binder, Michelle L. Harris, Antoine Ménoret and Pramod K. Srivastava2

Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Immunization with the heat shock protein (hsp)3-peptide complexes has been shown to elicit potent CD8+ cytotoxic T lymphocyte response against the peptide chaperoned by the hsp (1). This phenomenon has been demonstrated extensively in a number of models including cancers (2, 3, 4, 5, 6, 7, 8, 9), virus-infected cells (1, 10, 11), and cells expressing minor histocompatibility (12) or model Ags (13). A remarkable attribute of this phenomenon is the extremely small quantities of peptide required for generation of CD8+ T cell responses (1). As little as a few hundred picograms of peptide, if chaperoned by hsps, are sufficient for eliciting peptide-specific CD8+ T cells. It had been demonstrated earlier that immunization with hsp-peptide complexes is exquisitely dependent on the presence of functional APCs in the immunized host (14). Collectively, these two observations led to the suggestion that the unusually high immunogenicity of hsp-peptide complexes results from the presence of hsp receptors on APCs such as macrophage and dendritic cells (15). Indeed, a number of recent studies have suggested the presence of hsp-binding moieties on the surfaces of APCs (16, 17, 18). These studies, although consistent with the idea of a receptor(s) for hsps, fall short of defining the two minimal essential criteria for receptors, i.e., saturability and competibility of binding. The studies reported here make that incremental advance and show that the interaction of the cytosolic hsps, hsp70 and hsp90, and endoplasmic reticulum resident hsp gp96, with APCs is saturable. They further show that whereas hsp90 and gp96 compete with gp96 for binding to APCs, hsp70 does not, thus suggesting the presence of a shared receptors between hsp90 and gp96 and a unique receptor for hsp70.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cells and mice

Peritoneal exudate cells (PEC) were obtained from C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) injected 5–15 days earlier with 0.5 ml pristane (2,6,10,14-tetramethylpentadecane). CD11b+ cells were positively selected from mouse peritoneal exudates by the magnetic bead system (Miltenyi Biotec, Auburn, CA). Purifications of >93% CD11b+ (as measured by FACScan analysis) were always achieved. These cells are macrophages as defined by the following: MHC II+, CD11c-, phagocytic.

FITC labeling of proteins

Labeling of proteins with FITC was performed using FITC-conjugation kits and according to the manufacturer’s recommendations. Briefly, purified hsps or mouse serum albumin (SA, 1 mg/ml) were incubated with FITC in 0.1 M carbonate-bicarbonate buffer for 2 h at room temperature. Free unconjugated FITC was removed by passing the mixture over gel filtration columns. The number of FITC molecules bound per protein molecule was estimated by measuring the optical density at 280, 495, and 490 nm as recommended. Estimates show that between 4 and 5 FITC molecules bind to each protein molecule. All conjugated proteins were analyzed by SDS-PAGE and immunoblotting with the respective anti-hsp mAbs, anti-SA Ab or anti-FITC mAb. Abs used for the immunoblots were mouse mAb SPA-820 (clone N27F3-4 specific for both constitutive hsp73 and inducible hsp72 forms), rat mAb SPA-850 (clone 9G10 specific for gp96), all from StressGen Biotechnologies (Victoria, Canada). Anti-hsp90 Ab was a rabbit mAb (NeoMarkers, Lab Vision, Fremont, CA) specific for hsp84 and hsp86. Anti-SA and anti-FITC Abs were purchased from SIGMA (St. Louis, MO). All FITC-labeled proteins were centrifuged at 100,000 x g before use to remove any particulate matter.

Purification of hsps

hsp70 and gp96 were purified from liver cells of C57BL/6 mice according to previously described methods (19, 20). Supernatants of 100,000 x g centrifugations of cell lysates were subjected to stepwise ammonium sulfate precipitations. Pellets of 50% precipitations were used for hsp70 purification. Pellets of 80% precipitates were resuspended in PBS (with 2 mM MgCl2, 2 mM CaCl2, and 2 mM PMSF) and applied to preequilibrated Con A affinity columns. Con A eluents were exchanged into a phosphate buffer with PD-10 gel exclusion columns and applied to DEAE anion exchange columns. gp96 eluted from these columns as a purified protein and was identified by immunoblotting. Pellets from 50% ammonium sulfate precipitation were resuspended in buffer D (20 mM Tris with 20 mM NaCl, 3 mM MgCl, and 15 mM 2-ME) and applied to ADP-affinity columns as previously described. hsp70 was eluted with ADP-buffer D and applied to DEAE anion exchange columns in a phosphate buffer. hsp70 eluted off these columns as a purified protein shown by a single band on SDS-PAGE and identified in immunoblotting.

hsp90 was purified according to the protocol of Denis (21) with minor modifications. Briefly, 100,000 x g supernatants were obtained from liver cell lysates and applied to a Mono Q column (Mono Q HR 16/10, purchased from Pharmacia Biotech (Uppsala, Sweden) and attached to the BIOCAD Perseptives Biosystems, Cambridge, MA) in 200 mM sodium phosphate buffer and eluted with a gradient salt concentration to 600 mM. hsp90-positive fractions were collected and changed to Tris buffer, pH 8. Hsp90 was reapplied to the MonoQ column in the Tris buffer and eluted with a gradient salt concentration from 0 to 1 M NaCl. The Mono Q column was used according to the recommended conditions. hsp90 was eluted in a pure form as shown by SDS-PAGE and identified by immunoblotting.

Immunization with hsps

Mice were immunized with each of the three hsps as previously described (1).

Visualization of hsp receptors

Incubations of indicated amounts of FITC-labeled proteins and cells were done in the presence of Carnation 1% nonfat dry milk (Nestlé, Glendale, CA) in PBS for 20 min at 4°C. After repeated washing, cells were analyzed by flow cytometry (Becton Dickinson, La Jolla, CA). Cells were also labeled with propidium iodide just before FACScan analysis. Cells staining positive for propidium iodide were gated out of the events. No differences were observed in the binding of hsps to CD11b+ cells from pristaned or nonpristaned mice. For the saturation studies, mean fluorescent intensities were obtained from histogram plots. Competition studies were performed by mixing labeled and unlabeled competitor proteins together before incubation with cells. Excess protein was removed, and mean fluorescence intensities were measured by FACS analysis.

Confocal imaging

Paraformaldehyde-fixed or unfixed cells were labeled with FITC-labeled hsp as above. Labeled cells were visualized using a Zeiss LSM confocal microscope. For internalization studies, unfixed cells were used, and images were recorded at various intervals at the exact same focal plane and view.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
FITC-labeled hsps remain immunogenic

Homogeneous preparation of hsps gp96, hsp90, and hsp70 or serum albumin (Fig. 1GoA) were labeled with FITC as described in Materials and Methods. The labeling was confirmed by SDS-PAGE and immunoblotting with anti-hsp and anti-FITC Abs (Fig. 1Go, B and C). Each hsp or albumin molecule bound to an average of five FITC molecules. The resulting 2-kDa increase in the size of the proteins was detectable by SDS-PAGE (Fig. 1GoB). By immunoblotting with an anti-FITC mAb, conjugation of gp96 to FITC was confirmed (Fig. 1GoC). Similar results were obtained with other hsps (not shown). Because modification of proteins by conjugation to FITC could result in the loss of binding of hsp to their putative receptors, we tested whether the FITC-labeled hsps remained immunogenic. We used the unique ability of hsp-peptide complexes to generate peptide-specific CTLs as the functional assay (1). FITC-labeled gp96 was complexed in vitro to the Kb-binding OVA epitope SIINFEKL and used to immunize C57BL/6 mice. Spleens were removed from the immunized mice 1 wk later, stimulated in vitro with the OVA-expressing cell line E.G7, and tested for cytotoxicity of E.G7 or EL4 cells. It was observed that FITC-labeled gp96 complexed to SIINFEKL was able to elicit SIINFEKL-specific CTLs as effectively as unlabeled gp96-SIINFEKL and thus remained immunogenic (Fig. 2Go). Control preparations of FITC-labeled gp96 uncomplexed to peptide were not able to generate SIINFEKL specific CTLs. Identical experiments were conducted with FITC-labeled hsp70 and hsp90 with similar results (data not shown). The functionally active FITC-labeled hsp preparations were therefore used for further studies.



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  		FIGURE 1. Characterization of the hsp preparations used. A, Homogeneity and identity of gp96, hsp90, and hsp70 preparations used in the study. gp96, hsp90, and hsp70 were purified as described in Materials and Methods. hsp preparations were analyzed by SDS-PAGE and silver staining (left) and by immunoblotting with anti-gp96, anti-hsp 90, and anti-hsp72/73 mAbs (right). B, hsps and SA are complexed to FITC. Homogeneous preparations of hsps, gp96, hsp90, hsp70, and SA were complexed covalently to FITC. FITC-labeled proteins were analyzed by SDS-PAGE and silver staining (B) and by immunoblotting with anti-FITC or anti-gp96 mAbs (C).

 


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  		FIGURE 2. FITC-labeled hsps are functionally immunogenic. C57BL/6 mice were immunized twice, 1 wk apart, with 50 µg FITC-gp96-SIINFEKL, gp96-SIINFEKL, or FITC-gp96 complexes i.p., as described in Ref. 1 . Spleen cells were harvested from the mice, restimulated once with SIINFEKL-pulsed APCs, and tested for the presence of anti-SIINFEKL CTLs in a 51Cr release assay using the SIINFEKL-expressing cell line E.G7 or the control EL4 cells as targets. Two mice per group were immunized; each mouse is represented by a different symbol ({circ}, •, mouse 1; {square}, {blacksquare}, mouse 2).

 
hsps bind CD11b+ cells specifically and saturably

Peritoneum-derived CD11b+ cells can represent gp96-chaperoned antigenic peptides, suggesting that they express receptors capable of internalizing hsp-peptide complexes (10). CD11b+ cells were purified from peritoneal exudates of mice and were fixed in 2% paraformaldehyde so as to render them incapable of phagocytosis. Fixed cells were incubated with FITC-labeled gp96, hsp70, hsp90, or SA. Excess or unbound protein was removed by extensive washing, and cells were analyzed by FACS analysis. As shown in Fig. 3Go, FITC-labeled hsps could effectively label CD11b+ cells. Increasing quantities of hsps used for the incubations labeled an increasing percentage of cells. The labeling was specific to hsps because SA even at the highest concentration of protein used did not demonstrate significant labeling to CD11b+ cells (Fig. 3Go). The inability of FITC-labeled SA to label cells also shows that binding of hsps was not an artifact of the FITC label. FITC-labeled hsps and SA were also incubated with Meth A tumor cells (representative of CD11b- cells). No binding of hsps or SA to CD11b- cells was observed.



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  		FIGURE 3. hsps but not SA bind specifically to CD11b+ cells but not control cells. Paraformaldehyde-fixed cells were incubated with various doses of FITC-labeled hsp or SA as indicated. Cells were extensively washed to remove excess protein and analyzed by flow cytometry as described in Materials and Methods.

 
Saturability of hsp-APC interaction was tested. Paraformaldehyde-fixed cells were used as in the previous experiment to eliminate phagocytosis, turnover, internalization, and other vital phenomena. Increasing quantities of FITC-labeled gp96, hsp90, hsp70, or SA (as a negative control), between 0 and 200 µg/ml, were incubated with a constant number of fixed CD11b+ cells, as indicated earlier. The mean fluorescent intensity was measured by FACS analysis of cells. Increasing and saturable binding of gp96 and hsp90 to APCs was observed with higher concentrations. Interestingly, both hsp90 and gp96 reached saturable binding at 100 µg/ml, although the APCs bound nearly 5 times as much hsp90 as gp96 (Fig. 4Go). hsp70 did not reach saturable binding even at 200 µg/ml. We are not certain why hsp70 does not reach saturability; we presume that this is because it binds a receptor distinct from hsp90/gp96, one with a different binding capacity, or that if it is the same receptor, it has different affinities for binding the various hsps. As in the previous experiment, no detectable binding of SA with APCs was observed even at the highest concentration of SA used (200 µg/ml).



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  		FIGURE 4. Binding of hsps to CD11b+ cells is saturable. Paraformaldehyde-fixed CD1b+ cells were incubated with increasing doses of FITC-labeled gp96, hsp90, hsp70, or SA as indicated. Cells were extensively washed to remove excess protein and analyzed by flow cytometry. Mean fluorescent intensities of labeled stains are plotted against the concentrations of proteins.

 
gp96 and hsp90 but not hsp70 compete for binding of gp96 to CD11b+ cells

FITC-gp96 (100 µg/ml final concentration) was mixed with increasing quantities (100–1000 µg/ml final concentration) of competitor (unlabeled) gp96, hsp90, hsp70, or SA, followed by incubation of the mixtures with fixed CD11b+ cells. As shown in Fig. 5Go, increasing quantities of unlabeled gp96 were able to increasingly inhibit binding of FITC-gp96 to CD11b+ cells such that at the highest concentration tested (unlabeled:labeled ratio of 10:1), unlabeled gp96 could inhibit >50% of the binding of FITC-gp96. Unlabeled SA showed insignificant and untitratable competition with FITC-gp96 under the circumstances (p < 0.005). Interestingly, unlabeled hsp90 was also able to inhibit in a titratable manner, the binding of FITC-gp96 to CD11b+ cells quite effectively such that, at the highest concentration of hsp90 tested (unlabeled hsp90:labeled gp96 ratio of 10:1), it could reduce the binding of gp96 by nearly 90%. This observation suggests that gp96 and hsp90 share a common receptor. Unlabeled hsp70 did not inhibit binding of FITC-gp96 to CD11b+ cells at the scale that gp96 or hsp90 did. At the highest hsp70 concentration tested (unlabeled hsp70:labeled gp96 ratio of 10:1), the degree of inhibition of binding of FITC-gp96 to CD11b+ cells was almost identical with the inhibition observed by SA. Nonetheless, the inhibition by unlabeled hsp70 was titratable, whereas inhibition by unlabeled SA was not. This leads us to suggest ambiguously that although hsp70 may interact with C11b+ cells through a distinct receptor, it may at high concentrations interact with the gp96/hsp90 receptor as well. There may thus exist a mechanism of cross-talk among the various hsps and their receptors.



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  		FIGURE 5. Gp96 and hsp90 compete with FITC-gp96 for binding. Paraformaldehyde-fixed cells were incubated with gp96/competitor mixtures. Gp96 was used at a final and fixed concentration of 100 µg/ml. Competitor proteins were used at increasing doses, as indicated. Cells were extensively washed to remove excess protein and analyzed by flow cytometry. Mean fluorescent intensities are plotted as a function of increasing quantities of competitor proteins.

 
Binding of hsps occurs on the surface of APCs followed by internalization of hsps

To visualize the binding of hsps to APCs and the early downstream consequences of such binding, paraformaldehye-fixed CD11b+ cells incubated with FITC-gp96 were analyzed by confocal microscopy. Peripheral staining of CD11b+ cells with FITC-gp96 was observed (Fig. 6GoA). All CD11b+ cells were found to stain; i.e., no heterogeneity in the population was observed. No staining was observed when FITC-gp96 was incubated with P815 cells, an observation consistent with the FACS data in Fig. 3Go, that CD11b- cells do not interact with hsp. Also, no staining of CD11b+ cells was observed with FITC-SA. The specificity of the interaction of hsps with APC observed by FACS analysis was thus confirmed by confocal analysis. Similar to gp96, incubation of FITC-hsp90 or FITC-hsp70 led to peripheral staining of CD11b+ cells but not to CD11b- P815 cells (Fig. 6GoB).



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  		FIGURE 6. Confocal microscopy of CD11b+ cells stained with FITC-hsps. A, PEC (CD11b+) or P815 (CD11b-) cells were fixed with paraformaldehyde and incubated with FITC-labeled gp96 or FITC-labeled SA. Labeled cells were extensively washed and analyzed for transmission (right) and fluorescence (left) by confocal microscopy using a Zeiss LSM confocal microscope at x100 magnification. B, Similar fluorescence analysis was conducted using hsp90 and hsp70.

 
To further characterize the uptake, live unfixed CD11b+ cells were incubated with FITC-gp96 and were incubated at 25°C for 0, 5, 10, 15, 20, or 30 min. FITC-gp96 was visualized by confocal microscopy to first bind to the cell surface rather uniformly (0 and 5 min), then cluster at focal points on the cell surface (10 min), followed by internalization of FITC-gp96 molecules into the cells (20 min or later) (Fig. 7Go). Such internalization was not observed to occur with fixed cells (data not shown), arguing against a passive mechanism of uptake such as diffusion.



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  		FIGURE 7. Binding of gp96 to CD11b+ cells is followed by rapid internalization. Live, nonfixed CD11b+ cells were allowed to bind FITC-gp96 at 4°C and were then incubated at 25°C for the indicated periods of time. Cells were washed extensively and visualized by confocal microscopy as described.

 
The observations reported here allow a formal claim for the existence of hsp receptors on CD11b+ cells. Specificity of binding, saturability of interaction, and the ability of the ligand to compete with itself are essential attributes of ligand-receptor interaction and distinguish it from nonspecific aggregation, phagocytosis, adventitious adsorption, etc. Each of these characteristics has been examined. Specificity of binding of gp96, hsp90, and hsp70 to APCs is shown; saturability of binding is shown only for gp96 and hsp90. hsp70-APC interaction did not arrive at saturability within the range of concentrations tested. We are not certain why hsp70 does not reach saturability; we presume that this is because the receptor that hsp70 binds to is distinct from the receptor for hsp90/gp96, one with a different binding capacity. If the receptor for hsp70 and gp96/hsp90 is the same, then we assume it has different affinities for binding the various hsps. The ability of a ligand to compete with itself was shown directly in case of gp96-APC interaction; hsp90 and hsp70 were tested for competition with gp96 but not with themselves. Thus, the data speak most completely for gp96 (and by implication, hsp90) and its receptor(s) on APCs. The studies reported by Fujihara and Nadler (22), Basu et al. (23), and Asea et al. (24) have provided preliminary evidence for the existence of hsp70 receptors on APCs.

In addition to the above, the experiments shown here hint to some other novel aspects of hsp-APC interaction. They suggest that gp96 and hsp90 share a receptor and that hsp70 may have a distinct receptor, but it may also interact with the gp96/hsp90 receptor at relatively higher concentrations. This result is consistent with the close phylogenetic relationship between gp96 and hsp90 and with the lack of homology between them and hsp70 (9, 25, 26). Conserved regions in gp96 and hsp90 are most likely to be responsible for binding to this receptor. Secondly, our present results suggest that there is no obvious heterogeneity in the CD11b+ population with respect to binding any of the three hsps. This is in contrast to the suggestion from our previous study in which only a small subpopulation of CD11b+ cells were able to re-present gp96-chaperoned peptides (10). The differences between the two studies may lie in the events further downstream of binding such that binding of hsps to the cells may be necessary but not sufficient for re-presentation of hsp-chaperoned peptides. This remains to be examined. Structural characterization of the hsp receptor(s) is the obvious next step, which will provide a degree of finality to these suggestions and, hopefully, open a new chapter in our understanding of the role of APCs in priming of specific T cell responses.

Note added in proof. A receptor for heat shock protein gp96 has recently been identified as CD91 (27).


   Acknowledgments
 
We thank Sreyashi Basu, Kirsten Anderson, Toyoshi Matsutake, and Thiru Ramalingam (all of our laboratory) for reading the manuscript critically and Caroline Goupille for her input on the FACScan data.


   Footnotes
 
1 The work was supported by National Institutes of Health Grants CA64394 and CA44786, U.S. Department of Defense Grant BAA96024, and a research agreement with Antigenics, in which P.K.S. has a significant financial interest. Back

2 Address correspondence and reprint requests to Dr. Pramod K. Srivastava, University of Connecticut School of Medicine, MC1601, Farmington, CT 06030-1920. Back

3 Abbreviations used in this paper: hsp, heat shock proteins; PEC, peritoneal exudate cells; SA, mouse serum albumin. Back

Received for publication April 25, 2000. Accepted for publication June 19, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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