Immunology Letters, 30 (1991) 159- 164 Elsevier IMLET 01682
The heat shock response in human phagocytes Barbara S. Polla Allergy Unit, University Hospital, Geneva, Switzerland (Received 1 July 1991; accepted 23 July 1991)
1. Summary During the last 10 years the intriguing field of the heat-shock response and stress proteins has switched from a particular case to a phenomenon of general interest. Discovered in Drosophila, these proteins were observed in every living organism with a surprisingly high sequence homology. In addition, these proteins are not only inducible by stress or pathophysiological situations but are also expressed in unstressed cells. These are arguments for crucial roles of heat-shock proteins. Here we discuss some aspects of the heat shock/stress response that we observed in phagocytic cells after phagocytosis with regard to their physiologic functions such as oxygen-free radical generation, antigen processing and presentation. 2. Introduction Since the initial discovery of the heat-shock response by Ritossa, in 1962 [1], much progress has been made in the understanding of this peculiar cellular response to injury. There are two major fields in which important new discoveries have been made during recent years. First, it has been shown that heat-shock proteins (HSPs) are present in unstressed cells and that they exert important cellular functions, such as molecular chaperoning and Key words: Heat shock protein; Stress response; Phagocytosis; NADPH oxidase; Monocyte-macrophage; Antigen presenting cell
Correspondence to: H6pital Cantonal Universitaire de Gen~ve, D6partement de M6dicine, Division d'Immunologie et d'Allergologie, Unit~ d'Allergologie, 24 rue Micheli-du-Crest, 1211 Gen~ve 4, Switzerland.
translocation of proteins across subcellular membranes [2, 3]. HSPs also participate in protein folding and refolding and appear to protect unfolded proteins from aggregation [4]. Second, it has been appreciated that immunodominant antigens from a wide variety of bacteria and parasites are, by sequence homology, HSPs [5], and it has been suggested that because of molecular mimicry between host and pathogen HSPs, these antigenic proteins may participate either in autoimmunity or in protective immunity. The cellular stress response is induced under a number of conditions, including environmental stresses, pathophysiological states, and conditions usually considered as non-stressful such as cell cycling, exposure of cells to growth factors, differentiation and development [6]. Among the classical environmental stresses which induce HSPs, beside heat shock, one can mention inhibitors of energy metabolism, amino acid analogues, and heavy metals such as cadmium. We are interested in the role of HSPs in pathophysiological states, such as fever [7], ischemia [8], inflammation [7, 9], oxidant injury [10] and (bacterial) infection. 3. Heat shock proteins in host-pathogen interactions Whereas several groups have been extensively studying the stress response of bacteria and the role of stress proteins in bacterial virulence and in the immune responses to bacteria, we have focussed our attention on the stress response in the host cells, i.e., in human phagocytes. We are currently investigating the stress response in phagocytes in relationship to some of their relevant physiological functions, including phagocytosis, activation of the re-
0165 - 2478 / 91 / $ 3.50 © Elsevier Science Publishers B.V. All rights reserved.
159
spiratory burst, and, for monocytes-macrophages (m~), antigen presentation. The first question we have asked is whether stress proteins are induced during phagocytosis. We found indeed that phagocytosis of opsonized sheep red blood cells induces a striking stress response in human monocytes-macrophages, with induction of the classical HSPs, HSP70, together with HSP65, HSP83-90, HSP110, HSP47 and a 32-kDa oxidation-specific stress protein, heme oxygenase [11] (see Table 1 for a list of the stress proteins most relevant to our studies). In cells from patients with chronic granulomatous disease, although induction of the classical HSPs is maintained, the synthesis of heme oxygenase is barely detectable [7], suggesting that the regulation of HSPs and of heme oxygenase is, at least in part, different. The questions we next asked are three-fold: 1. is there any specificity for the second messenger systems involved in stress protein synthesis during phagocytosis? 2. what happens in terms of the stress response in phagocytes during phagocytosis of bacteria? and 3. do the stress proteins induced during phagocytosis interfere with the phagocyte's functions? 4. Induction of stress proteins in human monocytes-macrophages after heat shock and during erythrophagocytosis In human cells, the induction of stress proteins after exposure of cells to various types of injury is mediated by the phosphorylation and the binding of preformed heat-shock transcription factor(s) to the heat-shock consensus sequence (characterized by the repetitive motif GAA-TTC) and the coordinate transcriptional activation of all heat-shock genes [12]. The presence, within the cells, of abnormal, misfolded or unfolded proteins would represent the final common pathway for stress protein induction under the multiplicity of conditions mentioned above. It has, however, also been hypothesized that more classical second messengers, such as calcium or inositol phosphates, may represent a link between the initial cellular stress and the transcriptional activation of heat-shock genes [13]. Among these, we have investigated the role of calcium, and in the human premonocytic line U937, we 160
TABLE 1 Stress proteins induced during phagocytosis: classification and functions. Stress proteins HSPs families HSP 47
HSP 65
HSP 70
HSP 90
HSP 110
Enzymes Superoxide dismutase
Heme oxygenase
Chief characteristics
Glycoprotein associated with ER membrane, collagen binding activity. Mitochondrial protein analogous to E. coli GroEL and to major mycobacterial 65-kDa antigen, participates in proper folding and assembly of protein complexes. Cytoplasmic and nuclear proteins of 68 - 73 kDa, high affinity for ATP, uncoating ATPase, protein translocation, targeting for lysosomal degradation. Cytoplasmic protein of 8 3 - 90 kDa, interaction with tyrosine kinases and steroid hormone receptor Nucleoli located 110-kDa protein, may be involved in the transcription process.
Mitochondrial inducible form referred to as MnSOD, tetrameric complex of ~ 90 kDa responsible for the dismutation of superoxide anion to hydrogen peroxide. 32-kDa protein contained in the microsomal fraction, involved in heme catabolism.
have shown that calcium plays no role in HSP induction [14]. We are currently investigating, in these same cells, whether activation of protein kinase C participates in the sequence of stressful events leading to HSP synthesis. We have also attempted, in the erythrophagocytosis model, to ascertain whether phagocytosis itself, or the generation of oxygen-free radicals associated with phagocytosis (and more specifically the generation of the highly reactive hydroxyl radicals, whose production is catalyzed by the presence of iron) play the major role in the induction of stress proteins. To address this issue, we have used different phagocytic stimuli, as well as different cell types and various antioxidants or free radical scavengers,
including flavonoids. Flavonoids have recently been shown to inhibit the synthesis of HSPs after heat shock [15], and are, all together, radical scavengers, iron chelators, and inhibitors of protein kinase C. Our results suggest that both phagocytosis and the generation of toxic metabolites of oxygen are required for this induction: inert phagocytic stimuli such as latex beads do not affect protein synthesis in phagocytic cells, and phorbol esters, which are the most potent activators of the respiratory burst, induce only low levels of HSPs. Although it is generally accepted that antigen-presenting cells (APC) do not have the ability to distinguish between self and non-self proteins, heatshock proteins may represent a non-immunological tool to recognize and eventually to eliminate large amounts of intracellular foreign proteins such as is the case during erythrophagocytosis. 5. What happens in terms of the stress response during bacterial infection?
It has been known for a long time that viral infections induce stress protein synthesis in host cells [16, 17]. Recently, the possibility that cross-reactivity between bacteria and host HSPs may lead either to protective immunity or to autoimmunity has made it a crucial issue to know whether and how host stress proteins are available for T cell recognition after bacterial infection [18]. We are investigating the stress response to various bacteria and parasites and preliminary results suggest that different bacteria induce different stress responses. Bacteria which normally do not induce autoimmunity, such as Staphylococcus aureus, may lead to preferential synthesis of HSP70, whereas bacteria associated with autoimmunity (mycobacteria) appear to induce HSP65. Furthermore, when parasites do not induce a detectable respiratory burst, no stress response at all can be detected, despite phagocytosis of the pathogen, further emphasizing the role of reactive oxygen metabolites in HSP induction. The stress response associated with phagocytosis also appears to be dependent upon the state of differentiation of the phagocytic cells. Stress proteins are increased in 1,25-dihydroxyvitamin D3-treated cells and even more so in terminally differentiated tissue macrophages (alveolar macrophages).
6. What are the effects of stress proteins induced during phagocytosis on the phagocyte's functions?
6.1. Interactions of stress proteins with N A D P H oxidase We have shown that heat shock and other inducers of HSPs such as cadmium inhibit NADPH oxidase in human neutrophils [19]. This inhibition does not appear to be mediated by HSPs, inasmuch as heat shock also inhibited NADPH oxidase in the absence of any possible transcriptional activation and synthesis of HSPs, i.e., in enucleated neutrophils (cytoplasts) (Maridonneau-Parini et al., submitted for publication). If HSPs appear to play no role in the inhibition of the respiratory burst, they do, however, protect neutrophils from further inhibition of NADPH oxidase by a subsequent exposure to heat shock (Maridonneau-Parini et al., submitted). These data are in agreement with observations of Nguyen et al. [20] who showed that HSPs have the ability to protect enzymatic functions from degradation by heat shock. 6.2. Effects of liSPs (HSP70) in the A P C on subsequent T cell responses There is an important theoretical background for a role of HSPs on antigen processing and/or presentation: (i) Since HSP70 has the ability to "chaperone" other proteins through the cells it could also chaperone antigens. (ii) HSP70 is the clathrin uncoating ATPase and participates in endocytosis via coated pits [21]. (iii) A member of the HSPT0 family is involved in targeting for lysosomal degradation and may thus be involved in antigen processing [22]. (iv) Several genes for the HSP70 (there are at least five of them in the human system) have been mapped within the major histocompatibility complex [23] (close to the TNF genes). (v) Another member of the HSP70 family (PbP72/ 74) plays a role in processing and presentation of pigeon cytochrome c, possibly by facilitating the association of processed antigen with the MHC class II molecules [24]. (vi) HSP70 may also participate in the assembly of 161
MHC molecules themselves [25]. We investigated the effects of inducing a heatshock response in APCs on the T cell proliferative responses. Heat shock increased the expression of MHC class II molecules in a murine B cell line and in human peripheral blood monocytes (Rees et al., submitted for publication; Mari6thoz and Polla, unpublished data). Stress also enhanced the ability of B cells to stimulate both auto- and allo-reactive T cells but it was difficult to distinguish between an effect of stress on class II expression and a direct effect of HSPs on antigen processing a n d / o r presentation. In the human monocytes, preliminary data suggest that exposure of APC to heat shock does not increase T cell responses in mixed lymphocyte reactions, whereas in the autologous system, T cell responses are increased with only some, but not all, antigens tested. When the temperature to which monocytes were exposed was increased to 45 °C, T cell responses were abolished, as previously described by Goeken et al. [26]. Further studies will be required to precisely determine whether and how HSPs participate in antigen uptake, processing, M H C assembly, transport, and presentation. 6.3. HSPs and degradation material
of phagocytosed
Another important function of phagocytes is the production of high levels of proteases. HSPs may be considered as part of the proteolytic system: ubiquitin, a small 76-amino acid HSP, is involved in the targeting of cellular proteins for degradation and proteolysis. We are currently investigating whether in human phagocytes ubiquitin is, or is not, coordinately upregulated with other stress proteins during phagocytosis. Furthermore, a member of the HSP70 family has been shown to participate in targeting of intracellular proteins for lysosomal degradation [22]. This protein may also play a specific role in the degradation of phagocytosed material. 6.4. Protective or "'scavenging" role of liSPs We have shown that pre-exposure to heat shock protects human monocytic cells from DNA strand breaks (M. Perin et al., unpublished data) and cell death [27] secondary to oxidative injury (exposure 162
to hydrogen peroxide). We are investigating the mechanisms by which HSPs protect cells from oxidative damage. Our current hypothesis includes the prevention of DNA strand breaks (for a review, see the Proceedings of the Heat Shock Workshop, Naples, September 1990, in press). We also propose the hypothesis that cells which produce high levels of toxic substances such as reactive oxygen species or tumor necrosis factor ~ during their normal cell function (i.e., phagocytosis) synthesize HSPs as an autoprotective mechanism [28, 29].
Acknowledgements Work mentioned here was supported by the Swiss National Research Foundation (Grants 3.960-0.87 and 32-028645.90). I am grateful to Y. R. A. Donati for helpful discussions and critical review.
References [1] Ritossa, F. (1962) Experientia Basel 18,571. [2] Deshaies, R.J., Koch, B.D., Werner-Washburne, M., Craig, E.A. and Schekman, R. (1988)Nature 332,800. [3] Chirico, W. J., Waters, M.G. and Blobel, G. (1988) Nature 332, 805. [4] Pelham, H. R. B. (1986) Cell 46, 959. [5] Young, D., Lathigra, R., Hendrix, R., Sweetser, D. and Young, R.A. (1988) Proc. Natl. Acad. Sci. USA 85, 4267. [6] Morimoto, R. I. and Milarski, K. L. (1990)in: Cold Spring Harbor Monograph Series - Stress Proteins in Biology and Medicine (R. I. Morimoto, A. Tissibres and C. Georgopoulos, Eds.) Vol. 19, pp. 3 2 3 - 359, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [7] Polla, B.S. and Kantengwa, S. (1991) in: Current Topics in Microbiology and Immunology - Heat Shock Proteins and Immune Response (S. H. E. Kaufmann, Ed.) Vol. 167, pp. 93 - 105, Springer-Verlag, Berlin. [8] Polla, B.S., Mili, N., Donati, Y. and Bonventre, J.V. (1991) N6phrologie 12, 119, in press. [9] Polla, B. S. (1988) Immunol. Today 9, 134. [10] Donati, Y. R. A., Slosman, D.O. and Polla, B.S. (1990) Biochem. Pharmacol. 40, 2571. [11] Clerget, M. and Polla, B. S. (1990) Proc. Natl. Acad. Sci. USA 87, 1081. [12] Sorger, P. K. (1990) Cell 62, 793. [13] Calderwood, S.K., Stevenson, M.A. and Hahn, G.M. (1987) J. Cell. Physiol. 130, 369. [14] Kantengwa, S., Capponi, A. M., Bonventre, J. V. and Polla, B. S. (1990) Am. J. Physiol. 259, C77. [15] Hosokawa, N., Hirayoshi, K., Nakai, A., Hosokawa, Y., Marui, N., Yoshida, M., Sakai, T., Nishino, H., Aoike,
[16] [17] [18]
[19] [20] [21]
[22]
A., Kawai, K. and Nagata, K. (1990)Cell St ruct. Funct. 15, 393. Nevins, J. R. (1982) Cell 29, 913. Notarianni, E. L. and Preston, C. M. (1982) Virology 123, 113. Koga, T., Wand-W~irtenherger, A., DeBruyn, J., Munk, M.E., Schoel, B. and Kaufmann, S. H. E. (1989) Science 245, 1112. Maridonneau-Parini, I., Clerc, J. and Polla, B.S. (1988) Biochem. Biophys. Res. Commun. 154, 179. Nguyen, V.T., Morange, M. and Bensaude, O. (1989) J. Biol. Chem. 264, 10487. Chappell, T. G., Welch, W. J., Schlossman, D. M., Palter, K.B., Schlesinger, M. J. and Rothman, J.E. (1986) Cell 45, 3. Chiang, H. L., Terlecky, S. R., Plant, C. P. and Dice, J. F. (1989) Science 246, 382.
[23] Milner, C. M. and Campbell, R. D. (1990) Immunogenetics 32, 242. [24] VanBnskirk, A., Crump, B. L., Margoliash, E. and Pierce, S.K. (1989) J. Exp. Med. 170, 1799. [25] Pelham, H. R. B. (1989) Nature 340, 426. [26] Goeken, N. E., Ballas, Z. K. and Staggs, T. S. (1986) Hum. Immunol. 16, 234. [27] Polla, B.S., Bonventre, J.V. and Krane, S. M. 0988) J. Cell. Biol. 107,373. [28] Kantengwa, S., Donati, Y. R. A., Clerget, M., Maridonneau-Parini, 1., Sinclair, F., Mari6thoz, E., Perin, M., Rees, A. D. M., Slosman, D.O. and Polla, B.S. (1991) Semin. Immunol. 3, 49. [29] Fincato, G., Polentarutti, N., Sica, A., Mantovani, A. and Colotta, F. (1991) Blood 77,579.
163
The heat shock response in human phagocytes Barbara S. Polla Allergy Unit, University Hospital, Geneva, Switzerland (Received 1 July 1991; accepted 23 July 1991)
1. Summary During the last 10 years the intriguing field of the heat-shock response and stress proteins has switched from a particular case to a phenomenon of general interest. Discovered in Drosophila, these proteins were observed in every living organism with a surprisingly high sequence homology. In addition, these proteins are not only inducible by stress or pathophysiological situations but are also expressed in unstressed cells. These are arguments for crucial roles of heat-shock proteins. Here we discuss some aspects of the heat shock/stress response that we observed in phagocytic cells after phagocytosis with regard to their physiologic functions such as oxygen-free radical generation, antigen processing and presentation. 2. Introduction Since the initial discovery of the heat-shock response by Ritossa, in 1962 [1], much progress has been made in the understanding of this peculiar cellular response to injury. There are two major fields in which important new discoveries have been made during recent years. First, it has been shown that heat-shock proteins (HSPs) are present in unstressed cells and that they exert important cellular functions, such as molecular chaperoning and Key words: Heat shock protein; Stress response; Phagocytosis; NADPH oxidase; Monocyte-macrophage; Antigen presenting cell
Correspondence to: H6pital Cantonal Universitaire de Gen~ve, D6partement de M6dicine, Division d'Immunologie et d'Allergologie, Unit~ d'Allergologie, 24 rue Micheli-du-Crest, 1211 Gen~ve 4, Switzerland.
translocation of proteins across subcellular membranes [2, 3]. HSPs also participate in protein folding and refolding and appear to protect unfolded proteins from aggregation [4]. Second, it has been appreciated that immunodominant antigens from a wide variety of bacteria and parasites are, by sequence homology, HSPs [5], and it has been suggested that because of molecular mimicry between host and pathogen HSPs, these antigenic proteins may participate either in autoimmunity or in protective immunity. The cellular stress response is induced under a number of conditions, including environmental stresses, pathophysiological states, and conditions usually considered as non-stressful such as cell cycling, exposure of cells to growth factors, differentiation and development [6]. Among the classical environmental stresses which induce HSPs, beside heat shock, one can mention inhibitors of energy metabolism, amino acid analogues, and heavy metals such as cadmium. We are interested in the role of HSPs in pathophysiological states, such as fever [7], ischemia [8], inflammation [7, 9], oxidant injury [10] and (bacterial) infection. 3. Heat shock proteins in host-pathogen interactions Whereas several groups have been extensively studying the stress response of bacteria and the role of stress proteins in bacterial virulence and in the immune responses to bacteria, we have focussed our attention on the stress response in the host cells, i.e., in human phagocytes. We are currently investigating the stress response in phagocytes in relationship to some of their relevant physiological functions, including phagocytosis, activation of the re-
0165 - 2478 / 91 / $ 3.50 © Elsevier Science Publishers B.V. All rights reserved.
159
spiratory burst, and, for monocytes-macrophages (m~), antigen presentation. The first question we have asked is whether stress proteins are induced during phagocytosis. We found indeed that phagocytosis of opsonized sheep red blood cells induces a striking stress response in human monocytes-macrophages, with induction of the classical HSPs, HSP70, together with HSP65, HSP83-90, HSP110, HSP47 and a 32-kDa oxidation-specific stress protein, heme oxygenase [11] (see Table 1 for a list of the stress proteins most relevant to our studies). In cells from patients with chronic granulomatous disease, although induction of the classical HSPs is maintained, the synthesis of heme oxygenase is barely detectable [7], suggesting that the regulation of HSPs and of heme oxygenase is, at least in part, different. The questions we next asked are three-fold: 1. is there any specificity for the second messenger systems involved in stress protein synthesis during phagocytosis? 2. what happens in terms of the stress response in phagocytes during phagocytosis of bacteria? and 3. do the stress proteins induced during phagocytosis interfere with the phagocyte's functions? 4. Induction of stress proteins in human monocytes-macrophages after heat shock and during erythrophagocytosis In human cells, the induction of stress proteins after exposure of cells to various types of injury is mediated by the phosphorylation and the binding of preformed heat-shock transcription factor(s) to the heat-shock consensus sequence (characterized by the repetitive motif GAA-TTC) and the coordinate transcriptional activation of all heat-shock genes [12]. The presence, within the cells, of abnormal, misfolded or unfolded proteins would represent the final common pathway for stress protein induction under the multiplicity of conditions mentioned above. It has, however, also been hypothesized that more classical second messengers, such as calcium or inositol phosphates, may represent a link between the initial cellular stress and the transcriptional activation of heat-shock genes [13]. Among these, we have investigated the role of calcium, and in the human premonocytic line U937, we 160
TABLE 1 Stress proteins induced during phagocytosis: classification and functions. Stress proteins HSPs families HSP 47
HSP 65
HSP 70
HSP 90
HSP 110
Enzymes Superoxide dismutase
Heme oxygenase
Chief characteristics
Glycoprotein associated with ER membrane, collagen binding activity. Mitochondrial protein analogous to E. coli GroEL and to major mycobacterial 65-kDa antigen, participates in proper folding and assembly of protein complexes. Cytoplasmic and nuclear proteins of 68 - 73 kDa, high affinity for ATP, uncoating ATPase, protein translocation, targeting for lysosomal degradation. Cytoplasmic protein of 8 3 - 90 kDa, interaction with tyrosine kinases and steroid hormone receptor Nucleoli located 110-kDa protein, may be involved in the transcription process.
Mitochondrial inducible form referred to as MnSOD, tetrameric complex of ~ 90 kDa responsible for the dismutation of superoxide anion to hydrogen peroxide. 32-kDa protein contained in the microsomal fraction, involved in heme catabolism.
have shown that calcium plays no role in HSP induction [14]. We are currently investigating, in these same cells, whether activation of protein kinase C participates in the sequence of stressful events leading to HSP synthesis. We have also attempted, in the erythrophagocytosis model, to ascertain whether phagocytosis itself, or the generation of oxygen-free radicals associated with phagocytosis (and more specifically the generation of the highly reactive hydroxyl radicals, whose production is catalyzed by the presence of iron) play the major role in the induction of stress proteins. To address this issue, we have used different phagocytic stimuli, as well as different cell types and various antioxidants or free radical scavengers,
including flavonoids. Flavonoids have recently been shown to inhibit the synthesis of HSPs after heat shock [15], and are, all together, radical scavengers, iron chelators, and inhibitors of protein kinase C. Our results suggest that both phagocytosis and the generation of toxic metabolites of oxygen are required for this induction: inert phagocytic stimuli such as latex beads do not affect protein synthesis in phagocytic cells, and phorbol esters, which are the most potent activators of the respiratory burst, induce only low levels of HSPs. Although it is generally accepted that antigen-presenting cells (APC) do not have the ability to distinguish between self and non-self proteins, heatshock proteins may represent a non-immunological tool to recognize and eventually to eliminate large amounts of intracellular foreign proteins such as is the case during erythrophagocytosis. 5. What happens in terms of the stress response during bacterial infection?
It has been known for a long time that viral infections induce stress protein synthesis in host cells [16, 17]. Recently, the possibility that cross-reactivity between bacteria and host HSPs may lead either to protective immunity or to autoimmunity has made it a crucial issue to know whether and how host stress proteins are available for T cell recognition after bacterial infection [18]. We are investigating the stress response to various bacteria and parasites and preliminary results suggest that different bacteria induce different stress responses. Bacteria which normally do not induce autoimmunity, such as Staphylococcus aureus, may lead to preferential synthesis of HSP70, whereas bacteria associated with autoimmunity (mycobacteria) appear to induce HSP65. Furthermore, when parasites do not induce a detectable respiratory burst, no stress response at all can be detected, despite phagocytosis of the pathogen, further emphasizing the role of reactive oxygen metabolites in HSP induction. The stress response associated with phagocytosis also appears to be dependent upon the state of differentiation of the phagocytic cells. Stress proteins are increased in 1,25-dihydroxyvitamin D3-treated cells and even more so in terminally differentiated tissue macrophages (alveolar macrophages).
6. What are the effects of stress proteins induced during phagocytosis on the phagocyte's functions?
6.1. Interactions of stress proteins with N A D P H oxidase We have shown that heat shock and other inducers of HSPs such as cadmium inhibit NADPH oxidase in human neutrophils [19]. This inhibition does not appear to be mediated by HSPs, inasmuch as heat shock also inhibited NADPH oxidase in the absence of any possible transcriptional activation and synthesis of HSPs, i.e., in enucleated neutrophils (cytoplasts) (Maridonneau-Parini et al., submitted for publication). If HSPs appear to play no role in the inhibition of the respiratory burst, they do, however, protect neutrophils from further inhibition of NADPH oxidase by a subsequent exposure to heat shock (Maridonneau-Parini et al., submitted). These data are in agreement with observations of Nguyen et al. [20] who showed that HSPs have the ability to protect enzymatic functions from degradation by heat shock. 6.2. Effects of liSPs (HSP70) in the A P C on subsequent T cell responses There is an important theoretical background for a role of HSPs on antigen processing and/or presentation: (i) Since HSP70 has the ability to "chaperone" other proteins through the cells it could also chaperone antigens. (ii) HSP70 is the clathrin uncoating ATPase and participates in endocytosis via coated pits [21]. (iii) A member of the HSPT0 family is involved in targeting for lysosomal degradation and may thus be involved in antigen processing [22]. (iv) Several genes for the HSP70 (there are at least five of them in the human system) have been mapped within the major histocompatibility complex [23] (close to the TNF genes). (v) Another member of the HSP70 family (PbP72/ 74) plays a role in processing and presentation of pigeon cytochrome c, possibly by facilitating the association of processed antigen with the MHC class II molecules [24]. (vi) HSP70 may also participate in the assembly of 161
MHC molecules themselves [25]. We investigated the effects of inducing a heatshock response in APCs on the T cell proliferative responses. Heat shock increased the expression of MHC class II molecules in a murine B cell line and in human peripheral blood monocytes (Rees et al., submitted for publication; Mari6thoz and Polla, unpublished data). Stress also enhanced the ability of B cells to stimulate both auto- and allo-reactive T cells but it was difficult to distinguish between an effect of stress on class II expression and a direct effect of HSPs on antigen processing a n d / o r presentation. In the human monocytes, preliminary data suggest that exposure of APC to heat shock does not increase T cell responses in mixed lymphocyte reactions, whereas in the autologous system, T cell responses are increased with only some, but not all, antigens tested. When the temperature to which monocytes were exposed was increased to 45 °C, T cell responses were abolished, as previously described by Goeken et al. [26]. Further studies will be required to precisely determine whether and how HSPs participate in antigen uptake, processing, M H C assembly, transport, and presentation. 6.3. HSPs and degradation material
of phagocytosed
Another important function of phagocytes is the production of high levels of proteases. HSPs may be considered as part of the proteolytic system: ubiquitin, a small 76-amino acid HSP, is involved in the targeting of cellular proteins for degradation and proteolysis. We are currently investigating whether in human phagocytes ubiquitin is, or is not, coordinately upregulated with other stress proteins during phagocytosis. Furthermore, a member of the HSP70 family has been shown to participate in targeting of intracellular proteins for lysosomal degradation [22]. This protein may also play a specific role in the degradation of phagocytosed material. 6.4. Protective or "'scavenging" role of liSPs We have shown that pre-exposure to heat shock protects human monocytic cells from DNA strand breaks (M. Perin et al., unpublished data) and cell death [27] secondary to oxidative injury (exposure 162
to hydrogen peroxide). We are investigating the mechanisms by which HSPs protect cells from oxidative damage. Our current hypothesis includes the prevention of DNA strand breaks (for a review, see the Proceedings of the Heat Shock Workshop, Naples, September 1990, in press). We also propose the hypothesis that cells which produce high levels of toxic substances such as reactive oxygen species or tumor necrosis factor ~ during their normal cell function (i.e., phagocytosis) synthesize HSPs as an autoprotective mechanism [28, 29].
Acknowledgements Work mentioned here was supported by the Swiss National Research Foundation (Grants 3.960-0.87 and 32-028645.90). I am grateful to Y. R. A. Donati for helpful discussions and critical review.
References [1] Ritossa, F. (1962) Experientia Basel 18,571. [2] Deshaies, R.J., Koch, B.D., Werner-Washburne, M., Craig, E.A. and Schekman, R. (1988)Nature 332,800. [3] Chirico, W. J., Waters, M.G. and Blobel, G. (1988) Nature 332, 805. [4] Pelham, H. R. B. (1986) Cell 46, 959. [5] Young, D., Lathigra, R., Hendrix, R., Sweetser, D. and Young, R.A. (1988) Proc. Natl. Acad. Sci. USA 85, 4267. [6] Morimoto, R. I. and Milarski, K. L. (1990)in: Cold Spring Harbor Monograph Series - Stress Proteins in Biology and Medicine (R. I. Morimoto, A. Tissibres and C. Georgopoulos, Eds.) Vol. 19, pp. 3 2 3 - 359, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [7] Polla, B.S. and Kantengwa, S. (1991) in: Current Topics in Microbiology and Immunology - Heat Shock Proteins and Immune Response (S. H. E. Kaufmann, Ed.) Vol. 167, pp. 93 - 105, Springer-Verlag, Berlin. [8] Polla, B.S., Mili, N., Donati, Y. and Bonventre, J.V. (1991) N6phrologie 12, 119, in press. [9] Polla, B. S. (1988) Immunol. Today 9, 134. [10] Donati, Y. R. A., Slosman, D.O. and Polla, B.S. (1990) Biochem. Pharmacol. 40, 2571. [11] Clerget, M. and Polla, B. S. (1990) Proc. Natl. Acad. Sci. USA 87, 1081. [12] Sorger, P. K. (1990) Cell 62, 793. [13] Calderwood, S.K., Stevenson, M.A. and Hahn, G.M. (1987) J. Cell. Physiol. 130, 369. [14] Kantengwa, S., Capponi, A. M., Bonventre, J. V. and Polla, B. S. (1990) Am. J. Physiol. 259, C77. [15] Hosokawa, N., Hirayoshi, K., Nakai, A., Hosokawa, Y., Marui, N., Yoshida, M., Sakai, T., Nishino, H., Aoike,
[16] [17] [18]
[19] [20] [21]
[22]
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