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Cell-surface Expression of Heat Shock proteins in Dog Neutrophils after Oxidative Stress
Toxicology in Vitro 13 (1999) 437±443 www.elsevier.com/locate/toxinvit
Cell-surface Expression of Heat Shock Proteins in Dog Neutrophils after Oxidative Stress 1
A. CAMINS1,*, C. DIEZ-FERNANDEZ2 and P. PRIETO2
Laboratory of Pharmacology and Pharmacognosy, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain and 2European Commission Joint Research Centre Environment Institute, I-21020 Ispra (VA), Italy (Accepted 7 December 1998)
AbstractÐThe eect of oxidative stress induced by dierent concentrations of hydrogen peroxide on dog neutrophils was studied. This eect was measured using dichloro¯uorescein-diacetate (DCFH-DA) and by the cell surface membrane expression of heat shock protein (HSP) 27 kDa, HSP 72 kDa and HSP 90 kDa families. Hydrogen peroxide induced a concentration-dependent increase in DCFH oxidation (from 10ÿ6 M to 10ÿ4 M), and an increase in the cell surface expression of HSPs families. At a concentration of 10ÿ4 M, the percentage of positive cells that showed an oxidation of DCFH was 94.7%25.2 (n = 3). Only vitamin E (but not vitamin C) at a concentration of 0.5 mM was able to inhibit the intracellular oxidative stress induced by hydrogen peroxide. The percentage of positive cells that express these proteins after the treatment with hydrogen peroxide (10ÿ4 M) was: 74%23.5 for HSP 27, 72%22.6 for HSP 72 and 73%21.2 for HSP 90 (n = 3). This cell surface expression was not abolished by either vitamin C or vitamin E. Localization of HSPs in plasma membrane is of immunological interest because they have been implicated in autoimmune diseases. # 1999 Elsevier Science Ltd. All rights reserved Keywords: oxidative stress; heat shock proteins; neutrophils (dog). Abbreviations: DCFH = dichloro¯uorescein; DCFH-DA = dichloro¯uorescein-diacetate; HSP = heat shock protein; HBSS = Hanks' balanced salt solution; PBS = phosphate buered saline; ROS = reactive oxygen species.
INTRODUCTION
Heat shock proteins (HSPs) or stress proteins are a group of proteins, the amino acid sequences of which are highly conserved from prokaryotes to eukaryotes, and their synthesis is increased after heat shock, oxidative stress or UV radiation (Donati et al., 1990). Dierent families of HSPs could be classi®ed in constitutively expressed and stress-induced members (Young, 1990). In unstressed cells, constitutive HSPs exert a variety of functions such as the correct folding of new proteins, translocation of these new proteins between dierent cell compartments, and interacting with steroid receptors (Ciocca et al., 1993). An interesting ®nding is their immunological role as antigens. It has been recognized that some microorganisms bear antigens that are homologous to HSPs (Kaufman, 1990), therefore HSP families may *Corresponding author.
participate in autoimmunity or in immunity processes (Kaufman, 1992; Mollenhauer and Schulmeister, 1992). Furthermore, it has been proposed that inducible HSPs could play a role in cell protection (JaÈaÈtelaÈ and Wissind, 1993). For example, overexpression of HSP 70 or HSP 27 families in monocytes after heatshock protects them from oxidative stress, suggesting that these families of proteins may be useful in the repair of cell damage after heat treatment. Also, a role as antioxidants when cells are producing high concentrations of free radicals has been described (Polla, 1998). Human polymorphonuclear neutrophils induce HSP expression after exposure to heat (Eid et al., 1987), hepoxilin A3 (Lin et al., 1991), 12-HETE (Koller and KoÈning, 1991), after subphysiologic temperatures (Cox et al., 1993), bacterial toxins (Hensler et al., 1991) and they also are expressed in patients with tissue injury (Kindas-MuÈge et al., 1993).
0887-2333/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(99)00012-0
Fig. 1. Representative ¯uorescence histograms obtained from 10,000 cells incubated with DCFH-DA in the presence of hydrogen peroxide and hydrogen peroxide with vitamin C and vitamin E measured by ¯ow cytometry. Positive cells were those showing an increase in the intracellular ¯uorescence respect to the control. The ordinate is number of cells at each ¯uorescence intensity. The abcissa is arbitrary units of ¯uorescence intensity.
438 A. Camins et al.
Fig. 2. Representative experiment of the cell-surface expression of heat shock proteins induced by dierent concentrations of hydrogen peroxide. Positive cells were those showing an increase in the intracellular ¯uorescence respect to the control. The ordinate is number of cells at each ¯uorescence intensity. The abcissa is arbitrary units of ¯uorescence intensity.
HSP cell surface expression and oxidative stress 439
440
A. Camins et al.
In previous studies, we have demonstrated that peripheral-type benzodiazepine receptor ligands induce HSP expression in dog neutrophils (Camins et al., 1995). The aim of the present work was to study the eect of oxidative stress in dog neutrophils. We describe here the characteristics of cell surface expression of HSPs families (hsp27, hsp72 and hsp90) in dog neutrophils and also the eects of vitamin C (ascorbate) and vitamin E (a-tocopherol), two wellknown natural antioxidants, after oxidative stress.
MATERIALS AND METHODS
Chemicals Hydrogen peroxide was purchased from Janssen Chimica (Belgium). 2',7'-Dichloro¯uorescein diacetate (DCFH-DA) (Serva, Germany) was used as a 5 mM stock solution in dimethyl sulfoxide and was stored at ÿ208C. Propidium iodide (Molecular Probes, USA) was dissolved in Hanks' balanced salt solution (HBSS). Experiments with propidium iodide were performed in a dark room. Phosphate buered saline (PBS), vitamin C and vitamin E were purchased from Sigma Chemical Company (St Louis MO, USA). Mouse anti-HSP27, anti-HSP72 and anti-HSP90 were obtained from StressGen (Victoria, Canada). Second antibody was FICTlabelled and obtained from Amersham (UK). Culture medium (RPMI 1640 with HEPES 25 mM) and HBSS were obtained from Amimed (Muttenz, Switzerland). Preparation of dog neutrophils Neutrophils were isolated from fresh blood obtained from beagle dogs (Marshall, New York, USA) by Ficoll-Paque (Pharmacia, Uppsala, Sweden), following erythrocyte sedimentation in dextran (6% in HBSS). Brie¯y, 40 ml heparinized dog blood was added to 10 ml dextran solution. After 30 min, the buy coat sample was collected and centrifuged on a Ficoll-Paque gradient for 30 min at 500 g. Contaminating erythrocytes in the neutrophil pellet were removed by hypotonic lysis: 10 ml NaCl 0.2% was added for 20 sec and then 10 ml 1.6% NaCl, following centrifugation at 300 g for 10 min. Cells were washed in HBSS, and the resultant cell preparations contained more than 95% of viable neutrophils (measured by the propidium iodide assay). Flow cytometric assays A FACScan ¯ow cytometer (Becton Dickinson) was used for following determinations. In these experiments 10,000 cells from each sample were analysed. Cytotoxicity studies 1 ml RPMI 1640 containing 1 106 cells was treated with dierent concentrations of hydrogen
peroxide. After 2 hr at 378C and aeration with 95% O2/5% CO2, propidium iodide was added to a ®nal concentration of 0.1 mg/ml and incubated for 10 min at 48C. Samples were analysed for viability by ¯ow cytometry, which quanti®es the number of cells that have, or have not, incorporated the ¯uorochrome. Measurement of reactive oxygen species (ROS) The intracellular free radical production was measured using the non-¯uorescent compound 2',7'dichloro¯uorescein diacetate (DCFH-DA). This method (Bass et al., 1993) measures the formation of hydrogen peroxide generated by an oxidative metabolic burst. Viable cells can deacetylate DCFH-DA to 2',7'-dichloro¯uorescin, which is not ¯uorescent. This compound reacts quantitatively with oxygen species within the cell to produce the ¯uorescent dye 2',7'-dichloro¯uorescein, which remains trapped within the cell and can be measured to provide an index of intracellular oxidation. 2 106 neutrophils/ml were preincubated for 15 min with DCFH-DA (5 10ÿ6 M) in HBSS in a ®nal volume of 5 ml with horizontal agitation in a water-bath at 378C. After preincubation, cells were washed in HBSS and dierent concentrations of hydrogen peroxide were added to cell preparations for 30 min. Finally, the number of cells with or without ¯uorescence were measured by ¯ow cytometry. Surface expression of HSPs Neutrophil preparations were adjusted to 1 106 cells/ml in RPMI 1640 with HEPES 25 mM and incubated with dierent concentrations of hydrogen peroxide for 2 hr at 378C in a humidi®ed atmosphere of carbogen. Cells were washed in PBS and incubated at 48C for 30 min with dierent anti-HSP antibodies (®rst antibody) at a 1:200 dilution. All steps were performed with 0.05% sodium azide on ice to prevent internalization of antibodies. For washing, ice-cold RPMI 1640 was used. Fluorescein-labelled anti-mouse Ig was used as the second antibody at a 1:80 dilution, and this was incubated for 30 min in ice at 48C. Finally, cells were washed three times in cold RPMI 1640 and quanti®ed by ¯ow cytometry. Table 1. Flow cytometric analysis of DCFH oxidation after the treatment with dierent concentrations of hydrogen peroxide alone, and in the presence of vitamin C. Results are expressed as mean 2 SEM of three experiments performed separately Hydrogen peroxide (M) 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4
Alone
Vit C
37.12 2.7 76.32 2.4 92.12 3.8 94.72 5.2
9.67 20.3 62.8 21.2 79.6 22.7 89.4 24.2
HSP cell surface expression and oxidative stress
441
Table 2. Cell-surface expression of HSPs (% of cells that express HSPs on its surface) after the treatment with dierent concentrations of hydrogen peroxide alone, and in the presence of vitamin C and vitamin E. Results are expressed as mean 2SEM of three experiments performed separately Hydrogen peroxide (M) 0 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4 (In the presence of vitamin C) 0 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4 (In the presence of vitamin E) 0 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4
HSP 27 kDa
HSP 72 kDa
HSP 90 kDa
18.22 0.5 19.12 0.1 39.12 1.4 71.42 2.6 74.32 3.5
3.4 2 0.1 4.2 2 0.2 17.12 0.3 51.32 2.7 72.12 2.8
4.6 2 0.3 5.5 2 0.2 25.12 0.4 65.32 1.8 73.52 2.5
8.4 2 0.3 9.1 2 0.6 38.42 1.8 76.52 2.0 84.42 2.7
6.4 2 1.1 7.2 2 1.3 20.32 0.5 71.42 3.1 78.22 1.8
5.5 2 0.7 6.2 2 0.5 26.52 1.3 74.42 2.8 80.22 3.2
7.3 2 0.2 8.6 2 0.5 23.52 2.5 62.52 3.4 75.12 2.8
5.2 2 0.2 7.5 2 0.8 18.52 1.7 65.82 2.2 84.12 3.3
6.7 2 0.3 8.9 2 0.2 16.82 0.9 54.62 2.8 73.72 2.2
RESULTS
Cytotoxicity eect of hydrogen peroxide Neutrophils were exposed to dierent concentrations of H2O2 (ranging from 10ÿ6 M to 10ÿ4 M) at 378C for 2 hr. At these concentrations, hydrogen peroxide did not produce a decrease in neutrophil viability (at 10ÿ4 M the viability was 92.68 % 2 1.78) (n = 3).
vitamin C, the cell-surface expression of HSP families was unaected (Table 2). These experiments demonstrated a cell surface located form of HSPs families induced by hydrogen peroxide in dog neutrophils. Control experiments with vitamin E (0.5 mM) and vitamin C (2 mM) alone (without hydrogen peroxide) were performed and neither induced HSP cell surface-expression.
Eect of hydrogen peroxide on DCFH oxidation After preincubation with DCFH-DA, dog neutrophils were treated with dierent concentrations of hydrogen peroxide (ranging from 10ÿ6 M to 10ÿ4M) for 30 min. The ¯ow cytometric assay showed a marked increase in the number of cells with greatest ¯uorescence (Fig. 1). The percentage of positively stained cells following hydrogen peroxide treatment in the absence and in the presence of vitamin C, compared with control cells is given (Table 1). In the presence of vitamin E (0.5 mM), a decrease in intracellular DCFH oxidation was found. Only at the highest concentrations of hydrogen peroxide (5 10ÿ5 M and 10ÿ4 M) did we detect positive cells, which showed an intracellular DCFH oxidation, 13.5% 2 0.2 and 18.3% 2 0.9 (n = 3), respectively. Cell surface expression of HSP To determine whether the epitopes of three HSP families were expressed on the surface of neutrophils, ¯ow cytometric analysis was performed. As shown in Fig. 2, after the treatment with dierent concentrations of hydrogen peroxide (from 10ÿ6 M to 10ÿ4 M) for 2 hr at 378C, neutrophils were exposed to speci®c antibodies. Hydrogen peroxide at the concentration of 10ÿ4 M induced the maximal cell surface expression, and the percentage of positive cells was increased from 18.2 2 0.5 (control group, treated only with PBS) to 74.3% 2 3.5 for HSP 27, 72.1% 2 2.8 for HSP 72 and 73.5% 2 2.5 for HSP 90 (n = 3). In the presence of vitamin E or
DISCUSSION
In this study we describe the eects of oxidative stress on dog neutrophils. We provide evidence that dog neutrophils induce cell-surface expression of HSP families, 27 kDa, 70 kDa and 90 kDa after oxidative damage. The study of oxidative stress is of interest, because it has a key role in some diseases (Halliwell, 1987), and it is also believed that neuronal damage mediated by excitatory amino acids could be mediated by ROS (Coyle and Puttfarcken, 1993). Oxidative stress can damage cells by lipid peroxidation and alteration of protein and nucleic acid structure (Briehl and Baker, 1996; Gardner et al., 1996). ROS also play a role in physiological systems; they were shown to be responsible for the inducible expression of genes associated with in¯ammatory and immune responses. Current evidence indicates that dierent stimuli use ROS as signalling messengers to activate transcription factors, such as the cytoplasmatic factor NF-kB and AP-1 (Schreck and Baeuerle, 1991). Several observations of the induction of HSPs after oxidative stress have also been reported (Burdon et al., 1987; Donati et al., 1990). It has also been hypothesized that hydrogen peroxide induces a cellular stress and the expression of HSPs after the activation of a heat shock factor (Becker et al. 1990, 1991). Recently, it has been suggested that NF-kB and HSPs could be activated by several
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common inducers but are inversely regulated by intracellular redox (Kretz-Remy et al., 1998). Alternatively, ROS-induced DNA damage may stimulate synthesis of p53 protein, a eector molecule of apoposis. Of particular interest is the observation that proteins of hsp70 family form complexes with p53. This raises the possibility that HSPs may be involved in apoptosis, because p53 appears to be a key regulator of this process (Obeid and Hannun, 1995). The results of the present experiments with atocopherol at a concentration of 0.5 mM showed a decrease in DCFH oxidation, suggesting an inhibition of intracellular oxidative damage, but vitamin E did not modify HSPs expression. It could be speculated that free radicals are not the only mechanism by which hydrogen peroxide induces HSP cell-surface expression. However, these results could be in agreement with those of Werninghaus et al. (1991), who demonstrated that a-tocopherol had a protective eect on UV irradiation-induced epidermal damage. This protective eect is mediated by an increase of the gene expression of HSP 70 kDa. Furthermore, several observations suggest that antioxidants induce an increase in the transcriptional activation of hsp70 promoter in the presence of oxidative stress (Kretz-Remy et al., 1988). Increased expression of HSPs could be due to a role of HSP families as antioxidants (Polla, 1991), mediating an increase in cellular defence systems against oxidative damage. After the treatment with hydrogen peroxide, only vitamin E inhibited the intracellular oxidative stress. These results could be explained by the dierent localization of vitamin E and vitamin C in the neutrophil. Whereas, vitamin E is in the cellular membranes, vitamin C due to its polarity, is in the cytosol. On the other hand, vitamin C is accumulated into the neutrophil cytosol mediated transport system (Wasshko et al., 1989). Although the two vitamins have a scavenger mechanism, vitamin E is also an inhibitor of protein kinase C (a key enzyme in the activation of neutrophils) (Mahoney and Azzi, 1988). Recent studies demonstrated that HSPs have a protective role in phagocytic cells: they prevent the NADPH oxidase inhibition after heat shock and give support to the bactericidal activity of neutrophils and monocytes (Maridonneau-Parini et al., 1995). HSPs could be involved in the maintenance of functionality of phagocytic cells after dierent kinds of stress (Polla et al., 1995). It has been postulated that HSP families are among the major antigens recognized in immune response (Jacquier-Sarlin and Polla,1994; Kaufman, 1990). Experimental studies in animals and patients indicate that HSPs may have a role in some autoimmune diseases such as rheumatoid arthritis (Cohen, 1991) and systemic lupus erythematosus (Twomey et al., 1993).
HSP families are generally believed to be located intracellularly in unstressed cells. The cell-surface membrane expression of HSP families has been implicated in the pathology of Graves' disease, and could be a key mechanism of activation of the immune system (Heufelder et al., 1992). The exact mechanism of the cell-surface membrane expression of HSP is unknown. One candidate is a release of proteins from a stressed cell, which then bind to the cell surface (Heufelder et al., 1992). Finally, it is still unclear how the immune system can recognize HSPs expressed on the cell surface. However, it seems that T lymphocyte subpopulations, such as gamma-delta T cells, could recognize dierent HSP families (Kaufman, 1992).
REFERENCES
Bass D. A., Parce J. W., Dechatelet L. R., Szejda P., Seeds M. C. and Thomas M. (1983) Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. Journal of Immunology 130, 1910±1917. Becker J., Mezger V., Courgeon A. M. and BestBelpomme A. (1990) Hydrogen peroxide activates immediate binding of a Drosophila factor to DNA heatshock regulatory element in vivo and in vitro. European Journal of Biochemistry 189, 553±558. Becker J., Mezger V., Courgeon A. M. and BestBelpomme A. (1991) On the mechanism of action of H2O2 in the cellular stress. Free Radicals Research Communications , 12±13, 455±460. Briehl M. M. and Baker A. F. (1996) Modulation of the antioxidant defence as a factor in apoptosis. Cell Death and Dierentiation 3, 63±70. Burdon R. H., Gill V. M. and Rice-Evans C. (1987) Oxidative stress and heat shock protein induction in human cells. Free Radical Research Communications 3, 129±139. Camins A., DõÂ ez-FernaÂndez C., Camarasa J. and Escubedo E. (1995) Cell surface expression of heat shock proteins in dog neutrophils induced by mitochondrial benzodiazepine receptor ligands. Immunopharmacology 29, 159±166. Ciocca D. R., Oesterreich S., Chamness G. C., McGuire W. L. and Fuqua S. A. W. (1993) Biological and clinical implications of heat shock protein 27000 (Hsp27): a review. Journal of the National Cancer Institute 85, 1558±1570. Cohen I. R. (1991) Autoimmunity to chaperonins in the pathogenesis of arthritis and diabetes. Annual Review of Immunology 9, 567±589. Cox G., Mosely P. and Hunninghake G. W. (1993) Induction of heat-shock protein 70 in neutrophils during exposure to subphysiologic temperatures. Journal of Infectious Diseases 167, 769±771. Coyle J. T. and Puttfarcken P. (1993) Oxidative stress, glutamate and neurodegenerative disorders. Science 262, 689±695. Donati Y. R. A., Slosman, O and Polla B. S. (1990) Oxidative injury and the heat shock response. Biochemical Pharmacology 40, 2571±2577. Eid N. S., Kravath R. E. and Lanks K. W. J. (1987) Heat shock protein synthesis by human polymorphonuclear cells. Experimental Medicine 165, 1448±1452. Gardner A. M., Xu F. H., Fady C., Jacoby F. J., Duey D. C., Tu Y. and Lichtenstein A. (1996) Apoptotic VS.
HSP cell surface expression and oxidative stress nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radical Biology and Medicine 22, 73±83. Halliwell B. (1987) Oxidants and human disease: some new concepts. FASEB Journal 1, 358±364. Hensler B., KoÈller M., Alouf J. E. and KoÈning W. (1991) Bacterial toxins induce heat shock proteins in human neutrophils. Biochemical and Biophysical Research Communications 179, 872±879. Heufelder A. E., Wenzel B. E. and Bahn R. E. (1992) Cell surface localization of a 72 kilodalton heat shock protein in retroocular ®broblasts from patients with Graves' ophthalmopathy. Journal of Clinical Endocrinology and Metabolism 74, 732±736. JaÈaÈtelaÈ M. and Wissing D. (1993) Heat-shock proteins protect cells from monocyte cytotoxicity: possible mechanism of self protection. Journal of Experimental Medicine 177, 231±236. Jacquier-Sarlin M. R. and Polla B. S. (1994) ProteÂines de Stress: soi, non-soi et reÂponse immune. SyntheÁse 10, 31± 41. Kaufman S. H. E. (1990) Heat shock proteins and the immune response. Immunology Today 11, 129±136. Kaufman S. H. E. (1992) The cellular immune response to heat shock proteins. Experientia 48, 640±643. Kindas-MuÈgge I., Hammerle A. H., FroÈhlich I., OismuÈller C., Micksche M. and Trautinger F. (1993) Granulocytes of critically III patients spontaneously express the 72kD heat shock protein. Circulatory Shock 39, 247±252. KoÈller M. and KoÈning W. (1991) 12-Hydroxyeicosatetraenoic acid (12-HETE) induces heat shock proteins in human leukocytes. Biochemical and Biophysical Research Communications 175, 804±809. Kretz-Remy C., Bates E. E. M. and Patrick Arrigo A. (1998) Amino acid analogs activate NF-kB throug redox-dependent IkB-a degradation by the proteasome without apperent IkB-a phosphorylation. Journal of Biological Chemistry 273, 3180±3191. Lin Z., Laneuville, O and Pace-Asciak C. (1991) Hepoxilin A3 induces heat shock protein (HSP 72) expression in human neutrophils. Biochemical and Biophysical Research Communications 179, 52±56. Mahoney C. W. and Azzi A. (1988) Vitamin E inhibits protein kinase C activity. Biochemical and Biophysical Research Communications 154, 694±697.
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Maridonneau-Parini, I, Malawista S. E., Stubbe H., Russo-Marie F. and Polla B. S. (1995) Heat-Shock in human neutrophils: superoxide generation is inhibited by a mechanism distinct from heat-denaturation of NADPH oxidase and is protected by heat shock proteins in thermotolerant cells. Journal of Cell Physiology 156, 204±211. Mollenhauer J. and Schulmeister A. (1992) The humoral response to heat shock proteins. Experientia 48, 644± 649. Obeid L. M. and Hannun Y. A. (1995) Ceramide: A stress signal mediator of growth suppression and apoptosis. Journal of Cellular Biochemistry 58, 191±198. Polla B. S. (1988) A role for heat shock proteins in in¯ammation? Immunology Today 9, 134±137. Polla B. S. (1991) The heat shock response in human phagocytes. Immunology Letters 30, 159±164. Polla B. S., Stubbe H., Kantengwa S., MaridonneauParini I. and Jacquier-Sarlin M. R. (1995) Dierential induction of stress proteins and functional eects of heat shock in human phagocytes. In¯ammation 19, 363± 378. Schreck R. and Baeuerle P. A. (1991) A role for oxygen radicals as second messengers. Trends in Cell Biology 1, 39±42. Twomey B. M., Dhillon V. B., McCallum S., Isenberg D. A. and Latchman D. S. (1993) Elevated levels of the 90 kD heat shock protein in patients with systemic lupus erythematosus are dependent upon enhanced transcription of the hsp 90b gene. Journal of Autoimmunity 6, 495±506. Wasshko D., Rotrensen D. and Levine M. (1989) Ascorbic acid transport and accumulation in human neutrophils. Journal of Biological Chemistry 264, 18996± 19002. Werninghaus K., Handjani R. M. and Gilcherst B. A. (1991) Protective eect of alpha-tocopherol on ultraviolet-mediated human epidermal cell damage in vitro. Photodermatology, Photoimmunology and Photomedicine 8, 236±242. Young R. A. (1990) Stress proteins and immunology. Annual Review of Immunology 8, 401±420.
Cell-surface Expression of Heat Shock Proteins in Dog Neutrophils after Oxidative Stress 1
A. CAMINS1,*, C. DIEZ-FERNANDEZ2 and P. PRIETO2
Laboratory of Pharmacology and Pharmacognosy, Faculty of Pharmacy, University of Barcelona, 08028 Barcelona, Spain and 2European Commission Joint Research Centre Environment Institute, I-21020 Ispra (VA), Italy (Accepted 7 December 1998)
AbstractÐThe eect of oxidative stress induced by dierent concentrations of hydrogen peroxide on dog neutrophils was studied. This eect was measured using dichloro¯uorescein-diacetate (DCFH-DA) and by the cell surface membrane expression of heat shock protein (HSP) 27 kDa, HSP 72 kDa and HSP 90 kDa families. Hydrogen peroxide induced a concentration-dependent increase in DCFH oxidation (from 10ÿ6 M to 10ÿ4 M), and an increase in the cell surface expression of HSPs families. At a concentration of 10ÿ4 M, the percentage of positive cells that showed an oxidation of DCFH was 94.7%25.2 (n = 3). Only vitamin E (but not vitamin C) at a concentration of 0.5 mM was able to inhibit the intracellular oxidative stress induced by hydrogen peroxide. The percentage of positive cells that express these proteins after the treatment with hydrogen peroxide (10ÿ4 M) was: 74%23.5 for HSP 27, 72%22.6 for HSP 72 and 73%21.2 for HSP 90 (n = 3). This cell surface expression was not abolished by either vitamin C or vitamin E. Localization of HSPs in plasma membrane is of immunological interest because they have been implicated in autoimmune diseases. # 1999 Elsevier Science Ltd. All rights reserved Keywords: oxidative stress; heat shock proteins; neutrophils (dog). Abbreviations: DCFH = dichloro¯uorescein; DCFH-DA = dichloro¯uorescein-diacetate; HSP = heat shock protein; HBSS = Hanks' balanced salt solution; PBS = phosphate buered saline; ROS = reactive oxygen species.
INTRODUCTION
Heat shock proteins (HSPs) or stress proteins are a group of proteins, the amino acid sequences of which are highly conserved from prokaryotes to eukaryotes, and their synthesis is increased after heat shock, oxidative stress or UV radiation (Donati et al., 1990). Dierent families of HSPs could be classi®ed in constitutively expressed and stress-induced members (Young, 1990). In unstressed cells, constitutive HSPs exert a variety of functions such as the correct folding of new proteins, translocation of these new proteins between dierent cell compartments, and interacting with steroid receptors (Ciocca et al., 1993). An interesting ®nding is their immunological role as antigens. It has been recognized that some microorganisms bear antigens that are homologous to HSPs (Kaufman, 1990), therefore HSP families may *Corresponding author.
participate in autoimmunity or in immunity processes (Kaufman, 1992; Mollenhauer and Schulmeister, 1992). Furthermore, it has been proposed that inducible HSPs could play a role in cell protection (JaÈaÈtelaÈ and Wissind, 1993). For example, overexpression of HSP 70 or HSP 27 families in monocytes after heatshock protects them from oxidative stress, suggesting that these families of proteins may be useful in the repair of cell damage after heat treatment. Also, a role as antioxidants when cells are producing high concentrations of free radicals has been described (Polla, 1998). Human polymorphonuclear neutrophils induce HSP expression after exposure to heat (Eid et al., 1987), hepoxilin A3 (Lin et al., 1991), 12-HETE (Koller and KoÈning, 1991), after subphysiologic temperatures (Cox et al., 1993), bacterial toxins (Hensler et al., 1991) and they also are expressed in patients with tissue injury (Kindas-MuÈge et al., 1993).
0887-2333/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(99)00012-0
Fig. 1. Representative ¯uorescence histograms obtained from 10,000 cells incubated with DCFH-DA in the presence of hydrogen peroxide and hydrogen peroxide with vitamin C and vitamin E measured by ¯ow cytometry. Positive cells were those showing an increase in the intracellular ¯uorescence respect to the control. The ordinate is number of cells at each ¯uorescence intensity. The abcissa is arbitrary units of ¯uorescence intensity.
438 A. Camins et al.
Fig. 2. Representative experiment of the cell-surface expression of heat shock proteins induced by dierent concentrations of hydrogen peroxide. Positive cells were those showing an increase in the intracellular ¯uorescence respect to the control. The ordinate is number of cells at each ¯uorescence intensity. The abcissa is arbitrary units of ¯uorescence intensity.
HSP cell surface expression and oxidative stress 439
440
A. Camins et al.
In previous studies, we have demonstrated that peripheral-type benzodiazepine receptor ligands induce HSP expression in dog neutrophils (Camins et al., 1995). The aim of the present work was to study the eect of oxidative stress in dog neutrophils. We describe here the characteristics of cell surface expression of HSPs families (hsp27, hsp72 and hsp90) in dog neutrophils and also the eects of vitamin C (ascorbate) and vitamin E (a-tocopherol), two wellknown natural antioxidants, after oxidative stress.
MATERIALS AND METHODS
Chemicals Hydrogen peroxide was purchased from Janssen Chimica (Belgium). 2',7'-Dichloro¯uorescein diacetate (DCFH-DA) (Serva, Germany) was used as a 5 mM stock solution in dimethyl sulfoxide and was stored at ÿ208C. Propidium iodide (Molecular Probes, USA) was dissolved in Hanks' balanced salt solution (HBSS). Experiments with propidium iodide were performed in a dark room. Phosphate buered saline (PBS), vitamin C and vitamin E were purchased from Sigma Chemical Company (St Louis MO, USA). Mouse anti-HSP27, anti-HSP72 and anti-HSP90 were obtained from StressGen (Victoria, Canada). Second antibody was FICTlabelled and obtained from Amersham (UK). Culture medium (RPMI 1640 with HEPES 25 mM) and HBSS were obtained from Amimed (Muttenz, Switzerland). Preparation of dog neutrophils Neutrophils were isolated from fresh blood obtained from beagle dogs (Marshall, New York, USA) by Ficoll-Paque (Pharmacia, Uppsala, Sweden), following erythrocyte sedimentation in dextran (6% in HBSS). Brie¯y, 40 ml heparinized dog blood was added to 10 ml dextran solution. After 30 min, the buy coat sample was collected and centrifuged on a Ficoll-Paque gradient for 30 min at 500 g. Contaminating erythrocytes in the neutrophil pellet were removed by hypotonic lysis: 10 ml NaCl 0.2% was added for 20 sec and then 10 ml 1.6% NaCl, following centrifugation at 300 g for 10 min. Cells were washed in HBSS, and the resultant cell preparations contained more than 95% of viable neutrophils (measured by the propidium iodide assay). Flow cytometric assays A FACScan ¯ow cytometer (Becton Dickinson) was used for following determinations. In these experiments 10,000 cells from each sample were analysed. Cytotoxicity studies 1 ml RPMI 1640 containing 1 106 cells was treated with dierent concentrations of hydrogen
peroxide. After 2 hr at 378C and aeration with 95% O2/5% CO2, propidium iodide was added to a ®nal concentration of 0.1 mg/ml and incubated for 10 min at 48C. Samples were analysed for viability by ¯ow cytometry, which quanti®es the number of cells that have, or have not, incorporated the ¯uorochrome. Measurement of reactive oxygen species (ROS) The intracellular free radical production was measured using the non-¯uorescent compound 2',7'dichloro¯uorescein diacetate (DCFH-DA). This method (Bass et al., 1993) measures the formation of hydrogen peroxide generated by an oxidative metabolic burst. Viable cells can deacetylate DCFH-DA to 2',7'-dichloro¯uorescin, which is not ¯uorescent. This compound reacts quantitatively with oxygen species within the cell to produce the ¯uorescent dye 2',7'-dichloro¯uorescein, which remains trapped within the cell and can be measured to provide an index of intracellular oxidation. 2 106 neutrophils/ml were preincubated for 15 min with DCFH-DA (5 10ÿ6 M) in HBSS in a ®nal volume of 5 ml with horizontal agitation in a water-bath at 378C. After preincubation, cells were washed in HBSS and dierent concentrations of hydrogen peroxide were added to cell preparations for 30 min. Finally, the number of cells with or without ¯uorescence were measured by ¯ow cytometry. Surface expression of HSPs Neutrophil preparations were adjusted to 1 106 cells/ml in RPMI 1640 with HEPES 25 mM and incubated with dierent concentrations of hydrogen peroxide for 2 hr at 378C in a humidi®ed atmosphere of carbogen. Cells were washed in PBS and incubated at 48C for 30 min with dierent anti-HSP antibodies (®rst antibody) at a 1:200 dilution. All steps were performed with 0.05% sodium azide on ice to prevent internalization of antibodies. For washing, ice-cold RPMI 1640 was used. Fluorescein-labelled anti-mouse Ig was used as the second antibody at a 1:80 dilution, and this was incubated for 30 min in ice at 48C. Finally, cells were washed three times in cold RPMI 1640 and quanti®ed by ¯ow cytometry. Table 1. Flow cytometric analysis of DCFH oxidation after the treatment with dierent concentrations of hydrogen peroxide alone, and in the presence of vitamin C. Results are expressed as mean 2 SEM of three experiments performed separately Hydrogen peroxide (M) 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4
Alone
Vit C
37.12 2.7 76.32 2.4 92.12 3.8 94.72 5.2
9.67 20.3 62.8 21.2 79.6 22.7 89.4 24.2
HSP cell surface expression and oxidative stress
441
Table 2. Cell-surface expression of HSPs (% of cells that express HSPs on its surface) after the treatment with dierent concentrations of hydrogen peroxide alone, and in the presence of vitamin C and vitamin E. Results are expressed as mean 2SEM of three experiments performed separately Hydrogen peroxide (M) 0 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4 (In the presence of vitamin C) 0 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4 (In the presence of vitamin E) 0 1 10ÿ6 1 10ÿ5 5 10ÿ5 1 10ÿ4
HSP 27 kDa
HSP 72 kDa
HSP 90 kDa
18.22 0.5 19.12 0.1 39.12 1.4 71.42 2.6 74.32 3.5
3.4 2 0.1 4.2 2 0.2 17.12 0.3 51.32 2.7 72.12 2.8
4.6 2 0.3 5.5 2 0.2 25.12 0.4 65.32 1.8 73.52 2.5
8.4 2 0.3 9.1 2 0.6 38.42 1.8 76.52 2.0 84.42 2.7
6.4 2 1.1 7.2 2 1.3 20.32 0.5 71.42 3.1 78.22 1.8
5.5 2 0.7 6.2 2 0.5 26.52 1.3 74.42 2.8 80.22 3.2
7.3 2 0.2 8.6 2 0.5 23.52 2.5 62.52 3.4 75.12 2.8
5.2 2 0.2 7.5 2 0.8 18.52 1.7 65.82 2.2 84.12 3.3
6.7 2 0.3 8.9 2 0.2 16.82 0.9 54.62 2.8 73.72 2.2
RESULTS
Cytotoxicity eect of hydrogen peroxide Neutrophils were exposed to dierent concentrations of H2O2 (ranging from 10ÿ6 M to 10ÿ4 M) at 378C for 2 hr. At these concentrations, hydrogen peroxide did not produce a decrease in neutrophil viability (at 10ÿ4 M the viability was 92.68 % 2 1.78) (n = 3).
vitamin C, the cell-surface expression of HSP families was unaected (Table 2). These experiments demonstrated a cell surface located form of HSPs families induced by hydrogen peroxide in dog neutrophils. Control experiments with vitamin E (0.5 mM) and vitamin C (2 mM) alone (without hydrogen peroxide) were performed and neither induced HSP cell surface-expression.
Eect of hydrogen peroxide on DCFH oxidation After preincubation with DCFH-DA, dog neutrophils were treated with dierent concentrations of hydrogen peroxide (ranging from 10ÿ6 M to 10ÿ4M) for 30 min. The ¯ow cytometric assay showed a marked increase in the number of cells with greatest ¯uorescence (Fig. 1). The percentage of positively stained cells following hydrogen peroxide treatment in the absence and in the presence of vitamin C, compared with control cells is given (Table 1). In the presence of vitamin E (0.5 mM), a decrease in intracellular DCFH oxidation was found. Only at the highest concentrations of hydrogen peroxide (5 10ÿ5 M and 10ÿ4 M) did we detect positive cells, which showed an intracellular DCFH oxidation, 13.5% 2 0.2 and 18.3% 2 0.9 (n = 3), respectively. Cell surface expression of HSP To determine whether the epitopes of three HSP families were expressed on the surface of neutrophils, ¯ow cytometric analysis was performed. As shown in Fig. 2, after the treatment with dierent concentrations of hydrogen peroxide (from 10ÿ6 M to 10ÿ4 M) for 2 hr at 378C, neutrophils were exposed to speci®c antibodies. Hydrogen peroxide at the concentration of 10ÿ4 M induced the maximal cell surface expression, and the percentage of positive cells was increased from 18.2 2 0.5 (control group, treated only with PBS) to 74.3% 2 3.5 for HSP 27, 72.1% 2 2.8 for HSP 72 and 73.5% 2 2.5 for HSP 90 (n = 3). In the presence of vitamin E or
DISCUSSION
In this study we describe the eects of oxidative stress on dog neutrophils. We provide evidence that dog neutrophils induce cell-surface expression of HSP families, 27 kDa, 70 kDa and 90 kDa after oxidative damage. The study of oxidative stress is of interest, because it has a key role in some diseases (Halliwell, 1987), and it is also believed that neuronal damage mediated by excitatory amino acids could be mediated by ROS (Coyle and Puttfarcken, 1993). Oxidative stress can damage cells by lipid peroxidation and alteration of protein and nucleic acid structure (Briehl and Baker, 1996; Gardner et al., 1996). ROS also play a role in physiological systems; they were shown to be responsible for the inducible expression of genes associated with in¯ammatory and immune responses. Current evidence indicates that dierent stimuli use ROS as signalling messengers to activate transcription factors, such as the cytoplasmatic factor NF-kB and AP-1 (Schreck and Baeuerle, 1991). Several observations of the induction of HSPs after oxidative stress have also been reported (Burdon et al., 1987; Donati et al., 1990). It has also been hypothesized that hydrogen peroxide induces a cellular stress and the expression of HSPs after the activation of a heat shock factor (Becker et al. 1990, 1991). Recently, it has been suggested that NF-kB and HSPs could be activated by several
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common inducers but are inversely regulated by intracellular redox (Kretz-Remy et al., 1998). Alternatively, ROS-induced DNA damage may stimulate synthesis of p53 protein, a eector molecule of apoposis. Of particular interest is the observation that proteins of hsp70 family form complexes with p53. This raises the possibility that HSPs may be involved in apoptosis, because p53 appears to be a key regulator of this process (Obeid and Hannun, 1995). The results of the present experiments with atocopherol at a concentration of 0.5 mM showed a decrease in DCFH oxidation, suggesting an inhibition of intracellular oxidative damage, but vitamin E did not modify HSPs expression. It could be speculated that free radicals are not the only mechanism by which hydrogen peroxide induces HSP cell-surface expression. However, these results could be in agreement with those of Werninghaus et al. (1991), who demonstrated that a-tocopherol had a protective eect on UV irradiation-induced epidermal damage. This protective eect is mediated by an increase of the gene expression of HSP 70 kDa. Furthermore, several observations suggest that antioxidants induce an increase in the transcriptional activation of hsp70 promoter in the presence of oxidative stress (Kretz-Remy et al., 1988). Increased expression of HSPs could be due to a role of HSP families as antioxidants (Polla, 1991), mediating an increase in cellular defence systems against oxidative damage. After the treatment with hydrogen peroxide, only vitamin E inhibited the intracellular oxidative stress. These results could be explained by the dierent localization of vitamin E and vitamin C in the neutrophil. Whereas, vitamin E is in the cellular membranes, vitamin C due to its polarity, is in the cytosol. On the other hand, vitamin C is accumulated into the neutrophil cytosol mediated transport system (Wasshko et al., 1989). Although the two vitamins have a scavenger mechanism, vitamin E is also an inhibitor of protein kinase C (a key enzyme in the activation of neutrophils) (Mahoney and Azzi, 1988). Recent studies demonstrated that HSPs have a protective role in phagocytic cells: they prevent the NADPH oxidase inhibition after heat shock and give support to the bactericidal activity of neutrophils and monocytes (Maridonneau-Parini et al., 1995). HSPs could be involved in the maintenance of functionality of phagocytic cells after dierent kinds of stress (Polla et al., 1995). It has been postulated that HSP families are among the major antigens recognized in immune response (Jacquier-Sarlin and Polla,1994; Kaufman, 1990). Experimental studies in animals and patients indicate that HSPs may have a role in some autoimmune diseases such as rheumatoid arthritis (Cohen, 1991) and systemic lupus erythematosus (Twomey et al., 1993).
HSP families are generally believed to be located intracellularly in unstressed cells. The cell-surface membrane expression of HSP families has been implicated in the pathology of Graves' disease, and could be a key mechanism of activation of the immune system (Heufelder et al., 1992). The exact mechanism of the cell-surface membrane expression of HSP is unknown. One candidate is a release of proteins from a stressed cell, which then bind to the cell surface (Heufelder et al., 1992). Finally, it is still unclear how the immune system can recognize HSPs expressed on the cell surface. However, it seems that T lymphocyte subpopulations, such as gamma-delta T cells, could recognize dierent HSP families (Kaufman, 1992).
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