Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor alpha  Release by Murine Macrophages*

Baochong GaoDagger and Min-Fu Tsan

From the Institute for Clinical Research and Veterans Affairs Medical Center, Washington, D. C. 20422

Received for publication, August 26, 2002, and in revised form, October 25, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor alpha  (TNFalpha ) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70-1) with a LPS content of 1.4 pg/µg was unable to induce TNFalpha release by RAW264.7 murine macrophages at concentrations up to 5 µg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 µg/ml with a LPS content of 0.2 ng/µg was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNFalpha release was not because of defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFalpha -inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha -inducing activity as observed with rhHsp70-2. The TNFalpha -inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70-1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFalpha release from murine macrophages and that the observed TNFalpha -inducing activity in the rhHsp70-2 preparation is entirely due to the contaminating LPS.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 70-kDa heat shock proteins (Hsp70s)1 are highly conserved proteins expressed both constitutively (Hsc70) and under stressful conditions (Hsp70) in all prokaryotes and eukaryotes (1, 2). Members of the Hsp70 protein family play essential roles as molecular chaperones in the cytosol, mitochondria, and endoplasmic reticulum. Hsp70s are required for nascent or misfolded protein folding (3, 4), protein translocation into endoplasmic reticulum and mitochondria (5, 6), and uncoating of clathrin-coated vesicles (7-9). These molecular chaperone functions of Hsp70s require the enzymatic hydrolysis of ATP and cycles of bound nucleotide exchange.

Recently, Hsp70s were found to be present in circulation, and their levels were increased in a number of pathological conditions (10-12). Studies also show that Hsp70 is a potent activator of the innate immune system (13, 14). Using recombinant human Hsp70 (rhHsp70), it has been demonstrated that Hsp70 induces the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha ) and interleukin-6 via the CD14 and Toll-like receptor (TLR)-mediated signal transduction pathway (12, 15-17). Thus, it has been suggested that Hsp70, through its cytokine function, serves as a danger signal and that Hsp70 could be the endogenous ligand for the TLRs, both TLR-2 and TLR-4 (16, 17).

The CD14 and TLR complexes are pattern recognition receptors involved in the innate immunity for the pathogen recognition and host defense (18, 19). CD14, the endotoxin (lipopolysaccharide (LPS)) receptor, is a glycophosphatidylinositol-anchored membrane protein lacking transmembrane and intracellular domains (20, 21). TLRs are Type I transmembrane proteins with an extracellular domain containing a leucine-rich repeat and a cytoplasmic domain analogous to that of the interleukin-1 receptor family (18, 22). Together with CD14, TLR4 initiates signaling cascades in response to LPS, an abundant glycolipid of the outer membrane of the Gram-negative bacterial cell wall, whereas TLR2 initiates the signal cascades in response to bacterial lipoproteins, Gram-positive bacteria, mycoplasma, yeast, and spirochetes (18, 22-24).

Because rhHsp70 is produced by Escherichia coli expressing human Hsp70 cDNA, the final preparation may be contaminated with bacterial products such as LPS and lipoproteins. Contamination of rhHsp70 with LPS and/or lipoproteins could be responsible for the reported cytokine functions of Hsp70. In the current study, we demonstrated that the ability of commercially available rhHsp70 preparations to induce TNFalpha production by murine macrophages is solely due to the contaminating LPS.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant human Hsp70 proteins were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Recombinant Hsp70 was cloned from a human embryonic cDNA library and expressed in E. coli. Two preparations were available, catalog No. ESP-555 (previously ESP-755) and NSP-555 (previously SPP-755). None of the two rhHsp70 preparations contained the constitutively expressed Hsp70 (Hsc70). Both rhHsp70 preparations were assayed for their abilities to bind and hydrolyze ATP. The ESP-555 preparation was the low endotoxin preparation containing <50 EU (endotoxin units)/mg of rhHsp70 as determined using the Limulus Amebocyte lysate (LAL) assay, which was recommended for use in assays requiring low endotoxin. The NSP-555 preparation was not tested for endotoxin levels and was used in protein-binding assays. For the purpose of this report, the low endotoxin preparation, ESP-555, was designated rhHsp70-1, whereas the NSP-555 preparation was designated rhHsp70-2.

Protein-free LPS (from JM83 E. coli K-12, rough strain) was kindly provided by Dr. John E. Somerville of Bristol-Myers Squibb Co. Before use, LPS was dissolved in sterile, pyrogen-free water, sonicated on ice for 30 s with a sonic dismembrator (Fisher), and diluted with phosphate-buffered saline (Invitrogen). Polymyxin B sulfate (catalog No. P4932, cell culture-tested), phosphocreatine kinase, creatine phosphate, and ATP were purchased from Sigma. Polymyxin B-agarose (Detoxi Gel, catalog No. 20339) was obtained from Pierce. Enhanced chemiluminescence (ECL) Western blotting detection solutions were purchased from Amersham Biosciences.

Cell Culture-- RAW 264.7 murine macrophages (from American Tissue Culture Collection, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Subcultures of macrophages were prepared every 2 days by scraping cells into fresh medium.

Determination of TNFalpha Release by Murine Macrophages-- Murine macrophages were seeded in 24-well plates at 2.5 × 105 cells/well the day before the experiment. After washing 3 times with the medium, the cells were treated with or without rhHsp70 (0.1-5 µg/ml) and/or LPS (0.1-100 ng/ml) in 250 µl of medium containing 10% fetal bovine serum for 4 h at 37 °C. At the end of the treatment, media were collected and clarified by centrifugation at 10,000 rpm for 5 min in a microcentrifuge (Hermle-Labortechnik, Wehingm, Germany). TNFalpha content of the media was then determined by a quantitative sandwich enzyme-linked immunosorbent assay using the Quantikine M mouse TNFalpha immunoassay kit (catalog No. MTA00, R & D Systems, Minneapolis, MN) according to the manufacturer's recommendation. All experiments were done in duplicate.

In some experiments, rhHsp70 and LPS were heated for 60 min in a boiling water bath before being added to the cells. Likewise, in some experiments macrophages were preincubated with or without polymyxin B sulfate (10 µg/ml) for 30 min at 37 °C before the addition of rhHsp70 or LPS to inactivate LPS.

Measurements of Endotoxin Activity-- The endotoxin activities of rhHsp70 and LPS preparations were determined using the LAL assay kit (catalog No.50-648U, BioWhittaker, Walkersville, MD) according to the manufacturer's recommendation.

Quantification of rhHsp70 Protein by Gel Electrophoresis and Western Blotting-- The rhHsp70 preparations and fractions from polymyxin B-agarose columns (see "Removal of Endotoxin from rhHsp 70-2 Using Polymyxin B-agarose") were analyzed by SDS-PAGE using 7.5% polyacrylamide gels followed by staining with Coomassie Blue or by Western blotting using an antibody to recombinant human Hsp70 (catalog No. SPA-812, StressGen) followed by ECL detecting system as described previously (25). Densitometric quantification of rhHsp70 of the Coomassie Blue-stained SDS gels or immunoblots were performed using the FluorChem 8000 digital imaging system (Alpha Innotech Corp., San Leandro, CA).

Assay for the Activity of Removing Clathrin from Clathrin-coated Vesicles (Uncoating Reaction)-- This was performed as described previously (8). Briefly, the uncoating reaction mixture contained 35 µg/ml (0.5 µM) rhHsp70, bovine clathrin-coated vesicles containing 0.5 µM clathrin triskelions (kindly provided by Drs. Lois Greene and Evan Eisenberg of the NHLBI, National Institutes of Health, Bethesda, MD), 1 mM Mg-ATP, and an ATP-regenerating system consisting of 30 units/ml phosphocreatine kinase and 15 mM creatine phosphate. The mixtures were incubated at 25 °C for 10 min, and vesicles in the reaction mixture were removed by centrifugation at 1 × 105 rpm for 6 min in a TL-100 tabletop centrifuge (Beckman Instruments). Clathrin released from the coated vesicles in the supernatant was analyzed by SDS-PAGE, Coomassie Blue staining, and densitometric quantification. The results, clathrin-uncoating activities of rhHsp70, were expressed as optical density ratios of clathrin/rhHsp70.

Removal of Endotoxin from rhHsp70-2 Using Polymyxin B-agarose-- Endotoxin in rhHsp70-2 was removed using polymyxin B-agarose (Detoxi Gel, Pierce) according to manufacturer's recommendation. Briefly, aliquots of 0.5 ml of polymyxin B-agarose were poured into Poly-Prep disposable columns (Bio-Rad) and equilibrated in phosphate-buffered saline. Columns were washed with 5 volumes of 1% sodium deoxycholate followed by 10 volumes of phosphate-buffered saline. 250 µl of rhHsp70-2 at 100 µg/ml was loaded onto each 0.5-ml Detoxi column and incubated at room temperature for 60 min. The column was then eluted with phosphate-buffered saline in 250-µl fractions. The Detoxi column fractions were analyzed by SDS gels, stained with Coomassie Blue, and quantified as described above.

Statistical Analysis-- Results were expressed as the mean ± S.D. Levels of significance were determined using a two-tailed Student's t test (26), and a confidence level of greater than 95% (p < 0.05) was used to established statistical significance.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Induction of TNFalpha Release by rhHsp70 and LPS-- The reported induction of proinflammatory cytokine release by rhHsp70 was similar to the effect of LPS, i.e. mediated through the CD14 and TLR-4 receptor complex signal transduction pathway (12, 15-17). Because induction of manganese superoxide dismutase (MnSOD) by LPS is also dependent on CD14 and TLR-4 (27), we wanted to know whether rhHsp70 could also induce MnSOD. To avoid the potential confounding effect of LPS, we first studied the highly purified, low endotoxin preparation of rhHsp70, i.e. rhHsp70-1. We observed to our surprise that rhHsp70-1 not only did not induce manganese superoxide dismutase but also failed to induce TNFalpha production by RAW264.7 murine macrophages (data not shown). Failure to induce TNFalpha release from macrophages by rhHsp70-1 was in marked contrast to the reported effect of rhHsp70. For this reason, we decided to study the effects of both rhHsp70-1 and the less purified rhHsp70-2 preparation to determine whether rhHsp70 could in fact induce TNFalpha production by macrophages.

As shown in Fig. 1A, rhHsp70-1 at 1 µg/ml, as compared with control, did not cause an increase in TNFalpha release by murine macrophages. In contrast, rhHsp70-2 at the same concentration induced a marked increase in TNFalpha release to a similar extent as induced by 100 ng/ml LPS. Dose response studies revealed that rhHsp70-1 at concentrations up to 5 µg/ml failed to induce TNFalpha release. However, rhHsp70-2 at a concentration as low as 0.1 µg/ml induced a marked release of TNFalpha (Fig. 1B). Likewise, LPS at a concentration as low as 0.1 ng/ml induced a marked release of TNFalpha from murine macrophages (Fig. 1C).


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Fig. 1.   Induction of TNFalpha release from RAW264.7 murine macrophages by LPS and rhHsp70 preparations. Macrophages were treated with either LPS or rhHsp70 preparations at the indicated concentrations for 4 h. TNFalpha concentration in conditioned media was then determined. A, TNFalpha -inducing activities of rhHsp70 preparations and LPS. B, dose response of rhHsp70-induced TNFalpha release by murine macrophages. C, dose response of LPS-induced TNFalpha release by murine macrophages. Values in A and C represent the means ± S.D. of 3-6 experiments. *, p < 0.05 (versus control).

Endotoxin Contents and Functional Properties of rhHsp70-1 and rhHsp70-2-- There were two possible interpretations for the above observations. First, rhHsp70 had no effect on the TNFalpha release by macrophages; the observed effect of rhHSP70-2 was due to some contaminant(s) present in the rhHsp70-2 preparation. Second, rhHsp70 did have TNFalpha -inducing effect; failure of rhHsp70-1 to induce TNFalpha release by macrophages was due to the presence of functionally inactive rhHsp70 in the rhHsp70-1 preparation. To distinguish these two possibilities, we determined the functional characteristics as well as endotoxin contents of the rhHsp70-1 and rhHsp70-2 preparations.

As shown in Fig. 2, A and B, SDS-PAGE and immunoblot analyses of rhHsp70-1 and rhHsp70-2 revealed that both preparations were identical in rhHsp70 protein content, molecular weight, and ability to interact with an anti-rhHsp70 antibody. In addition, both preparations had similar molecular chaperone activities as determined by the uncoating of clathrin-coated vesicles (Fig. 2C). In contrast, rhHsp70-2 contained a markedly higher content of endotoxin than rhHsp70-1 as determined using the LAL assay. As shown in Fig. 2D, the endotoxin activity of rhHsp70-1 was 4.1 ± 0.2 EU/mg, that of rhHsp70-2 was 577.0 ± 74.2 EU/mg, and that of E. coli LPS was (2.9 ± 0.7) × 106 EU/mg (n = 3). Thus, the endotoxin content in the rhHsp70-2 preparation was 140 times higher than that in the rhHsp70-1 preparation. The calculated equivalent LPS concentration in the rhHsp70-1 was 1.4 pg/µg, whereas it was 0.2 ng/µg in the rhHsp70-2. As shown in Fig. 1C, the concentration of LPS present in 1 µg/ml rhHsp70-2, i.e. 0.2 ng/ml, was sufficient to cause the observed TNFalpha release from macrophages by rhHsp70-2.


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Fig. 2.   Enzymatic and endotoxin activities of two rhHsp70 preparations. A, 2 µg of rhHsp70-1 (lane 1) or rhHsp70-2 (lane 2) were analyzed on SDS gels and stained with Coomassie Blue. B, 0.5 µg of rhHsp70-1 (lane 1) or rhHsp70-2 (lane 2) was analyzed by Western blotting using an antibody to rhHsp70. C, both rhHsp70 preparations were tested for their activities in removing clathrin from coated vesicles. The uncoating reaction was carried out with clathrin-coated vesicles containing 0.5 µM clathrin triskelion, 0.5 µM either rhHsp70-1 or rhHsp70-2, 1 mM ATP-Mg2+, and an ATP-regenerating system. After removing the coated vesicles by centrifugation, the amount of clathrin released and rhHsp70 in the supernatant were quantified by SDS-PAGE and densitometric scanning. The relative uncoating activities of the two rhHsp70 preparations were plotted as optical density (OD) ratios of clathrin/rhHsp70. D, the endotoxin activity of LPS or rhHsp70 preparations was determined by LAL assay. Values in bar graphs shown in C and D represent the means ± S.D. of three experiments.

Role of LPS in the Induction of TNFalpha Release by rhHsp70-2-- To determine whether the contaminating LPS in the rhHsp70-2 preparation was responsible for its TNFalpha -inducing activity, we first used polymyxin B-agarose (Detoxi gel) to remove LPS from rhHsp70-2. The concentration of rhHsp70 in the fractions collected from polymyxin B-agarose columns was determined using SDS-PAGE and densitometric quantification. The same amounts of rhHsp70 present in the polymyxin B column fractions as in the rhHsp70-2 preparation before passing through the column (Fig. 3A) were then used to determine the endotoxin activity as well as the TNFalpha -inducing activity.


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Fig. 3.   Effect of removing endotoxin with polymyxin B-agarose on the endotoxin activity and TNFalpha -inducing activity of rhHsp70-2. The endotoxin in rhHsp70-2 was removed by passing through a polymyxin B-agarose column. Two fractions were collected from the column, PB-1 and PB-2. rhHsp70 concentrations in polymyxin B column fractions were determined by Coomassie Blue-stained SDS gels and densitometric scanning. A, 1 µg of rhHsp70-2 before polymyxin B column, PB-1 and PB-1, containing 1 µg of rhHsp70-2 were analyzed by SDS-PAGE. B, the endotoxin activities of rhHsp70-2 or polymyxin B (PB) column fractions were determined by LAL assay. C, the TNFalpha -inducing activity of 0.5 µg/ml rhHsp70-2 or polymyxin B column fractions containing 0.5 µg/ml rhHsp70 was determined. the values in the bar graphs shown in B and C represent the means ± S.D. of three experiments. *, p < 0.01 (versus rhHsp70-2).

As shown in Fig. 3B, the polymyxin B column removed more than 95% of the endotoxin activity from rhHsp70-2. The endotoxin activity of rhHsp70-2 before the polymyxin B column was 577.0 ± 74.2 EU/mg, whereas it was 26.0 ± 14.2 EU/mg and 22.0 ± 2.8 EU/mg in the two fractions of the polymyxin B column. Likewise, polymyxin B column removed most of the TNFalpha -inducing activity of rhHsp70-2 (Fig. 3C). Thus, polymyxin B column removed LPS from the rhHsp70-2 preparation along with its TNFalpha -inducing activity.

In several similar studies (12, 15, 17), polymyxin B has been directly added into the incubation medium to determine whether the observed effect is due to LPS. Therefore, we also tested the effect of polymyxin B in the incubation medium. In these experiments, macrophages were preincubated with polymyxin B at 10 µg/ml before rhHsp70-2 or LPS was added to the incubation media. To make the comparison relevant, we used an amount of LPS (0.2 ng/ml) with an endotoxin activity equivalent to that found in 1 µg/ml rhHsp70-2. As shown in Fig. 4, polymyxin B markedly inhibited the TNFalpha -inducing activities of both LPS and rhHsp70-2.


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Fig. 4.   Effect of preincubation with polymyxin B on TNFalpha -inducing activities of LPS and rhHsp70-2. Macrophages were preincubated with (solid bars) or without (open bars) 10 µg/ml polymyxin B (PB) for 30 min at 37 °C before LPS or rhHsp70-2 was added at the indicated concentrations. Macrophages were incubated for an additional 4 h before the media were collected and analyzed for TNFalpha concentration. Values represent the means ± S.D. of three experiments. *, p < 0.01 (versus no polymyxin B).

The above findings strongly suggest that the observed TNFalpha -inducing effect of rhHsp70-2 was due to the contaminating LPS. If this were the case, one would expect that the addition of LPS at a concentration found in rhHsp70-2 to the rhHsp70-1 should result in the same TNFalpha -inducing activity as the rhHsp70-2. As shown in Fig. 5, rhHsp70-1 at 1 µg/ml alone had no TNFalpha -inducing activity. However, the addition of LPS to the rhHsp70-1 at a final concentration of 0.2 ng/ml resulted in a similar TNFalpha -inducing activity as that of 0.2 ng/ml LPS or 1 µg/ml rhHsp70-2.


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Fig. 5.   The TNFalpha -inducing activity of LPS added to rhHsp70-1. Macrophages were treated with 1 µg/ml rhHsp70-1, 0.2 ng/ml LPS, 1 µg/ml rhHsp70-2, or 1 µg/ml rhHsp70-1 + 0.2 ng/ml LPS for 4 h. TNFalpha concentrations in the culture media were then determined. Values represent the means ± S.D. of three experiments.

Effect of Heat Inactivation on TNFalpha -inducing Activities of LPS and rhHsp70-2-- One of the characteristics of LPS is its relative heat resistance. Previous studies show that the TNFalpha -inducing activity of rhHsp70 was heat-sensitive, whereas that of LPS was heat-resistant (12, 15-17). It was concluded that the TNFalpha -inducing activity of rhHsp70 could not have been due to the effect of contaminating LPS. As shown in Fig. 6, we also demonstrated that the TNFalpha -inducing activity of rhHsp70-2 at 1 µg/ml, but not that of LPS at 100 ng/ml, was sensitive to prior heating in a boiling water bath for 60 min.


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Fig. 6.   Effect of heat inactivation on TNFalpha -inducing activities of rhHsp70-2 and LPS. Stock solutions of rhHsp70-2 at 100 µg/ml and LPS at 10 µg/ml were placed in boiling water for 1 h. Macrophages were treated with or without rhHsp70-1, rhHsp70-2, heated rhHsp70-2, LPS, or heated LPS at the indicated concentrations for 4 h. TNFalpha concentrations in media were then determined. Values represent the means ± S.D. of three experiments. *, p < 0.01 (versus rhHsp70-2).

The demonstrated heat sensitivity of rhHsp70-2 in inducing TNFalpha release appeared in contradiction to our above conclusion that the TNFalpha -inducing activity of rhHsp70-2 was due to the contaminating LPS. However, the following evidence suggests that LPS at low concentrations, such as 20 ng/ml, is heat-sensitive and that heat sensitivity by itself is not sufficient to distinguish whether an observed effect is due to LPS.

As shown in Fig. 7A, the addition of 0.2 ng/ml LPS to rhHsp70-1 (1 µg/ml) resulted in a TNFalpha -inducing effect similar to that of 0.2 ng/ml LPS or 1 µg/ml rhHsp70-2 (as previously demonstrated in Fig. 5). However, the TNFalpha -inducing effect of the combination of LPS and rhHsp70-1 was as heat-sensitive as that of the rhHsp70-2. Analysis of rhHsp70 using SDS-PAGE revealed that heat inactivation removed most of Hsp70 from the rhHsp70-1 and rhHsp70-2 solutions (Fig. 7B). Thus, it was possible that heat inactivation removed the rhHsp70 along with LPS from the solution, resulting in the observed lost of TNFalpha -inducing activity.


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Fig. 7.   Effect of heat inactivation on the TNFalpha -inducing activity of LPS added in rhHsp70-1. Stock solutions of 100 µg/ml rhHsp70-2 and 20 ng/ml LPS added to 100 µg/ml rhHsp70-1 were heated in boiling water for 1 h. A, macrophages were treated with either rhHsp70 or LPS at the indicated concentrations for 4 h. TNFalpha concentrations in media were determined. B, 2 µg of each rhHsp70 preparation before and after heat inactivation were analyzed on SDS gels stained with Coomassie Blue. Lane 1, rhHsp70-1; lane 2, heated rhHsp70-1; lane 3, rhHsp70-2; lane 4, heated rhHsp70-2. Values in A represent the means ± S.D. of three experiments. *, p < 0.05 (versus non-heated rhHsp70).

To eliminate the potential confounding effect of rhHsp in the above-observed heat sensitivity of LPS, we heated LPS alone at two concentrations, 20 ng/ml and 20 µg/ml, and then determined the endotoxin activity and TNFalpha -inducing activity of 0.2 ng/ml LPS before and after heating. As shown in Fig. 8, heating 20 ng/ml LPS in a boiling water bath for 60 min markedly reduced its endotoxin activity as well as its TNFalpha -inducing activity. Likewise, heating LPS at 20 µg/ml also reduced its endotoxin activity and TNFalpha -inducing activity, but to a lesser degree. Thus, LPS at low concentrations is highly heat-sensitive.


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Fig. 8.   Effect of heat inactivation on the endotoxin activity and TNFalpha -inducing activity of LPS. LPS stock solution at 20 µg/ml or 20 ng/ml was heated in boiling water for 1 h. Both heated and non-heated LPS were tested for endotoxin activity using LAL assay (A) and for TNFalpha induction in macrophages (B) at the same concentration (0.2 ng/ml). Values represent the means ± S.D. of three experiments. *, p < 0.05 (versus non-heated controls).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Results presented in the current study demonstrated that rhHsp-70 did not induce TNFalpha release from murine macrophages and that the TNFalpha -inducing activity of the rhHsp70-2 preparation was entirely due to the contaminating LPS. This conclusion was derived from the following evidence. First, the highly purified rhHsp70 preparation (rhHsp70-1) with an LPS content of 1.4 pg/µg was unable to induce TNFalpha release from macrophages at concentrations up to 5 µg/ml, whereas the less purified rhHsp70 preparation (rhHsp70-2), at 1 µg/ml, with a LPS content of 0.2 ng/µg was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS (Figs. 1 and 2D). Second, failure of rhHsp70-1 to induce TNFalpha release was not due to defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical Hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody (Fig. 2, A and B). Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles (Fig. 2C). Third, removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFalpha -inducing activity of rhHsp70-2 (Figs. 3 and 4). Fourth, the addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha -inducing activity as observed with rhHsp70-2 (Fig. 5).

Using rhHsp70 obtained from the same source as the one we used in the current study (StressGen Biotechnologies Corp.), a number of laboratories reported that rhHsp70 markedly induced TNFalpha production by monocytes and macrophages (12, 15-17). However, with the exception of one report by Dybdahl et al. (12), none of the reports indicated which rhHsp70 preparation from StressGen was used in the studies. Using the less purified preparation of rhHsp70 (NSP-555/SPP-755 or rhHsp70-2), Dybdahl et al. (12) report that their rhHsp70 preparation had an endotoxin activity of about 100 EU/mg. They demonstrated that the rhHsp70 preparation could induce TNFalpha and interleukin-6 production by murine macrophages and human monocytes at rhHsp70 concentrations of 3-10 µg/ml. The endotoxin activities present in 3-10 µg/ml rhHsp70, e.g. 0.3-1 EU/ml (equivalent to that of 0.1-0.3 ng/ml LPS), were sufficient to induce the cytokine production as demonstrated in the current study. Other studies (15-17), although not specifying which rhHsp70 preparation was studied, used two criteria to rule out the possibility that the observed cytokine-inducing effect was due to the contaminating LPS. These included the differential sensitivity of rhHSP70 and LPS to heat inactivation and the specific LPS inhibitory effect of polymyxin B. However, as demonstrated in the current study, these criteria are insufficient to rule out the possibility of LPS being responsible for the observed effects.

The current study demonstrates that LPS is heat-sensitive, particularly at low concentrations, e.g. 20 ng/ml. This appeared to be contrary to the popular belief that LPS is heat-resistant (12, 15-17). However, the immunosuppressive activity of Shigella sonnei LPS has also been shown to be heat-sensitive (28). In the current study, we show that the results of heat sensitivity experiments may vary depending on the concentrations of LPS used to activate cells. As shown in Fig. 6, when 100 ng/ml LPS was used to stimulate TNFalpha release from murine macrophages, heat inactivation of LPS appeared to have no effect on TNFalpha induction. Results in Fig. 1C showed, however, LPS at 0.1 ng/ml was sufficient to induce significant TNFalpha release from murine macrophages. Thus, even if heating in boiling water for 60 min inactivated more than 99% of LPS at 100 ng/ml, there would still be sufficient active LPS left to induce TNFalpha release, giving the impression that TNFalpha -inducing activity of LPS was heat-resistant. Results in Fig. 8 showed that heating LPS at 20 ng/ml, the same concentration found in 100 µg/ml rhHsp70-2 when it was heated, resulted in an 80% decrease in TNFalpha -inducing activity. This explains why the observed TNFalpha -inducing activity of rhHsp70-2 was heat-sensitive.

In the current study, we demonstrated that removal of the contaminating LPS from rhHsp70-2 by polymyxin B-agarose column or by preincubation of macrophages with polymyxin B almost completely eliminates the TNFalpha -inducing activity of rhHSP70-2. In contrast, others observed no significant effect of preincubation or co-incubation with polymyxin B on cytokine-inducing activity of the rhHsp70 (12, 15, 17). The reason for this discrepancy is not clear. The effect of polymyxin B may depend on the concentrations of polymyxin B and rhHsp70 and the amount of contaminating LPS present in the final incubation medium. Further studies are necessary to clarify this discrepancy.

In addition to rhHsp70, recombinant chlamydial Hsp60 and recombinant human Hsp60 have all been shown to have cytokine-inducing effects (29-32). Hsp70, Hsp90, and gp96 isolated from mouse liver are also shown to induce cytokine production (33), although at concentrations 1-2 orders of magnitude higher than the recombinant Hsp70s. In view of the current study, further investigation is required to determine whether these observed cytokine-inducing effects of heat shock proteins (Hsps) was indeed due to the Hsps themselves or the contamination of LPS and/or bacterial lipoproteins in heat shock protein preparations.

    FOOTNOTES

* This material is based upon work supported by the Medical Research Service, Office of Research and Development, Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Veterans Affairs Medical Center (10R), 50 Irving St., N. W., Washington, D. C. 20422. Tel.: 202-745-8000 (ext. 5426); Fax: 202-745-8538; E-mail: baochong.gao@med.va.gov.

Published, JBC Papers in Press, October 27, 2002, DOI 10.1074/jbc.M208742200

    ABBREVIATIONS

The abbreviations used are: Hsp70, heat shock protein 70; rhHsp70, recombinant human Hsp70; TNF, tumor necrosis factor; LPS, lipopolysaccharide; EU, endotoxin units; LAL, Limulus amebocyte lysate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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