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The Journal of Immunology, 1999, 162: 3212-3219.
Copyright © 1999 by The American Association of Immunologists

Human 60-kDa Heat-Shock Protein: A Danger Signal to the Innate Immune System1

Wei Chen, Ulrike Syldath, Kerstin Bellmann, Volker Burkart and Hubert Kolb2

Diabetes Research Institute, Heinrich-Heine-University, Düsseldorf, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mammalian 60-kDa heat-shock protein (hsp60) is a key target of T cell and Ab responses in chronic inflammation or atherosclerosis. We show in this study that human hsp60 is also an Ag recognized by cells of the innate immune system, such as macrophages. Both mouse and human macrophages respond to contact with exogenous human hsp60 with rapid release of TNF-{alpha}; mouse macrophages in addition produce nitric oxide. The proinflammatory macrophage response is hsp60 dose dependent and similar in kinetics and extent to LPS stimulation. Human hsp60 was found to synergize with IFN-{gamma} in its proinflammatory activity. Finally, human hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and IL-15. These findings identify autologous hsp60 as a danger signal for the innate immune system, with important implications for a role of local hsp60 expression/release in chronic Th1-dependent tissue inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Heat-shock proteins of the hsp603 family are molecular chaperones that guide several steps during synthesis, transportation, and degradation of proteins 1, 2, 3 . As a consequence, hsp60 proteins are abundant in the cell and highly conserved during evolution 4, 5 . During stress, synthesis is rapidly up-regulated, in microbial as well as in eukaryotic cells, and changes in intracellular location are noted, including some expression on the cell surface 6, 7, 8, 9 . Microbial hsp60 is a major target of the immune defense in infection 10 . Interestingly, mammalian hsp60 is also an important autoantigen during chronic inflammation or atherosclerosis 11, 12, 13, 14 . Both Ab and T cell responses to autologous hsp60 have been reported in a variety of inflammatory conditions.

In recent years, evidence has been accumulating for an important role of the innate immune system in initiating and guiding responses of the adaptive immune system, as carried by T and B cells 15, 16, 17, 18, 19 ; therefore, we wondered whether the innate immune system may recognize endogenous hsp60 and respond to it.

In the present study, we analyzed the outcome of interactions between mammalian hsp60 and mouse or human macrophages. To our surprise, we observed that human hsp60 has potent immunostimulatory properties, when added to macrophage cultures. Hsp60 thus may serve as danger signal to the innate immune system when expressed on or released from stressed autologous cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
J774 cells

The mouse macrophage cell line J774 A.1 was purchased from German Collection of Microorganism and Cell Culture (Braunschweig, Germany). J774 cells were cultured in RPMI 1640 medium (Sigma, Deisenhofen, Germany) supplemented with 10% (v/v) FCS (Life Technologies, Eggeustein, Germany), ampicillin (25 mg/L), penicillin (120 mg/L), streptomycin (270 mg/L), 1 mM sodium pyruvate, 2 mM L-glutamine, nonessential amino acids (10 ml/L, 100x), 24 mM NaHCO3, and 10 mM HEPES.

Mouse bone marrow-derived macrophages

Mice of both sexes of C57BL/6JBom mice purchased from Breeding & Research Center A/S (Bomholtgård, Ry, Denmark) were used. Bone marrow cells were obtained by flushing femurs and tibias with ice-cold PBS under sterile conditions and washed by centrifugation (500 x g, 10 min). A total of 2.5 x 106 bone marrow cells was incubated in tissue culture dishes with 10 ml of Pluznik medium (5% heat-inactivated horse serum, 15% FCS, 15% L929 cell-conditioned medium 20 , and 65% RPMI 1640 supplemented as described above) at 37°C in humidified 5% CO2 in air. After 7 or 8 days, adherent bone marrow-derived macrophages were detached by incubation with ice-cold Ca2+-Mg2+-free HBSS for 10 min, followed by twice washing with HBSS (500 x g, 5 min).

Mono Mac 6 cell

Mono Mac 6, a human monocyte line 21 , was kindly provided by Dr. H. W. Ziegler-Heitbrock (Institute for Immunology, University of Munich, Munich, Germany). Mono Mac 6 cells were cultured in RPMI 1640 medium containing the OPI (oxaloacetate, pyruvate, insulin) supplement (Sigma), 2 mM L-glutamine, antibiotics (120 mg/L penicillin and 200 mg/L streptomycin), and 10% FCS.

Stimulation of macrophage

For analysis of TNF-{alpha} or nitrite production, mouse macrophages were adjusted to a density of 1 x 106/ml and placed on a flat-bottom 96-well plate (200 µl/well). Mono Mac 6 cells were cultured at a concentration of 2 x 106/ml in 24-well plates (total volume 500 µl). For analysis of cytokine gene expression, 1.5 x 106 of macrophages per 2 ml were seeded in 12-well plates. After incubation at 37°C and 5% CO2 for 18 h, macrophages were incubated in the presence or absence of recombinant human hsp60 (StressGen Biotechnologies, Victoria, Canada), Escherichia coli LPS (Sigma), or recombinant mouse IFN-{gamma} (Sigma). To test for endotoxin contaminations, 0.1 µg/ml polymyxin B sulfate (PmB; Sigma) was added to cell cultures. At the end of experiments, culture supernatants were collected and stored at -20°C until further analyses.

TNF-{alpha} measurements

TNF-{alpha} released into culture supernatants was determined by sandwich ELISA, as described previously 22 . A 96-well NUNC-Immuno-plate (Nunc, Wiesbaden, Germany) was coated overnight at 4°C with 50 µl rat anti-mouse TNF-{alpha} mAb (5 µg/ml) or mouse anti-human TNF-{alpha} mAb (2 µg/ml) (PharMingen, San Diego, CA) diluted with coating buffer (0.1 mM NaHCO3, pH 8.2). After discarding the coating solution and two washes with washing buffer (PBS/0.05% Tween-20, pH 7.4), the wells were blocked with 250 µl of blocking buffer (PBS/2% (w/v) milk powder) for 1 h at 37°C. The plates were washed twice with washing buffer thereafter. Binding of TNF-{alpha} was performed by incubation of 50 µl of culture supernatants diluted 1/5- and 1/10-fold with PBS/2% (w/v) milk powder or serial standard dilutions of rTNF-{alpha} (Genzyme, Kent, U.K.) for 2 h at 37°C. Subsequently, the wells were washed four times. A total of 50 µl of matching biotinylated rat mAb against mouse TNF-{alpha} (2 µg/ml) or biotinylated mouse mAb against human TNF-{alpha} (1 µg/ml) was placed in each well. The plates were incubated for 1 h at 37°C. After extensive washing, the samples were incubated for 45 min at 37°C with 50 µl of 1 µg/ml peroxidase-conjugated avidin (Dianova, Hamburg, Germany) in blocking buffer and subsequently washed six times. A total of 100 µl of 1 mM ABTS (2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid)diammonium salt)/0.1 M citrate buffer, pH 4.35, containing 0.02% H2O2 was added to each well for determining the residual peroxidase activity as a measure of Abs bound. After 15–30 min of incubation at 37°C, the OD was measured at 405 nm against a reference wavelength of 492 nm on a microplate reader. The TNF-{alpha} content was calculated by using a standard curve of rTNF-{alpha} with RPMI 1640 medium alone as a blank.

Measurement of nitrite production

The amount of nitrite (NO2-) released by macrophages was detected in cell-free supernatants by the colorimetric Griess reaction, as described previously 23 . Briefly, 50 µl of the supernatant and serial dilutions of NaNO3 standard solution was placed in 96-well plastic plate, and then mixed with equal volume of Griess reagent containing 1% sulfanilamide, 0.1% naphthylethylene-diamide-dihydrochloride, and 2.5% H3PO4. After incubation for 10 min at room temperature, the OD of reaction products reflecting the concentration of NO2- was assessed at 550 nm on a Microplate reader. The results were expressed in micromoles of NO2- per ml.

Cytokine mRNA analysis

RNA was isolated immediately after termination of the experiments by adding 1 ml Trizol-Reagent (Life Technologies) to the culture plates, and following the procedure suggested by the manufacturer. Determination and quantification of specific mRNA were performed by RT-PCR, as described elsewhere 24 . In brief, first-strand cDNA synthesis was performed using the Life Technologies RT-PCR kit. Specific primers for ß-actin, IL-12 p40, and IL-12 p35 were as described previously 25 . The specific primers for IL-15 were as follows: 5' primer, 5'-CCATCTCGTGCTACTTGTGT-3'; 3' primer, 5'-CTGTTTGCAAGGTAGAGCAC-3'. A total of 4-µl aliquots of cDNA was added to 23.4-µl reaction mixture containing MgCl2 (1.25 mM final concentration, for IL-15, only 0.8 mM), all four dNTPs (4 mM), and specific primers for IL-12p40, IL-12p35, IL-15, and ß-actin. After hot start at 78°C, 1 U of Taq polymerase was added to the reaction pool. The first cycle of each PCR was started after 3 min at 95°C of denaturation time. In the next cycles, denaturation was performed for 1 min. Primer annealing was done for 1 min at 60°C (ß-actin), 58°C (IL-12p35), 55°C (IL-12p40), and 45°C (IL-15). The elongation steps were done at 72°C for 80 s (ß-actin), 40 s (IL-12p35), 30 s (IL-12p40), and 45 s (IL-15). The PCR products were separated on a 2% agarose gel in 0.5x TBE buffer (Tris, boric acid, EDTA). PCR products were blotted onto nylon membrane, followed by hybridization with specific 32P-labeled probes binding at sites between the primer sequences. Signals were quantified by measuring 32P-stimulated luminescence (PSL) with a phosphor imager. Relative PSL of cytokine mRNA was calibrated to the strength of ß-actin signal, which was set equal to 1. Controls verified a linear relationship between mRNA quantity analyzed and signals obtained.

Statistical analysis

Data were expressed as mean ± SEM. Statistical analysis was performed using the Student’s t test, two-sided. Differences were considered statistically significant with p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of TNF-{alpha} production by human hsp60 in mouse macrophages

When mouse macrophages J774 were exposed to 10 ng/ml of LPS for 7 h, substantial amounts of TNF-{alpha} were detected in culture supernatants. Culture of cells with 10 µg/ml of human hsp60 elicited a TNF-{alpha} response of similar magnitude (Fig. 1GoA). TNF-{alpha} secretion in response to human hsp60 was found dose dependent, with 1 µg/ml being the lowest effective concentration. Neutralization of LPS by 0.1 µg/ml PmB completely suppressed the TNF-{alpha} response to LPS, but not to hsp60 (Fig. 1GoA). The suppression of the response to LPS by PmB was observed down to a concentration of 0.01 µg PmB/ml. There was no suppression of the response to human hsp60 by 0.01–0.1 µg/ml PmB. Only the highest concentration is depicted in Fig. 1GoA. This finding ruled out a contribution of endotoxin to the macrophage-stimulatory property of human hsp60. Heat treatment destroyed hsp60 activity (not shown).



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  		FIGURE 1. Production of TNF-{alpha} from human hsp60-stimulated J774 cells. A, Dose dependency. J774 cells were incubated for 7 h with medium alone or with medium supplemented with different concentration of hsp60 or LPS, as indicated. Endotoxin was neutralized by the addition of 0.1 µg/ml PmB. The data represent the mean concentrations of TNF-{alpha} in the supernatant ± SEM of five independent experiments, each performed in triplicate. Significant differences to the control are indicated as ***, p < 0.001. B, Time course of human hsp60 induced secretion of TNF-{alpha}. J774 cells were incubated with or without various concentrations of hsp60 and LPS for 0–48 h. The data represent mean TNF-{alpha} concentrations in the supernatant ± SEM of five different experiments. Significant differences to the medium control were identical to A and omitted for better readability.

 
To investigate the time kinetics of TNF-{alpha} production induced by human hsp60, we analyzed the amounts of TNF-{alpha} produced by J774 cells after 2, 7, 24, and 48 h of incubation. Fig. 1GoB illustrates the kinetics of TNF-{alpha} production in response to human hsp60. TNF-{alpha} was demonstrable in culture supernatants as early as 2 h after addition of human hsp60 (Fig. 1GoB). After stimulation with the highest hsp60 concentration, TNF-{alpha} production peaked at 24 h, with TNF-{alpha} still demonstrable at 48 h. With the lower dose of hsp60, peak levels were seen earlier, at 7 h, and TNF-{alpha} concentrations were close to control levels already by 24 h of culture. The kinetics of the TNF-{alpha} response to LPS was similar to the one seen with 10 µg/ml human hsp60 (Fig. 1GoB).

Induction of NO2- production by human hsp60 in mouse macrophages

To determine whether human hsp60 affected inducible NO formation in mouse macrophages, experiments were conducted examining the effect of human hsp60 on NO2- production, including dose dependency and kinetics. As shown in Fig. 2GoA, J774 cells treated with 1 and 10 µg of human hsp60/ml released significant amounts of NO2- after incubation for 24 h (8.4 ± 0.6 and 13.5 ± 0.5 µM, respectively; p < 0.001 compared with the control group). At a concentration of 0.1 µg/ml hsp60/ml, NO2- formation was not induced. J774 cells treated with 10 ng/ml LPS also responded with nitrite production at levels comparable with those seen for human hsp60 (Fig. 2GoA). The stimulatory effect of human hsp60 could not be inhibited by the addition of PmB, while the effect of 10 ng/ml of LPS was almost neutralized by the same amount of PmB.



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  		FIGURE 2. Human hsp60 induces nitrite production in J774 cells. A, Dose dependency. J774 cells were incubated for 24 h with medium alone or with medium supplemented with different concentration of hsp60 or LPS, as indicated. Endotoxin was neutralized by the addition of 0.1 µg/ml PmB. The data represent mean nitrite levels in the culture supernatant ± SEM of four experiments. Significant differences to the medium controls were indicated as **, p < 0.01 or ***, p < 0.001. B, Time course of hsp60 induced nitrite production. J774 cells were incubated for 0–48 h with or without hsp60 and LPS. The data represent mean nitrite levels in culture supernatant ± SEM of four independent experiments.

 
Subsequently, the kinetics of NO2- production was investigated in J774 cells. Accumulation of nitrite in the supernatant was demonstrable after an incubation time of 12 h for both LPS and human hsp60. There was a steady rise of nitrite levels in both groups until the end of the observation period of 48 h. Mean nitrite levels in these experiments were higher than in Fig. 2GoA, demonstrating the biological variability of the nitrite response (Fig. 2GoB).

Human hsp60 also stimulates primary mouse macrophages and synergizes with IFN-{gamma}

Next, we analyzed whether the results obtained with a transformed mouse macrophage line could be reproduced in primary cultures of mouse macrophages. Bone marrow-derived macrophages from C57BL/6 mice were stimulated with human hsp60 or LPS, as above. As shown in Fig. 3GoA, treatment with 1 or 10 µg/ml of human hsp60 elicited dose-dependent formation of nitrite. Again, the response was similar as induced by 10 ng/ml LPS. Incubation of macrophages with IFN-{gamma} induced less nitrite than in response to hsp60. There was a strong synergistic effect between human hsp60 and IFN-{gamma} on nitrite production. Culture of macrophages with 1 µg of hsp60/ml plus IFN-{gamma} also resulted in significantly enhanced production of nitrite (32.8 ± 1.1 µM) compared with that produced by 1 µg of hsp60/ml (3.3 ± 0.5 µM) or by IFN-{gamma} alone (3.6 ± 0.1 µM, Fig. 3GoB; p < 0.001). The synergistic effect was also observed in the presence of PmB, excluding a contribution of endotoxin (Fig. 3GoB).



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  		FIGURE 3. Hsp60 induces nitrite production in mouse bone marrow-derived macrophages. A, Dose dependency. Bone marrow-derived macrophages were incubated for 24 h with medium alone or with medium supplemented with different concentration of hsp60 or LPS, as indicated. The data represent mean concentrations of nitrite in the supernatant ± SEM of four to five experiments performed in triplicate. Significant differences to medium control are indicated as ***, p < 0.001. B, Synergistic effects of human hsp60 and IFN-{gamma}. Bone marrow-derived macrophages were incubated for 24 h with medium alone or with medium supplemented with IFN-{gamma} or hsp60 and both together. Endotoxin was neutralized by the addition of 0.1 µg/ml PmB. The data represent mean nitrite levels in culture supernatant ± SEM of four to five experiments performed in triplicate. Significant differences to stimulation with hsp60 alone are indicated as ***, p < 0.001.

 
Human hsp60 is stimulatory in human monocytes

To determine whether the stimulatory properties of human hsp60 are confined to a xenogeneic system, we also analyzed the response of a human monocyte line, Mono Mac 6. Fig. 4GoA shows that human hsp60 dose dependently induced the production of TNF-{alpha} in Mono Mac 6 cells. The two higher concentrations of human hsp60 (3 and 10 µg/ml) induced significant TNF-{alpha} production in Mono Mac 6 (p < 0.001 and p < 0.0001, respectively, compared with the control group). Slightly elevated levels of TNF-{alpha} were also found in cultures with 1 µg/ml hsp60 (p < 0.05). The amount of TNF-{alpha} produced by macrophages incubated with 0.1 µg of hsp60 did not differ from that produced by the controls (p > 0.05).



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  		FIGURE 4. Effects of human hsp60 on TNF-{alpha} production in human Mono Mac 6 cells. A, Dose dependency. Mono Mac 6 cells were incubated for 6 h with medium alone or with medium supplemented with different concentration of hsp60 or LPS, as indicated. The data represent mean TNF-{alpha} concentrations in the supernatant ± SEM of three experiments performed in duplicates. Significant differences to the medium control are indicated as **, p < 0.01 or ***, p < 0.001. B, Time course of human hsp60-induced secretion of TNF-{alpha}. Mono Mac 6 cells were incubated with or without hsp60 or LPS for 0–24 h. The data represent mean concentrations of TNF-{alpha} in the supernatant ± SEM of four experiments. Significant differences to the medium control were p < 0.001 and omitted for better readability.

 
Mono Mac 6 cells released substantial amounts of TNF-{alpha} within 2 h of exposure to human hsp60 (Fig. 4GoB). Peak levels were reached at 6 h, and levels declined rapidly thereafter. These kinetics were similar to the ones obtained for stimulation with LPS (Fig. 4GoB).

Induction of IL-12 gene expression by human hsp60

In a further series of experiments, we determined whether human hsp60 might not only induce mediators characteristic of nonspecific immune defense, such as TNF-{alpha} or NO, but also mediators linking the primitive immune system to T cell responses, such as IL-12. Bone marrow-derived macrophages from C57BL/6 were cultured for 24 h in the presence of varying concentration of human hsp60. Subsequently, RNA was isolated and the amount of message for IL-12p35 and IL-12p40 was determined by semiquantitative RT-PCR, which included quantitation of PCR products by phosphor imaging and calibration to the amount of ß-actin mRNA observed. As shown in Fig. 5Go, A and B, the mRNAs encoding both IL-12 subunits were up-regulated in a dose-dependent manner following exposure to 10 µg/ml of human hsp60. In macrophages cultured only in the presence of medium, minimal IL-12 mRNA was detectable. Mean increases in IL-12p35 and IL-12p40 mRNA expression in cultures treated with 10 µg/ml human hsp60 were 13- and 9.8-fold, respectively, compared with levels in medium controls. In the presence of 1 µg/ml human hsp60, the increase of IL-12p35 mRNA was 5.8-fold and that of IL-12p40 mRNA was 5-fold. The induction of IL-12 gene expression by human hsp60 could not be ascribed to contamination with endotoxin, since the effect induced by 10 µg/ml of human hsp60 could not be inhibited by the addition of PmB. As a positive control, 10 ng/ml of LPS induced IL-12p35 and IL-12p40 mRNA production to a similar extent as seen for hsp60 (Fig. 5Go, A and B). To illustrate the mean data of several experiments presented in Fig. 5Go, A and B, bands of a single experiment obtained after gel electrophoresis and blotting are demonstrated in Fig. 5GoC.



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  		FIGURE 5. Effects of human hsp60 on IL-12 gene expression in mouse bone marrow-derived macrophages. A, IL-12p40 mRNA; B, IL-12p35 mRNA. Bone marrow-derived macrophages were incubated with medium alone, with hsp60 ± PmB, or with LPS for 24 h at 37°C. Subsequently, RNA was isolated. The data represent mean quantities of RT-PCR signals for IL-12p40 mRNA, as determined by PSL, followed by normalization to the signals of ß-actin mRNA of individual samples of three to four experiments ± SEM. Significant differences to the control are indicated as **, p < 0.01 or ***, p < 0.001. C, The blots shown are of one experiment of dose dependency of hsp60-induced IL-12 mRNA expression.

 
To investigate the kinetics of hsp60-induced IL-12 gene mRNA expression, macrophages were incubated for varying times in the presence of 10 µg/ml human hsp60. Expression of IL-12p35 and IL-12p40 was then analyzed by semiquantitative RT-PCR as before. As shown in Fig. 6Go, A and B, message for both IL-12 subunits increased within 7 h. While mRNA levels for IL-12p40 remained high at 24 and 72 h (Fig. 6GoA), there was a more transient production of IL-12p35 mRNA, which was strongly decreased already at 24 h (Fig. 6GoB).



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  		FIGURE 6. Kinetics of IL-12 mRNA expression induced by human hsp60 in bone marrow-derived macrophages. A, IL-12p40 mRNA; B, IL-12p35 mRNA. Bone marrow-derived macrophages were incubated with or without hsp60 (10 µg/ml) for 0–72 h. Total RNA was isolated from cell culture at the indicated time points. Analysis of mRNA was done as described in Fig. 5Go. The data represent mean PSL levels ± SEM of three experiments.

 
Induction of IL-15 gene expression by human hsp60

To identify whether human hsp60 would induce IL-15 gene expression, we repeated the PCR reaction with IL-15-specific primers and quantified the amount of mRNA as described for IL-12. IL-15 mRNA was up-regulated about fourfold following exposure to 10 µg/ml of human hsp60 with or without PmB (Fig. 7GoA). In response to 1 µg/ml of hsp60, there was a 2.5-fold increase of mRNA detectable. IL-15 mRNA expression was also induced by 10 ng/ml of LPS.



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  		FIGURE 7. Effects of human hsp60 on IL-15 gene expression of mouse bone marrow-derived macrophages. A, Dose dependency. B, Time course. RNA from the same experiments as in Fig. 5Go and 6Go were analyzed, and IL-15 mRNA levels were determined by RT-PCR, as described above. Significant differences to the medium control are indicated as **, p < 0.01 or *, p < 0.05. C, The blots represent the dose dependency of hsp60-induced IL-15 mRNA expression from the same experiment as that shown of Fig. 5GoC.

 
Fig. 7GoB presents the kinetics of hsp60-induced IL-15 mRNA expression. After incubation for 7 h, IL-15 mRNA levels had increased about threefold. Maximal message expression was observed at 24 h, and increased mRNA expression was still noted at 72 h. Fig. 7GoC depicts bands obtained for PCR of a single experiment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings show that human hsp60 directly induces nitrite production and cytokine synthesis in macrophages. Control experiments using PmB for neutralization of endotoxin or heat denaturing of hsp60 ruled out that the stimulatory activity of the recombinant human hsp60 preparation could be ascribed to contamination with LPS.

The initial studies were performed with a mouse macrophage line, allowing for the possibility that the inflammatory response toward homologous mammalian hsp60 may be an abnormal property acquired during cell transformation and adaption to long-term in vitro propagation. However, the same response to human hsp60 was observed with primary macrophages. Finally, human hsp60 was also added to a human monocyte line, and induction of proinflammatory cytokine secretion was seen also in this nonxenogeneic system.

The stimulatory action of human hsp60 in mammalian macrophages may be related to a similar property of bacterial hsp60 26, 27, 28, 29, 30, 31, 32 . The latter studies concur that the minimal effective dose for eliciting a cytokine response is in the microgram range, as found in this study for human hsp60. In addition, these studies excluded a role of endotoxin contamination in the effects observed. We conclude that mammalian hsp60 shares with the microbial homologues the ability to activate macrophages, and probably other cells of the innate immune system, as has been reported for microbial hsp60 31, 32 .

The mechanisms involved in the stimulatory action of microbial hsp60 have not yet been identified so that we could not test for an analogous mode of action for human hsp60. Two opposing mechanisms may be considered. Hsp60 may act as chaperone by binding to immature or partially denatured proteins 33 on the surface of macrophages or in the cell interior, after endocytosis. Hence, hsp60 would exert its physiologic function 1, 2, 3, 4 , only that the site of action and target molecules is different for exogenous hsp60 in comparison with its endogenously synthesized counterpart. The other conceivable mechanism would be that macrophages express a receptor on the cell surface, which recognizes a defined epitope of hsp60. This epitope would have to be common between bacterial and mammalian hsp60, and therefore associated with highly conserved parts of the molecule. Indeed, human hsp60 is more than 50% identical to mycobacterial hsp65 5 . Because of the similarity in action to LPS, the LPS receptor may be involved. However, it has been reported that the stimulatory action of mycobacterial hsp65 could not be inhibited by an Ab to the LPS receptor CD14 26 .

The physiologic or pathophysiologic relevance of the proinflammatory macrophage response to either microbial or autologous hsp60 presently has not been determined. In eukaryotic cells, most of the hsp60 is localized to mitochondria; cell surface expression was found increased in inflammatory conditions or during wound healing 9, 34, 35, 36, 37, 38, 39, 40, 41, 42 . Both hsp60 on cell surfaces or in mitochondria becoming accessible during cell necrosis might provide high local concentrations sufficient for stimulation of an innate immune response. Further insight will come from analyzing the stimulatory effect of membrane-bound versus free hsp60. Hsp60 may also be released from cells in soluble form, and the increased expression of hsp60 in peritoneal fluids from women with endometriosis was found associated with proinflammatory cytokines and activated macrophages 43 .

In nonstimulated monocytes or macrophages, the induction of cytokine or NO production requires prior transcriptional activity 44, 45, 46 so that it is safe to assume that human hsp60 exerts at least some of its stimulatory effects at the level of gene transcription. Direct evidence comes from the observation of significantly elevated IL-12 and IL-15 mRNA levels in macrophages exposed to hsp60. Analysis of the time course revealed peak or close to peak mRNA levels after 7 h of incubation with human hsp60, which fits with the time course of classical receptor-mediated signal transduction pathway as reported for the LPS-induced transcription of the IL-12 47 and IL-15 genes 48 .

Taken together, the available evidence supports the concept that macrophages respond to autologous hsp60 when it becomes accessible to their cell surface. Such may happen when hsp60 is set free during necrosis of tissue cells during inflammation or when hsp60 is partially translocated to the plasma membrane 8, 9 in response to diverse types of stress. Increased local expression of autologous hsp60 has been noted in many inflammatory conditions, such as rheumatoid arthritis, insulitis, Crohn’s disease, or atherosclerosis 49, 50, 51, 52, 53, 54 . Furthermore, the local overexpression of autologous hsp60 is found simultaneously with the formation of cellular infiltrates in tissues 50, 53, 54, 55 .

Autologous hsp60 when translocated to the cell surface or released during cell necrosis hence may qualify as danger Ag, as proposed by P. Matzinger 56 , which alerts the innate immune system to sites of cell stress or nonprogrammed cell death, i.e., necrosis. Local exposure of hsp60 would account for an early-onset response, which eventually would be followed by activation of the adaptive T cell-dependent immune system. In this context, it is noteworthy that human hsp60 did not only induce nonspecific proinflammatory mediators such as NO or TNF-{alpha}, but also the expression of IL-12 and IL-15. The latter two cytokines play a key role in the induction of cellular Th1-type immune responses and link the innate immune system to T cell immunity 57, 58 . Interestingly, human hsp60 was found to synergize with IFN-{gamma} in its stimulatory action of macrophages. This finding is in accord with previous studies of mycobacterial hsp65 28 and underscores a role of hsp60 in regulatory circuits between innate and adaptive immunity.

It may therefore not come as a surprise that hsp60 is a key Ag also of adaptive immune responses. This holds true for both the immune defense against infectious pathogens 10 , as well as the chronic tissue inflammation of assumed autoimmune nature 11, 12, 13, 14 . Experimentally induced sterile inflammation readily induces self hsp60-specific T cells 59 .

In conclusion, we show in this study that human hsp60 induces a proinflammatory response in mouse macrophages as well as in human monocytes. Both proinflammatory mediators as well as Th1-inducing cytokines were induced by human hsp60, which identifies hsp60 as a danger Ag on autologous cells, eliciting an innate immune response and preparing for an adaptive cellular, Th1-type, immune reaction.


   Acknowledgments
 
We thank Dr. Helga Rothe and Dr. Jügen Radons for help with some experiments, and Dr. Victoria Kolb-Bachofen for reading the manuscript.


   Footnotes
 
1 This work was supported by the Bundesminster für Gesundheit, the Minister für Forschung und Wissenschaft des Landes Nordrhein-Westfalen, the Deutsche Forschungsgemeinschaft, and the Deutscher Akademischer Austauschdienst. Back

2 Address correspondence and reprint requests to Dr. Hubert Kolb, Clinical Department, Diabetes Research Institute, Auf'm Hennekamp 65, D-40225 Düsseldorf, Germany. E-mail address: Back

3 Abbreviations used in this paper: hsp, heat-shock protein; NO, nitric oxide; PmB, polymyxin B; PSL, 32P-stimulated luminescence. Back

Received for publication August 24, 1998. Accepted for publication December 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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