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(Journal of Leukocyte Biology. 2000;68:729-736.)
© 2000 by Society for Leukocyte Biology

Differential expression of heat shock protein 70 (hsp70) in human monocytes rendered apoptotic by IL-4 or serum deprivation

Detlef Lang*, Andreas Hubrich*, Frank Dohle*, Martin Terstesse*, Hilmi Saleh*, Michael Schmidt*, Hans-Gerd Pauels{dagger} and Stefan Heidenreich*

* Department of Medicine and
{dagger} Institute of Immunology, University of Münster, Münster, Germany

Correspondence: Dr. Detlef Lang, Department of Medicine D, University Hospital Münster, Albert-Schweitzer-Str. 33, D-48129 Münster, Germany. E-mail: langd{at}uni-muenster.de


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ABSTRACT
 
Apoptosis of monocytes is regulated by the balance between pro- and antiapoptotic triggers and pathways and may strongly influence inflammatory disorders. The major heat shock protein, hsp70, is an effective inhibitor of apoptosis in lymphocytic and monocytic tumor cell lines, but the implications in the regulation of apoptosis of freshly isolated human monocytes have not been elucidated. In this study, we examined whether two different triggers of monocyte apoptosis, serum deprivation and IL-4, respectively, altered hsp70 expression and whether expression levels correlated with monocyte survival. Monocyte apoptosis was determined quantitatively by flow cytometry detecting annexin V binding or nuclear stainability with propidium iodide (PI). Hsp70 expression was analyzed by semiquantitative RT-PCR and immunoblotting. Exposing monocytes to heat shock (47°C, 20 min) induced a rapid and marked upregulation of hsp70 without evoking injury or apoptosis, suggesting that hsp70 conferred protection and survival. In accordance, when monocytes were rendered apoptotic by serum deprivation, a drastic downregulation of hsp70 occurred, which was accompanied by a reduced synthesis of the constitutive family member hsc70. However, induction of monocyte apoptosis by IL-4 increased hsp70 expression in a concentration and time-dependent fashion. A neutralizing antibody against IL-4 abolished hsp70 expression and apoptosis induction after IL-4 treatment and so excluded indirect effects. LPS rescued monocytes from apoptosis but did not alter hsp70 formation significantly. These findings suggest that, in monocytes, distinct apoptotic triggers induce different responses of hsp70 so that this molecule does not exert protection against cell death directly or in general.

Key Words: apoptosis • heat shock protein • IL-4 • LPS • monocytes


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INTRODUCTION
 
Heat shock proteins (hsp) represent a highly conserved family of proteins whose expression is induced in situations involving cell stress and other forms of injury [1 2 3 ]. They are normally localized in the cytoplasm and nucleus. In humans, the major group of hsp is the 70-kDa hsp (hsp70) family, which comprises constitutively expressed and inducible proteins acting as chaperones [3 , 4 ]. Molecular chaperones facilitate protein folding and organize the transport and degradation of proteins [5 ]. The recovery of hsp is associated with a state of stress tolerance of cells, e.g., cells become resistant to lethal temperatures. Induction of hsp70 in different cell lines also increases the resistance to apoptotic triggers. Cells overexpressing hsp70 were protected against cytotoxicity induced by tumor necrosis factor (TNF), nitric oxide (NO), oxidative stress, chemotherapeutic agents, ceramide, or radiation [6 7 8 9 ]. On the other hand, not all studies support the notion that hsp generally confer cellular protection. So, it has been shown that overexpression of hsp70 enhanced the Fas-mediated apoptosis in Jurkat cells [10 ]. In monocytic U937 cells, an excess of hsp90 was associated with increased apoptosis after induction with TNF-{alpha} and cycloheximide [11 ].

For freshly isolated human monocytes, the significance of hsp expression for the protection against injury and counterregulation of apoptotic pathways has not been elucidated. Monocytes/macrophages play a central role in innate and acquired immunity of the host being engaged in the defence against infectious microorganisms and tumors. It has been shown that monocytes in culture were rescued from constitutively occurring apoptosis by serum supplementation or growth factors [12 , 13 ]. Activation in response to lipopolysaccharides (LPS) or inflammatory cytokines such as TNF or interleukin (IL)-1, acting in an autocrine or paracrine way, antagonized apoptitic processes effectively [14 ]. On the other hand, antiinflammatory mediators such as IL-4 [15 , 16 ] or crosslinking of the cell-surface death receptor Fas [17 , 18 ] are stimuli able to evoke monocyte apoptosis. Previously, we could show that downregulation of the CD14 cell-surface receptor is an early effector mechanism preceding and inducing monocyte apoptosis [19 ]. Survival or early apoptosis of monocytic cells can be crucial steps to determine spread or resolution of an inflammatory disease. The understanding of the mechanisms involved in the induction or inhibition of apoptotic events could offer therapeutic implications. In the present study, we investigated the role of hsp70 as a putative protection factor against monocyte apoptosis induced by serum deprivation or IL-4.


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MATERIALS AND METHODS
 
Abs and reagents
Phycoerythrin (PE)-conjugated mouse anti-human Leu M3 monoclonal antibody [mAb; anti-CD14, clone P9, immunoglobulin G (IgG)2b] and control mAb of appropriate Ig isotypes were obtained from Becton Dickinson (Palo Alto, CA); fluorescein isothiocyanate (FITC)-labeled annexin V, from Bender Medsystems (Vienna, Austria); and FITC-labeled F(ab')2 fragments of goat anti-mouse IgG, from Dianova (Hamburg, Germany). mAb, against the inducible form of 70-kDa hsp (mouse anti-human IgG2b mAb, clone 5G10) and the constitutive form of 73-kDa hsp (hsc70; mouse anti-human IgG2a mAb, clone 1B5), was purchased from PharMingen (Los Angeles, CA). Human rIL-4 (specific activity 5.0x105 U/mg) was purchased from Boehringer Mannheim (Mannheim, Germany); neutralizing polyclonal sheep anti-human IL-4 ab, from Genzyme (Cambridge, MA); reverse transcriptase (RT), from Stratagene (Heidelberg, Germany); Taq DNA polymerase, from Gibco BRL (Karlsruhe, Germany); dNTPs, from New England Biolabs (NEB; Beverly, MA); pd(N)6, from Boehringer Mannheim; and agarose, from AGS (Heidelberg, Germany). LPS was from Escherichia coli, strain 0127:B8 (Sigma, Deisenhofen, Germany). All other reagents were obtained from Sigma, unless otherwise indicated.

Monocytes and cell culture
Human monocytes were isolated from leukocyte buffy coats or healthy volunteers. Mononuclear cells were obtained by Ficoll-Hypaque density-gradient centrifugation (400 g, 30 min), washed, and purified further by centrifugation on a hypotonic Percoll density gradient [54% in phosphate-buffered saline (PBS); 400 g, 20 min]. Two interphases were found, in which the upper phase contained the enriched monocytes. Cells were collected, washed three times in cold PBS, and seeded out in 24-well culture plates (Greiner, Nürtingen, Germany) in RPMI 1640 culture medium (CM) containing 2 mM L-glutamine, 50 µg/ml penicillin/streptomycin, and 5 mM HEPES at 37°C in a 5% CO2/95% air atmosphere. Monocytes were purified further by the adherence to the culture plates, which finally gave a purity of >85%, as assessed by flow cytometry on a FACScan flow cytometer (Becton Dickinson) defined by forward- and side-light scatter properties, detection of the CD14 surface molecule, and staining for nonspecific esterase. Monocytes (2x106/ml) were incubated in CM without or with fetal calf serum [FCS; 0.5–5% (v/v), endotoxin content<0.01 ng/ml]. When monocytes were treated with heat shock (47°C, 20 min; in a water bath), IL-4 (2.5–25 U/ml) or LPS (10 ng/ml), 5% ICS containing CM, was used. Incubation times are given in Results or in the figure legends.

Quantification of apoptosis by flow cytometry
Monocyte apoptosis was determined by flow cytometry by detecting CD14 expression and annexin V binding simultaneously, as described previously [19 ]. In this previous study, we could show that monocytes first down-regulate CD14 expression before becoming apoptotic. So monocytes were identified as apoptotic when CD14 expression was low, and annexin V binding was high, after gating out lymphocytes by forward- and side-scatter properties using flow cytometry. This assumption was confirmed by DNA electrophoresis and electron microscopy studies [19 ]. Monocytes, prepared and treated as described above, were double-labeled with PE-conjugated Leu M3 mAb and annexin V-FITC in staining buffer [SB; containing 1% bovine serum albumin (BSA) in 50 mM HEPES buffer, pH 7.4] for 15 min on ice. PE- and FITC-conjugated murine IgG mAb of unrelated specifities were used as controls. After staining, cells were washed and fixed in 4% paraformaldehyde to apply flow cytometry.

A second flow cytometric method detecting apoptosis by propidium iodide (PI) staining of nuclei was performed, which is based on the principle that, after DNA fragmentation, permeabilized cells exhibit a reduced chromatin stainability and accessibility to fluorochromes [20 ]. Monocytes were washed in SB, fixed with 4% paraformaldehyde, and permeabilized with 0.1% saponin. For staining, PI (5 µg/ml) was applied for 15 min before cells were washed again in SB containing 0.1% saponin.

Evaluation of cell necrosis
Viability of monocytes after different treatments was determined by trypan blue exclusion or PI uptake of nonpermeabilized cells using flow cytometry by standard protocols.

Western blotting
Cells were washed in PBS; resuspended at 106 cells/100 µl of sample buffer containing 2% sodium dodecyl sulfate (SDS), 62.5 mM Tris HCl (pH 6.8), 10% glycerol, and 5% ß-mercaptoethanol and bromphenol blue; heated at 95°C for 10 min; and stored at -20°C until analysis. Samples were separated by a NuPAGE Gel 4–12% Bis-Tris (Novex, Frankfurt, Germany) and transferred to nitrocellulose membranes [BA83 (0.2 µm) Schleicher and Schuell, Dassel, Germany] by a Western-blot modul (Novex). Membranes were washed three times with potassium-phosphate buffer (0.05 M K3PO4, pH 8.5) and incubated with digoxigenin-3-O-methylcarbonyl-{varepsilon}-aminocapron acid-N-hydroxy-succinimidester (DIG-NHS-ester) and Nonidet P-40 (0.01% v/v) for 2 h. Then, membranes were blocked with 5% fat-free milk powder in TTBS buffer (Tween 20 0.01%, Tris HCl 0.05 M, NaCl 0.15 M, pH 7.5) and afterward incubated with the indicated antibodies. Reactive bands were visualized after incubation with anti-mouse IgG peroxidase-labeled Fab fragments and staining with bone marrow (BM) Teton. As an isotype-matched control for the primary Ab, mouse IgG2b or IgG2a was used. Bio-imager Master 3D was used for analysis of visualized bands.

Semiquantitative RT-polymerase chain reaction (PCR)
RNA isolation from monocytes after stimulation was conducted by RNeasy Kit (Quiagen, Hilden, Germany), according to the manufacturer’s instructions. Before transcribing into cDNA, DNase (DNase I, RNase-free, Boehringer Mannheim) digestion was performed. cDNA was synthesized after the addition of 5 µM random primers [pd(N)6, Boehringer Mannhein], 1 mM dNTPs (NEB), and incubation at 37°C with moloney murine leukemia virus RT (Stratagene). Contamination with DNA was excluded by performing PCR from templates incubated without RT. The primers used for PCR amplification were 5'-ATG GAT GAT GAT ATC GCC GCG-3' and 5'-TCT CCA TGT CGT CCC AGT TG-3' (human ß-actin, 248 bp), as well as 5'-CAC CAC CTA CTC CGA CAA CCA-3' and 5'-GCC CCT AAT CTA CCT CCT CAA TG-3' (human hsp70, 644 bp). The PCR reaction mixture (40 µl) contained 2 mM MgCl2, 0.2 mM dNTP, 1 µM primers, and 1 U Taq DNA polymerase. Samples were amplified during 30 cycles by 60 sec denaturation at 94°C, 30 sec annealing at 62°C (hsp70) or 55°C (ß-actin), and 60 sec elongation in a Peltier thermal cycler (Biometra Uno II Thermocycler, Biometra, Göttingen, Germany).

For semiquantitative PCR, the relation between the expression of ß-actin and hsp70 was analyzed. Signal intensity, as measured by PCR products, was analyzed on a 1.5% agarose gel and visualized by ethidium bromide staining. Densitometric quantification of PCR signals was performed by the Bio Image Intelligent Quantifier program (Bio Image, Ann Arbor, MI).

Statistical analysis
Results are given as means ± SD. For statistical analysis, Mann-Whitney U test was used; for paired comparisons, Wilcoxon signed rank test was performed. P < 0.05 was considered statistically significant.


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RESULTS
 
Induction and inhibition of monocyte apoptosis by different treatments and effect of heat shock on cell viability
Monocyte apoptosis was quantitatively determined by flow cytometry detecting CD14 expression and annexin V binding simultaneously (Fig. 1 ). Cells, which expressed low levels of CD14 and were positively stained by annexin V, were indicated as apoptotic monocytes, as previously demonstrated [19 ]. Figure 1A shows that only 5% of monocytes cultured in medium supplemented with 5% FCS (control) for 60 h were CD14low/annexin Vhigh monocytes representing apoptotic cells (lower right quadrangle). However, when monocytes were cultured in medium without serum, 39% of cells underwent apoptosis (Fig. 1B) ; in monocytes treated with IL-4 (10 U/ml), apoptosis occurred in 21% of analyzed cells (Fig. 1C) . Monocytes treated with LPS (10 ng/ml) were protected against constitutively occurring apoptosis so that only 1% of cells were apoptotic 60 h after preparation (Fig. 1D) . When monocytes were stressed by heat (47°C, 20 min), no injury occurred, and the apoptosis rate was similar, as compared with control (Fig. 1E) . These results were confirmed by the evaluation of nuclear alterations indicative for apoptosis, as measured by low PI staining of cells after permeabilization (Table 1 ). Again, serum deprivation and IL-4 treatment, respectively, induced significant levels of apoptosis, whereas LPS rescued monocytes from apoptosis. Heat shock did not evoke apoptosis or necrosis, as shown by trypan blue exclusion (Table 1) .



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  		Figure 1. Effects of serum deprivation, IL-4, LPS, and heat shock on monocyte CD14 expression and annexin V binding determined by flow cytometry. Cells depicted in the lower right quadrangle (CD14low/annexin Vhigh) were identified as apoptotic monocytes. Monocytes were cultured in medium supplemented with 5% FCS (control, A), without FCS (without serum, B), with IL-4 (10 U/ml) in 5% FCS (C), with LPS (10 ng/ml) in 5% FCS (D) for 60 h, or stressed by heat (47°C; 20 min) prior to the 60 h culture under control conditions (E). Significant induction of apoptosis occurred by serum deprivation or IL-4 and rescue from apoptosis, by LPS. Repesentative tracings out of 12 independent experiments are shown.


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  		Table 1. Degree of monocyte apoptosis and necrosis after heat shock, LPS, or IL-4 treatment, or after serum deprivation without or with heat shock. Apoptosis was determined by low PI staining using flow cytometry, necrosis by trypan blue exclusion

Expression of hsp70 after apoptosis induction, LPS-dependent rescue from apoptosis, or heat shock
Because hsp70 has been attributed to protection against injury and apoptosis in monocytic and lymphocytic cell lines, as well as in tumor cells, we determined hsp70 expression of monocytes after the above-mentioned treatment protocols on mRNA and protein levels by RT-PCR and immunoblotting, respectively. The main objective of our study was to correlate hsp70 expression with monocyte viability to assess the significance of hsp70 as an antiapoptotic protein. As shown in Figure 2A , monocytes expressed low levels of hsp70-specific mRNA under control conditions (5% FCS) after a 60 h culture, which were not significantly altered when monocytes were rescued from apoptosis by LPS. Heat shock significantly upregulated hsp70. When apoptosis was induced by serum deprivation (0% FCS) or IL-4, respectively, contrary hsp responses were determined. Serum deprivation completely abolished hsp70 mRNA expression, whereas IL-4 markedly increased expression. These results obtained by RT-PCR were confirmed completely by immunoblotting detecting hsp70 protein expression (Fig. 2B , lower panel). When the constitutive hsc70 protein was detected by immunoblotting, expression was similar in monocytes treated with 5% FCS, IL-4, heat shock, or LPS. However, hsc70 expression was markedly reduced in cells after serum starvation (0% FCS), suggesting that, by this mode of treatment, apoptosis was accompanied or induced by a down-regulation of the complete protein synthesis (Fig. 2B , upper panel). Densitometric analysis of hsp70-specific RT-PCR signals confirmed the results of the examples given in Figure 2 , indicating that IL-4 and heat shock significantly enhanced expression, whereas 0% FCS abolished hsp70 expression (Fig. 3 ).



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  		Figure 2. Effects of serum deprivation (0% FCS), control medium (5% FCS), IL-4 (10 U/ml), heat shock (47°C; 20 min), or LPS (10 ng/ml) on hsp70 expression of monocytes 60 h after isolation as determined on an mRNA level by RT-PCR (A) or a protein level by immunoblotting (B). Western blotting was done with an anti-hsc70 (73 kDa) antibody to determine the constitutive family member (upper panel) and an anti-hsp70 antibody (lower panel). Increased hsp70 expression for IL-4 and heat, and abolished hsp70 expression after serum deprivation as compared with control (5% FCS) were found. M, marker {phi}X174 HaeIII Digest in A, and 70 kDa protein marker in B. A representative blot out of six independent experiments is depicted.



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  		Figure 3. Densitometry of hsp70-specific RT-PCR signals of monocytes treated by serum deprivation (0% FCS), control medium (5% FCS), IL-4 (10 U/ml), heat shock (47°C; 20 min), or LPS (10 ng/ml) for 60 h. Hsp70 expression was enhanced significantly by IL-4 and heat shock. LPS did not change the signal intensity significantly. Signal intensities are given in arbitrary units as means ± SD from six independent experiments. *P < 0.05 as compared with unstimulated monocytes in 5% FCS medium.

Recovery of monocytes after an incubation of 60 h was always above 90% and was not affected by the different modes of treatment.

Time course of hsp70 expression after heat shock and implications for protection against injury
When monocytes were stressed by heat, upregulation of hsp70-specific mRNA occurred within minutes (0.1 h) and remained increased during a culture of up to 60 h, as compared with cells not treated by heat shock (control; Fig. 4A ). These data were corroborated on a protein level (Fig. 4B) . As shown in Figure 1 and Table 1 , heat shock even in the applied sublethal range did not evoke injury or apoptosis so that monocyte viability was maintained most likely by hsp70 induction. This assumption was supported by the detection of marked apoptosis and necrosis (Table 1) when monocytes were first cultured in medium without serum over 24 h and consecutively heat-stressed, which prevented hsp70 induction (unpublished results).



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  		Figure 4. Effect of heat shock (47°C; 20 min) and time of monocyte culture after heat shock (0.1–60 h) on hsp70 expression as determined by RT-PCR (A) or immunoblotting (B). Maximum hsp70 expression was found when monocytes were harvested 0.1 h after heat shock. Densitometric signal intensity in arbitrary units for RT-PCR: control, 16.1 ± 1.2; 0.1 h, 66.4 ± 5.3; 12 h, 41.2 ± 3.7; 36 h, 37.5 ± 1.8; 60 h, 25.5 ± 2.9. M, marker {phi}X174 HaeIII Digest in A, and 70 kDa protein marker in B. Data out of six independent experiments are summarized.

Concentration-dependent effect of serum depletion on monocyte apoptosis and hsp70 expression
Figure 5A shows that reduction of the FCS concentration in CM from 5%–0% enhanced apoptosis of monocytes cultured for 60 h in a concentration-dependent manner. Depletion of serum and increase of apoptosis were significantly paralleled by a linear reduction of hsp70-specific mRNA (Fig. 5B) , suggesting that hsp70 might function as a protection factor against apoptosis.



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  		Figure 5. Effect of FCS concentration in CM on monocyte apoptosis as determined by annexin V binding (A) and hsp70-specific mRNA levels (B) after a 60 h culture. Reducing FCS concentration from 5% (control) to 0% increased monocyte apoptosis significantly but reduced and abolished hsp70 expression in a linear fashion. Densitometric signal intensity in arbitrary units for RT-PCR: 0% FCS, 2.1 ± 1.2; 0.5% FCS, 4.8 ± 0.7; 1% FCS, 8.1 ± 0.8; 5% FCS, 12.2 ± 2.1. Five independent experiments were performed, and under B, a representative blot is depicted. M, marker {phi}X174 HaeIII Digest. *P < 0.05 as compared with 5% FCS in medium.

Concentration-dependent effect of IL-4 on monocyte apoptosis and hsp70 expression
Figure 6A shows that IL-4 in the concentration range between 0.5 U/ml and 25 U/ml dose-dependently enhanced monocyte apoptosis when applied for 60 h. Increasing concentrations of IL-4 and levels of apoptosis were accompanied by a linear increase of hsp70-specific mRNA (Fig. 6B) . This finding suggests that hsp70 induced by IL-4 was not able to counterregulate the apoptotic process of human monocytes.



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  		Figure 6. Effect of IL-4 concentration on monocyte apoptosis as determined by annexin V binding (A) and hsp70-specific mRNA levels (B) after 60 h as determined by RT-PCR. A dose-dependent increase of apoptosis and of hsp70 by IL-4 was found. ctl, Control without IL-4; M, marker {phi}X174 HaeIII Digest. Five independent experiments were performed, and under B, a representative blot is depicted. Densitometric signal intensity in arbitrary units for RT-PCR: control, 14.1 ± 1.3; 2 U/ml IL-4, 28.4 ± 3.3; 10 U/ml IL-4, 32.2 ± 3.7; 25 U/IL-4, 45.5 ± 5.8. *P < 0.05 as compared with medium without (0) IL-4.

Time course of hsp70 expression in response to IL-4
The time dependency of hsp70 expression by IL-4 was analyzed for incubation times of 1 h, 10 h, and 60 h (Fig. 7 ). Although IL-4-dependent hsp70 expression was maximal after 60 h, a significant increase in comparison with medium control was already noted after 1 h. The late hsp70 peak after IL-4 treatment stands in contrast to the immediate stimulation in response to heat and can be explained by a slow induction of apoptosis and injury by the cytokine.



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  		Figure 7. Time course of hsp70 mRNA expression by IL-4. Densitometry of hsp70 mRNA expression detected in RT-PCR from unstimulated monocytes (open bars) and monocytes stimulated with IL-4 (10 U/ml) (solid bars). IL-4 increases the expression of hsp70 mRNA significantly (*P<0.05 as compared with unstimulated monocytes in 5% FCS medium) in a time-dependent fashion. Three independent experiments were performed.

Effect of a neutralizing antibody against IL-4 on monocyte apoptosis and hsp70 expression induced by IL-4
Administration of a neutralizing anti-IL-4 antibody on monocytes treated with IL-4 did not only reduce apoptosis to baseline levels (Fig. 8A ) but also abrogated hsp70-specific mRNA expression completely (Fig. 8B) . This result underlines a direct effect of IL-4 on hsp70 and widely excludes indirect cytokine-coupled effects.



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  		Figure 8. Induction of apoptosis (A) and hsp70 mRNA (B) by IL-4 is specifically blockable by a mAb against IL-4 ({alpha}IL-4). Apoptosis and hsp70-specific mRNA expression induced by IL–4 are reduced to control levels by {alpha}IL-4 as determined by fluorescein-activated cell sorter (FACS) analysis (A) or RT-PCR (B). Three independent experiments were performed. *P < 0.05 as compared with control.


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DISCUSSION
 
Monocytes are centrally involved in the pathogenesis of various inflammatory disorders with sepsis syndrome and shock, representing the most harmful conditions. In vitro, monocyte apoptosis can be induced easily by heterogeneous stimuli such as serum- or growth-factor deprivation, cytokines (e.g., IL-4 or IL-10), ligation of the Fas surface receptor, or chemotherapeutic drugs [12 13 14 15 16 17 18 19 ]. The interregulation between pro- and antiapoptotic mechanisms of monocytes may be disturbed during inflammatory or septic diseases with a reduced sensitivity against proapoptotic triggers or a predominance of antiapoptotic pathways. A recent study by Perera and Waldmann [21 ] showed that the LPS-dependent resistance of human monocytes against apoptosis was mediated by a down-regulation of caspase-8 and a dramatic induction of the bfl-1 gene. Aside from the different antiapoptotic members of the bcl-2 gene family, hsps have been shown also to exert antiapoptotic properties and even to prevent necrotic damage [6 7 8 9 ]. The present study was conducted to elucidate the significance of hsp70 for the protection of freshly isolated human monocytes against apoptosis induced by serum deprivation or IL-4, respectively. Our data show that human monocytes markedly upregulated hsp70 a few minutes after a heat shock of 47°C over 20 min. This result confirmed that the primer and mAb detecting hsp70 were specific for the inducible form of hsp70. Expression of hsp70 declined with time but after 60 h of culture expression, was still enhanced, as compared with cells not exposed to heat stress. The rapid and massive induction of hsp70 after heat shock allowed survival of monocytes, even after a stress with a sublethal temperature, without exerting increased levels of apoptosis. This notion is supported by the detection of necrosis and pronounced apoptosis when monocytes were preincubated in medium without serum for 24 h and consecutively stressed by heat, which prevented hsp70 induction in parallel with evoking necrotic damage (Table 1) . These findings suggest that hsp70 counterregulates injury and apoptosis of monocytes effectively. We chose a high and rather short thermal stress, because under lower temperatures (e.g., 42°C for 30–40 min), hsp70 expression was not as high. The effectiveness of hsp70 in the protection of monocytes against apoptosis was underlined by our data, indicating that serum deprivation induced significant apoptosis after 60 h in parallel with a drastic downregulation of hsp70. Serum starvation reduced hsp70 not specifically but similarly down-regulated hsc70. This treatment seems to paralyze protein synthesis as a whole and differs from active proapoptotic triggers. We detected hsp70 expression on a mRNA level by semiquantitative RT-PCR and on a protein level, by immunoblotting because previous studies have shown that heat and chemical substances, such as phorbol esters, can regulate hsp expression on different cellular levels. Heat increased hsp70 and hsp90 transcriptionally, whereas phorbol esters stabilized mRNA and acted on a posttranscriptional level [22 ]. Our results show that PCR-detected mRNA levels and protein accumulation went in parallel. All these data are in accordance with previous studies mostly analyzing cell lines, which confirmed the role of hsp as proteins conferring resistance against heat and other physical stress factors or apoptotic triggers [5 , 6 , 23 ].

The most striking finding of our study was that IL-4 induced significant apoptosis in conjunction with a concentration-dependent upregulation of hsp70. Maximum hsp70 stimulation occurred late after 60 h of IL-4 treatment. IL-4-dependent effects on hsp70 were direct because a neutralizing ab against IL-4 blocked hsp70 expression completely. IL-4 has been described extensively as a factor able to deactivate monocytes and to suppress most monocytic-effector functions. This lymphokine inhibits transcription and formation of IL-1, TNF-{alpha}, IL-6, and prostaglandins [24 , 25 ] and down-regulates most cell-surface receptors such as CD14 [26 ]. All these effector functions have been implicated with monocyte apoptosis because enhanced monokine production [14 ] or CD14 expression [19 ] may be involved in antiapoptotic pathways. However, several receptors, enzymes, and mediators can be upregulated also in monocytes by IL-4. It has been shown that IL-4 increased 15-lipoxygenase [27 ], IL-1 receptor-antagonist formation [28 ], or expression of CD23 [29 ], type I and type II, IL-1 receptors (IL-1R) [30 ]. Probably, these factors may contribute to IL-4-dependent monocyte apoptosis also. Induction of hsp by IL-4 has been described only for lymphocytes or tumor cells and not for monocytes so far [31 ]. In the renal carcinoma cell line ACHN, IL-4 upregulated hsp27 and exerted, together with IFN-{gamma}, an additive effect on chaperone formation [32 ]. In human T-cells, IL-4 strongly upregulated hsp90 via so-called heat shock factors by activating heat shock elements [33 ]. However, these data cannot be transferred to monocytes because IL-4 induces survival or proliferation in lymphocytes, which may be supported by the activation of hsp but not apoptosis, as shown for monocytes. Our study clearly shows that hsp70, upregulated in response to IL-4, is not sufficient to rescue monocytes from apoptosis, probably because proapoptotic pathways predominate or because hsp effects are antagonized. When monocytes were exposed to heat and consequently treated with IL-4 for 60 h, we found a highly enhanced hsp70 expression but no modulation of apoptosis, as compared with cells treated with IL-4 without heat shock (unpublished results). Serum-deprived monocytes that were treated consecutively with IL-4 showed low hsp70 levels and marked signs of apoptosis, suggesting that IL-4 is not a dominant trigger for chaperone formation. From hematopoietic cells, we know that Janus protein kinases (Jak), which can be stimulated in human monocytes by IL-4 [34 ], can antagonize apoptosis in conjunction with hematopoietic growth factors by the induction of bcl-2 and bcl-xL [35 ]. Because monocytes become apoptotic only when IL-4 acts alone or together with LPS [15 ], but differentiate into dendritic cells (DC) when IL-4 acts together with granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF [36 , 37 ], it can be speculated that hsp induction in monocytes by IL-4 is coupled with and is probably necessary for the differentiation into DC when growth factors are present.

Taken together, our study shows that for human monocytes, hsp70 stimulated by heat protects cells from injury and enables survival. Two distinct apoptotic triggers induce contrary responses of hsp70 with a drastic downregulation by serum deprivation and an upregulation by IL-4, suggesting that hsp70 is not directly coupled with antiapoptotic pathways but rather is engaged in a complex regulatory network.

Received September 1, 1999; revised March 28, 2000; accepted May 14, 2000.


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