key molecules
Heat shockproteinsin host-parasiteinteractions Barbara S. Polla For most living organisms, beat shock represents an unusual stress situation, but for parasites that are transmitted between invertebrate vectors and mammalian hosts it is a frequent physiological occurrence. Because of the extraordinary conservation ofheat shock proteins (HSPs) and their potential immunogenicity, much attention has recently focused on the role of HSPs in infection and immunity. In parasites, HSPsappear to play specific functions in differentiation, in protection from the host cell’s killing mechanisms, including oxygen free radicals, and even in virulence. In this article, Barbara Polla uses the example of malaria to illustrate the possible role of HSPs in host-parasite relationships. Exposure of any living cell or organismto environmental stress,including elevatedtemperature, chemicalstressor oxidative injury, resultsin the transcription of a highly conservedset of genesand the synthesisof the so-called heat shock proteins (HSPs)‘.There hasbeenan explosion of interest in theseproteins recently. Before 1988, most papers on the subiect stated in their introductory paragraph (1) that HSPs were produced when cells were stressed,(2) that they were highly evolutionarily conserved,(3) that they, or closelyrelatedproteins, were also present in unstressedcellsand (4) that their function(s) was unknownl. In the lVVOs, HSPs tend to have a different emphasis:‘... over the last few years it has become clear that “chaperonins” [i.e. HSPs] provide attractive targets for specific recognition both by antibodies and by T cells’3.Clearly HSPs have becomethe domain of the immunologist.Immunodominant antigens from a wide variety of bacteria and parasiteshave been identified by sequencehomology as HSPs”. The role of HSPsin infection and immunity is currently receiving a great dealof attention and a major function for the stress responseduring inflammation has been postulateds-‘. ParasiteHSPsappearon the one hand to play important rolesin adaptation to the mammalianhost both in parasite differentiation and infectivity, while on the other hand they function as prominent antigenic proteins. Whether the host cell is stressedby the invading parasite so that it synthesizesHSPs, whether and how parasite and host HSPscan affect parasite-host interactions, and whether HSPsparticipate in protective immunity and/or autoimmunity are asyet unresolvedissues. Stressproteins are usually classifiedaccording to their apparent molecular mass, as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), and their inducing agents.A list of some eukaryotic stressproteins is presentedin Table 1, and their multiplicity is illustrated in Fig. 1.
related proteins were already present.This extraordinary conservatism supports the concept that HSPs are endowed with functions essentialfor cell survival. HSP70 is important in protein-protein interactions and by binding to unfolded, denatured or abnormal proteins may function as a molecular chaperone, allowing proteins to locate and fold (or refold) properly in their definitive cellular compartment. This function is assisted by the ability of HSP70 to translocateacrossmembranes and participate in proteolysis. Under conditions of massive protein unfolding, as may occur after heating or free radical exposure, HSPs that recognize and bind to the altered proteins within the cell are induced. Chaperoning of damagedproteins may contribute to cellular protection and repair processes. The accumulation of abnormal proteins has been suggestedasan important signal for the induction of the stressresponse;foreign proteins from intracellular para-
Table 1. Eukaryotic stressproteins ClassicalHSPs”
Other stressproteins
HSPllO HSP83-90 HSP70 (multiple genesand isoforms) HSP6.S HSPS81Pl HSP47 (collagen binding) Low molecular weight HSPs Glucose-relatedproteins GRP96 (related to HSPVO) GRP78-BiP (related to HSP70)
Ubiquitin Metallothioneins Enzymes Superoxide dismutaseb Heme oxygenase TCP- 1?
“In Leishmania theapparentmolecular masses of stress proteinsare 88,74 and 54 kDa (afterexposure to heatshock)and94, 78 and Regulation and functions of HSPs 56kDa (majorstress proteins)and70,45, 22 and18 kDa (minor HSPs are among the most evolutionarily conserved stress proteins)(afterexposure to arsenite) (Ref.15). bSuperoxide proteins. Recentwork by Guptassuggeststhat even in the dismutase isan HSP in bacteria but it is questionable whether it is an progenote, the common universal ancestor of pro- HSP in eukaryotes. BiP: heavy chain immunoglobulin binding karyotes, eukaryotes and archaebacteria, chaperonin- protein;TCP-1: mouse T-cell-complex protein 1. 0 1991, Elscvicr kicnce Publisbcrr
Ltd. UK. 0167-4919/91/502.00
A38
110 kDa 90 kDa 70 kDa 65 kDa -
actin 615
715
-’ 515
ur
. 615
716
pH Gradient
32 kDa -
Fig, 1. Pattern of protein synthesis after heat shock and during erytbrophagocytosis in human monocytes. Normal human peripheral blood monocytes (PBMs)were isolated by gradient centrifugation, purified by adherence and then cultured, labeled and processed as &.T:‘ FJ \ ‘i. .,t _ described %rdetail elsewhereg. Proteins labeled with /3sSjmethionine wereanalysed by (a) -one-dimensional or [b) two-dimensional gel electrophoresis. (a) Multiple proteins are kDa and less well characterized proteins. Erythrophagocytosis induced by heat shock (44°C 20 min; lane I), tire classical HSPs,65,70,83-90,110 (lanes 2 and 3) induces the classical HSPsand HO (32 kDa) which in human cells is not induced by heat shock. Lane 4 is the control. (6) Twodimensional electrophoresis from samples corresponding to lane 4 (1) and 2 (2) in (a) respectively. Besides the highly induced HSP70 and HO (arrows), multiple proteins are induced or increased in macrophages during erythrophagocytosis.
sites may also act as an inducing signal for the activation of HSP genes. The transcriptional control of mammalian HSP genes is mediated by heat shock transcription factor(s) acting on 5’ heat shock consensus sequences (GAAITC). Parasite HSP genes, for example Typanosoma brucei HSP70 and HSP83, T. crtlzi HSP83 and ubiquitin genes, have a’single copy of a similar sequence in their 5’ untranscribed region y. Stress proteins expressed within the host cell may then participate in the elimination of pathogens, either by a nonimmunological self-nonself discrimination mechanism, as hypothesized by Forsdyke’e, or during processing and presentation. Indeed, there is some evidence that HSPs contribute to antigen processing and presentation; for example a 72-74 kDa peptide-binding protein, involved in the presentation of pigeon cytochrome c has recently been found to be a member of the HSP70 gene family”, but whether or not host HSPs are involved in the same way during immune responses to parasites in vivo remains to be determined. Specific HSP functions in parasites During their life cycles, parasites are exposed to major temperature differences when moving from invertebrate vectors to homeothermic mammalian hosts. Most parasites undergo a classical heat shock response after temperature shifts, or exposure to other types of stress such as metabolic poisons t2. This response is associated with the transcriptional activation of heat shock genes and the inhibition of normal protein synthesis. However, some of the proteins identified as stressproteins in parasites differ in size from the classical HSPs (for example in
Leishmanidz). Besides the universal phenomenon of increased synthesis of HSPs in response to a rise in temperature,
parasite HSPs appear to play unique roles in differentiation. HunterI first suggested that heat shock was involved in the transformation of promastigotes to amastigotes. Van Der Ploeg et al. I4 then established that a temperature shift can induce differentiation of L. major in vitro. Evidence has now accumulated supporting a role for heat shock in the differentiation of many parasites, including Schistosoma mansoni and Naegleria gruberi, although modifications of growth or environmental conditions other than heat shock can cause a heat shock response and also induce differentiationy. Other interesting functions of HSPs examined in parasites concern mtp70, a mitochondrial member of the HSP70 family isolated in T. cruzi, and ubiquitin’sJ6. Mtp70 is located in the trypanosome mitochondrion in the region where kDNA replication occurs and it has been suggested that mtp70 participates in this replicationIs. T. crud also possessesover 100 ubiquitin genes organized into a single cluster that contains both polyubiquitin and ubiquitin fusion gene@. Ubiquitin may either sensitize proteins to proteases or act by itself as a protease. It has been suggested that parasite ubiquitin could be secreted into the cytoplasm of the host so that sufficient amounts of small peptides or amino acids derived from the host’s proteins are provided for nourishment and survival of the parasitel6. A role for HSPs in parasite virulence and resistance has been suggested by several observations. In the case of Leishmania spp, promastigotes subjected to heat shock convert to amastigotes, leading to an increase in infectivity (as determined by lesion size on the base of the tail of hamsters injected intradermally with samples of parasites exposed, or not, to heat shock, that is to 34°C)“. After heat shock in vitro promastigote morphology
A39
key molecules indirect heat shock response by the elevation of body temperature or the generation of oxygen free radicals, evidence for direct induction of HSPs by TNF-a has been provided only in macrophages2’. Since it has been shown that heat shock decreases cellular sensitivity to TNF-ainduced cytolysis 2s, TNF-a may contribute, via the induction of HSPs, to protective mechanisms against its own toxicity. This may be of particular importance to monocytes and macrophages, the ‘professional’ producers of TNF-a. Oxygen free radicals are generated either by phagocytes after activation of the respiratory burst enzyme, NADPH oxidase, or as a consequence of ATP depletion and xanthine metabolism by xanthine oxidase during ischemia and reperfusion. Both mechanisms may occur during malaria (brain damage during cerebral malaria being analogous to ischemia) and may contribute to HSPs in malaria disease. On the other hand, oxygen free radicals proHSPs have been suggested as missing links in hostparasite relationships 19. One link that can be made duced by macrophages represent a major first-line debetween malaria and the stress response is the recent fence among the anti-parasite mechanisms used by the observation that several immunodominant antigens host. Here, oxygen free radicals will be considered solely of Plasmodium fulcipurum, and indeed of many other from the point of view of their interactions with HSPs. Macrophage-derived oxygen free radicals are proparasites, belong to the HSP family; examples include a duced during phagocytosis and are known to induce a 75 kDa cytoplasmic antigen homologous to Drosophila stress response in bacteria. In human macrophages, both melunoguster HSP70, a 75 kDa protein associated with the merozoite surface and an HSP antigen belonging exogenous free radicals and erythrophagocytosis induce to the HSPSO familyli~ZO-l~. But, unlike other parasites, HSP synthesis” and, in turn, pre-exposure to heat inthe genome of P. fulcipurtlm contains at least five HSP70- duces partial tolerance to oxidative injuryrg. HSPs synlike genes, located on four different chromosomesy. thesized during phagocytosis may thus be part of an P. fulcipurum HSP70 has been detected in intra- autoprotective mechanism for the phagocyte, against the erythrocytic stages but not on free merozoites, although free radicals (or TNF-a) that it is producing. As already Renia et ~1.‘~ have now shown that its synthesis occurs mentioned, induction of a heat shock response protects during intrahepatic development. Even more interesting monocytes from HzOz-induced injury’y, and heme oxyis the observation that this HSP-like protein is expressed genase, induced during erythrophagocytosis, has definite on the surface of infected hepatocytesZ3 and is immunoanti-oxidant potential 30. In the brain, HSPs are induced genic. in viva in several animal models for cerebral ischemia and During the course of malarial infection, phagocytosis reperfusion injury, and by elevations in body temperaof parasitized erythrocytes is one of the host’s defence tures similar to those observed in malaria. In the nervous mechanisms. Phagocytic cells release soluble factors, system, HSPs are transported by slow axonal transincluding oxygen free radicals, other reactive oxygen port but are also transferred from glia to axon, allowing species, such as hydrogen peroxide, and tumor necrosis rapid supply of HSPs to any part of an injured axon31. factor cx(TNF-a), that may cause intraerythrocytic death During cerebral malaria, host HSPs may also be induced, of the parasites. Reactive oxygen species and TNF pro- and play a significant role in protection from brain duced by monocytes-macrophages are involved in intradamage. erythrocytic killing of the malarial parasites but both also cause tissue injury. HSPs, free radicals and parasites It has been shown that phagocytosis of sheep Toxic oxygen species probably induce HSP production erythrocytes by human macrophages induces synthesis of both in parasites and in host cells where they may serve the classical HSPs and of a 32 kDa oxidation-specific similar protective functions. Log phase, avirulent, prostress protein, heme oxygenase (Ref. 24 and Fig. 1) in the mastigotes of L. donovani chugusi appeared to be more macrophages. Whether or not phagocytosis of para- sensitive to HzOz than stationary, virulent, prositized red blood cells induces a distinct, specific stress mastigotes32. Pre-exposure of avirulent promastigotes to response in the phagocytes is currently being examined in heat shock induces a similar level of resistance to HzOz as our laboratory. In any event, stress proteins synthesized that found in stationary promastigotes; both the heat by macrophages during phagocytosis of parasitized red shocked and stationary promastigotes showed increased blood cells or in association with the host’s fever induced HSP70 transcriptiod2, and transcriptional activity was a by the infection, provides a second link between malaria prerequisite for H202 resistance induced by heat shock. and the stress response. These data suggest a role for parasite HSPs in protection The role of TNF-a in malaria has been well docu- from oxidative damage. Likewise, a major antigen of S. mented both in the mouse model and in humans25 and is mansoni is a glutathione transferase, which may also be discussed in detail by Titus and colleagues (this issue). part of parasite defences against nonspecific, oxidationWhereas TNF-a does induce the production of super- mediated, immune attack, since it both inhibits lipid oxide dismutase (Table 1 and Ref. 26) and perhaps an peroxidation and scavenges hydroxyalkenalsss.
changes to resemble that of intracellular amastigotes, but even before the changes in morphology are observed, the infectivity of heat-shocked L. brusiliensis panamensis promastigotes increases 17.This finding suggests a role for HSPs in virulence and in the adaptation of these organisms to a change in environment when they invade mammalian tissues or the insect vector. In bacteria, a similar role for HSPs has recently been established. Macrophagesensitive mutant Salmonellu fail to synthesize a subset of stress proteins normally induced during the infection of macrophages, indicating that these proteins contribute to bacterial survival within the macrophagest8. HSPs may thus have contributed to the evolutionary selection of pathogens, which may in part explain the conservation and immunodominance of these proteins.
A40
key molecules Work mentioned here was supported by the Swiss National Research Foundation (Grant 3.960-0.87). The author is grateful to S. Kantengwa for helpful discussions and critical review of the manuscript.
Another peculiarity of oxidative injury to parasites is that it is also mediated by eosinophils, which besides their unique, prolonged release of superoxide and eosinophil peroxidase, characteristically secrete toxic granule proteins such as major basic protein, which are involved in parasite killing. This may be of particular relevance with respect to the stress response. Indeed, selective spontaneous HSP synthesis by human alveolar mactophages recovered by bronchoalveolar lavage from patients with interstitial lung diseases and alveolar eosinophilia has been observed (B. Polla et al., unpublished). It thus appears that when macrophages are infected by parasites, both host and pathogen HSPs may be induced. The net effect (if any) of HSP synthesis on host-parasite interactions (for example pathogen clearance, immunogenicity or virulence) may result from a subtle balance between the amount, type and availability for immune recognition of the HSPs produced by the host and the pathogen. Similarly, HSP synthesis by parasites during anti-parasite therapy, as described for Giurdiu fumbliu in response to mettonidazole and quinacrine3” may reflect either the stress imposed on the pathogens by the drugs ot a putative escape mechanism. However, if HSPs are assumed to be essentially good for the host, drugs would be expected to induce HSPs in host cells, as seen when gold compounds and penicillamine are used for the treatment of rheumatoid atthritis3’. Chloroquine may have a similar effect as gold on host cell HSP synthesis but via another mechanism, in this case alterations of the cytoskeleton (inhibition of phagosome-lysosome fusion), which ate known to play a role in the stress response. Parasite HSPs as immunogens Finally, HSPs have to be discussed with respect to their antigenicity. The possibility has been raised that molecular mimicry between host and pathogen HSPs could be involved in protective immunity and/or autoimmunityG6,9J9. The potential role of HSPs, mainly HSP65, in autoimmunity has been particularly developed with tespect to mycobactetia and rheumatoid arthritis. However, in parasites, although HSPs ate prominent immunogens, there appears to be a sufficient lack of homology between parasite and host HSPs to permit a specific host immune response to attack the parasite HSPs selectively3b38. HSP70 is a major antigen in chronic T. crwzi infection (Chagas disease, a condition in which autoimmune processes may be involved in pathogenesis), but despite a 73% similarity at the amino acid level between human and T. crtrzi HSP70, antibodies to T. crtrzi do not teact with human HSP70 (Ref. 39). f. fulcipurum HSP70 is expressed on infected hepatocytes and becomes a target for antibody-dependent cellmediated cytotoxicity 23.The 1Cll epitope preferentially recognized by sera from immune monkeys is located in a poorly conserved region of the HSP family. Thus the potential of HSP-like antigens for the development of anti-malaria vaccines remains13. Finally, the role of HSPs as selective targets for y6 T cells40 remains even more speculative than the role of HSPs in immunology and parasitology. We shall thus await more data in this rapidly moving field, perhaps discussing them in a future combined issue of Purusitology Today and Immunology Today.
Barbara Pollu is at the Allergy Unit, University Hospital, CH 1211 Geneva 4, Switzerland. References 1 Lindquist, S. and Craig, E.A. (1988) Annu. Rev. Genet. 22, 63l-677 2 Pelham,H. (1988)Nature 332, 776-777 3 Young, D.B. (1990)Semis.CellBio/. 1, 27-35 4 Young, D.B. (1988)Proc. Nat/ Acad. Sci. USA 85, 42674270 5 Young, R.A. and Elliott, T.J. (1989)Cell59, S-8 6 Kaufmann,S.H.E. (1990)Immunol. Today 11, 129-136 7 Polla,B.S.andKantengwa,S. Curr. Top. Microbial. hmtnol. (in press) 8 Gupta, R.S.(1990)Biochem. Int. 20, 833-841 9 Newport,G. et al. (1988) Parasitol. Today 4, 306-312 10 Forsdyke,D.R. (1985)1. Tbeor. Biol. 115,471-473 11 VanBuskirk,A. et al. (1989)/. Exp. Med. 170, 1799-1805 12 Lawrence,F. and Robert-Gero,M. (1985)Proc.Nat1 Acad. Sci. USA 82,4414-4417 13 Hunter, K. et al. (1984)Biockem. Biophys. Res.
Commun.125,755-760 14 Van Der Ploeg,L.H.T. et al. (1985)Science 228, 1443-1445 15 Engman,D.M. et al. (1989)Mol. Cell. Biol. 9, 5163-5168 16 Swindle,J. et ai. (1988)EMBO]. 7, 1121-l 127 17 Smejkal,R.M. et al. (1988)Exp. Parasitol. 65, l-9 18 Buchmeier,N.A. and Heffron, F. (1990)Science 248, 730-732 19 Kaufmann,S.H.E. (1990)Med. Microbial. lmmunol. 179, 61-66 20 Bianco,A.E. et al. (1986)Proc. Nat/ Acad. Sci. USA 83, 8713-8717 21 Ardeshir,F. et al. (1987)EMBO 1. 6,493499 22 Jendoubi,M. and Bonnefoy,S. (1988)Nucleic Acids Res. 16, 10928 23 RCnia,L. et al. (1990)Eur. 1. Immunol. 20, 1445-1449 24 Clerget,M. and Polla,B.S.(1990)Proc. Nat1 Acad. Sci. USA 87,108 l-1085 25 Grau, G.E. et al. (1989)New Engl. /. Med. 320, 1586-1591 26 Wang, G.H.W. et al. (1989)CeU58,923-931 27 Fincato.G. et al. Blood (in Dress) 28 JZittel$ M., Saksela,K.‘andSaksela,E. (1989)Eur. /. Immunol. 19. 1413-1417 29 Polla,B.S:et al. (1988)1. Ceil Bioi. 107,373-380 30 Keyse,S.M. andTyrrell, R.M. (1989)Proc. Nat1 Acad. Sci. USA 86, 99-103
31 Clark, B.D. and Brown, I.R. (1985)Proc. Nat1 Acad. Sci. USA 82, 1281 32 Zarley, J.H. et al. (1990)Clin. Res. 305A 33 Taylor, J. et al. (1988)EMBOJ. 7,465-472 34 Lindley,T.A. et al. (1988)Mol. Biochem. Parasitol. 28, 135-144 35 Caltabiano,M.M. et al. (1986)Biochem. Biophys. Res. Commun. 138,1074-1080 36 Hedsttom,R. et al. (1988)Mol. Biochem. Pnrasitol. 29,
275-282 37 Robertson,N.P. et al. (1988)/. Parasitol. 74, 1004-1008 38 Selkirk,M.E. et al. (1989)/. lmmunol. 143,299-308 39 Engman,D.M. et al. (199O)j. lmmunol. 144,3987-3991 40 O’Brien, R. et al. (1989)Cell57, 667-674
A41
Heat shockproteinsin host-parasiteinteractions Barbara S. Polla For most living organisms, beat shock represents an unusual stress situation, but for parasites that are transmitted between invertebrate vectors and mammalian hosts it is a frequent physiological occurrence. Because of the extraordinary conservation ofheat shock proteins (HSPs) and their potential immunogenicity, much attention has recently focused on the role of HSPs in infection and immunity. In parasites, HSPsappear to play specific functions in differentiation, in protection from the host cell’s killing mechanisms, including oxygen free radicals, and even in virulence. In this article, Barbara Polla uses the example of malaria to illustrate the possible role of HSPs in host-parasite relationships. Exposure of any living cell or organismto environmental stress,including elevatedtemperature, chemicalstressor oxidative injury, resultsin the transcription of a highly conservedset of genesand the synthesisof the so-called heat shock proteins (HSPs)‘.There hasbeenan explosion of interest in theseproteins recently. Before 1988, most papers on the subiect stated in their introductory paragraph (1) that HSPs were produced when cells were stressed,(2) that they were highly evolutionarily conserved,(3) that they, or closelyrelatedproteins, were also present in unstressedcellsand (4) that their function(s) was unknownl. In the lVVOs, HSPs tend to have a different emphasis:‘... over the last few years it has become clear that “chaperonins” [i.e. HSPs] provide attractive targets for specific recognition both by antibodies and by T cells’3.Clearly HSPs have becomethe domain of the immunologist.Immunodominant antigens from a wide variety of bacteria and parasiteshave been identified by sequencehomology as HSPs”. The role of HSPsin infection and immunity is currently receiving a great dealof attention and a major function for the stress responseduring inflammation has been postulateds-‘. ParasiteHSPsappearon the one hand to play important rolesin adaptation to the mammalianhost both in parasite differentiation and infectivity, while on the other hand they function as prominent antigenic proteins. Whether the host cell is stressedby the invading parasite so that it synthesizesHSPs, whether and how parasite and host HSPscan affect parasite-host interactions, and whether HSPsparticipate in protective immunity and/or autoimmunity are asyet unresolvedissues. Stressproteins are usually classifiedaccording to their apparent molecular mass, as estimated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), and their inducing agents.A list of some eukaryotic stressproteins is presentedin Table 1, and their multiplicity is illustrated in Fig. 1.
related proteins were already present.This extraordinary conservatism supports the concept that HSPs are endowed with functions essentialfor cell survival. HSP70 is important in protein-protein interactions and by binding to unfolded, denatured or abnormal proteins may function as a molecular chaperone, allowing proteins to locate and fold (or refold) properly in their definitive cellular compartment. This function is assisted by the ability of HSP70 to translocateacrossmembranes and participate in proteolysis. Under conditions of massive protein unfolding, as may occur after heating or free radical exposure, HSPs that recognize and bind to the altered proteins within the cell are induced. Chaperoning of damagedproteins may contribute to cellular protection and repair processes. The accumulation of abnormal proteins has been suggestedasan important signal for the induction of the stressresponse;foreign proteins from intracellular para-
Table 1. Eukaryotic stressproteins ClassicalHSPs”
Other stressproteins
HSPllO HSP83-90 HSP70 (multiple genesand isoforms) HSP6.S HSPS81Pl HSP47 (collagen binding) Low molecular weight HSPs Glucose-relatedproteins GRP96 (related to HSPVO) GRP78-BiP (related to HSP70)
Ubiquitin Metallothioneins Enzymes Superoxide dismutaseb Heme oxygenase TCP- 1?
“In Leishmania theapparentmolecular masses of stress proteinsare 88,74 and 54 kDa (afterexposure to heatshock)and94, 78 and Regulation and functions of HSPs 56kDa (majorstress proteins)and70,45, 22 and18 kDa (minor HSPs are among the most evolutionarily conserved stress proteins)(afterexposure to arsenite) (Ref.15). bSuperoxide proteins. Recentwork by Guptassuggeststhat even in the dismutase isan HSP in bacteria but it is questionable whether it is an progenote, the common universal ancestor of pro- HSP in eukaryotes. BiP: heavy chain immunoglobulin binding karyotes, eukaryotes and archaebacteria, chaperonin- protein;TCP-1: mouse T-cell-complex protein 1. 0 1991, Elscvicr kicnce Publisbcrr
Ltd. UK. 0167-4919/91/502.00
A38
110 kDa 90 kDa 70 kDa 65 kDa -
actin 615
715
-’ 515
ur
. 615
716
pH Gradient
32 kDa -
Fig, 1. Pattern of protein synthesis after heat shock and during erytbrophagocytosis in human monocytes. Normal human peripheral blood monocytes (PBMs)were isolated by gradient centrifugation, purified by adherence and then cultured, labeled and processed as &.T:‘ FJ \ ‘i. .,t _ described %rdetail elsewhereg. Proteins labeled with /3sSjmethionine wereanalysed by (a) -one-dimensional or [b) two-dimensional gel electrophoresis. (a) Multiple proteins are kDa and less well characterized proteins. Erythrophagocytosis induced by heat shock (44°C 20 min; lane I), tire classical HSPs,65,70,83-90,110 (lanes 2 and 3) induces the classical HSPsand HO (32 kDa) which in human cells is not induced by heat shock. Lane 4 is the control. (6) Twodimensional electrophoresis from samples corresponding to lane 4 (1) and 2 (2) in (a) respectively. Besides the highly induced HSP70 and HO (arrows), multiple proteins are induced or increased in macrophages during erythrophagocytosis.
sites may also act as an inducing signal for the activation of HSP genes. The transcriptional control of mammalian HSP genes is mediated by heat shock transcription factor(s) acting on 5’ heat shock consensus sequences (GAAITC). Parasite HSP genes, for example Typanosoma brucei HSP70 and HSP83, T. crtlzi HSP83 and ubiquitin genes, have a’single copy of a similar sequence in their 5’ untranscribed region y. Stress proteins expressed within the host cell may then participate in the elimination of pathogens, either by a nonimmunological self-nonself discrimination mechanism, as hypothesized by Forsdyke’e, or during processing and presentation. Indeed, there is some evidence that HSPs contribute to antigen processing and presentation; for example a 72-74 kDa peptide-binding protein, involved in the presentation of pigeon cytochrome c has recently been found to be a member of the HSP70 gene family”, but whether or not host HSPs are involved in the same way during immune responses to parasites in vivo remains to be determined. Specific HSP functions in parasites During their life cycles, parasites are exposed to major temperature differences when moving from invertebrate vectors to homeothermic mammalian hosts. Most parasites undergo a classical heat shock response after temperature shifts, or exposure to other types of stress such as metabolic poisons t2. This response is associated with the transcriptional activation of heat shock genes and the inhibition of normal protein synthesis. However, some of the proteins identified as stressproteins in parasites differ in size from the classical HSPs (for example in
Leishmanidz). Besides the universal phenomenon of increased synthesis of HSPs in response to a rise in temperature,
parasite HSPs appear to play unique roles in differentiation. HunterI first suggested that heat shock was involved in the transformation of promastigotes to amastigotes. Van Der Ploeg et al. I4 then established that a temperature shift can induce differentiation of L. major in vitro. Evidence has now accumulated supporting a role for heat shock in the differentiation of many parasites, including Schistosoma mansoni and Naegleria gruberi, although modifications of growth or environmental conditions other than heat shock can cause a heat shock response and also induce differentiationy. Other interesting functions of HSPs examined in parasites concern mtp70, a mitochondrial member of the HSP70 family isolated in T. cruzi, and ubiquitin’sJ6. Mtp70 is located in the trypanosome mitochondrion in the region where kDNA replication occurs and it has been suggested that mtp70 participates in this replicationIs. T. crud also possessesover 100 ubiquitin genes organized into a single cluster that contains both polyubiquitin and ubiquitin fusion gene@. Ubiquitin may either sensitize proteins to proteases or act by itself as a protease. It has been suggested that parasite ubiquitin could be secreted into the cytoplasm of the host so that sufficient amounts of small peptides or amino acids derived from the host’s proteins are provided for nourishment and survival of the parasitel6. A role for HSPs in parasite virulence and resistance has been suggested by several observations. In the case of Leishmania spp, promastigotes subjected to heat shock convert to amastigotes, leading to an increase in infectivity (as determined by lesion size on the base of the tail of hamsters injected intradermally with samples of parasites exposed, or not, to heat shock, that is to 34°C)“. After heat shock in vitro promastigote morphology
A39
key molecules indirect heat shock response by the elevation of body temperature or the generation of oxygen free radicals, evidence for direct induction of HSPs by TNF-a has been provided only in macrophages2’. Since it has been shown that heat shock decreases cellular sensitivity to TNF-ainduced cytolysis 2s, TNF-a may contribute, via the induction of HSPs, to protective mechanisms against its own toxicity. This may be of particular importance to monocytes and macrophages, the ‘professional’ producers of TNF-a. Oxygen free radicals are generated either by phagocytes after activation of the respiratory burst enzyme, NADPH oxidase, or as a consequence of ATP depletion and xanthine metabolism by xanthine oxidase during ischemia and reperfusion. Both mechanisms may occur during malaria (brain damage during cerebral malaria being analogous to ischemia) and may contribute to HSPs in malaria disease. On the other hand, oxygen free radicals proHSPs have been suggested as missing links in hostparasite relationships 19. One link that can be made duced by macrophages represent a major first-line debetween malaria and the stress response is the recent fence among the anti-parasite mechanisms used by the observation that several immunodominant antigens host. Here, oxygen free radicals will be considered solely of Plasmodium fulcipurum, and indeed of many other from the point of view of their interactions with HSPs. Macrophage-derived oxygen free radicals are proparasites, belong to the HSP family; examples include a duced during phagocytosis and are known to induce a 75 kDa cytoplasmic antigen homologous to Drosophila stress response in bacteria. In human macrophages, both melunoguster HSP70, a 75 kDa protein associated with the merozoite surface and an HSP antigen belonging exogenous free radicals and erythrophagocytosis induce to the HSPSO familyli~ZO-l~. But, unlike other parasites, HSP synthesis” and, in turn, pre-exposure to heat inthe genome of P. fulcipurtlm contains at least five HSP70- duces partial tolerance to oxidative injuryrg. HSPs synlike genes, located on four different chromosomesy. thesized during phagocytosis may thus be part of an P. fulcipurum HSP70 has been detected in intra- autoprotective mechanism for the phagocyte, against the erythrocytic stages but not on free merozoites, although free radicals (or TNF-a) that it is producing. As already Renia et ~1.‘~ have now shown that its synthesis occurs mentioned, induction of a heat shock response protects during intrahepatic development. Even more interesting monocytes from HzOz-induced injury’y, and heme oxyis the observation that this HSP-like protein is expressed genase, induced during erythrophagocytosis, has definite on the surface of infected hepatocytesZ3 and is immunoanti-oxidant potential 30. In the brain, HSPs are induced genic. in viva in several animal models for cerebral ischemia and During the course of malarial infection, phagocytosis reperfusion injury, and by elevations in body temperaof parasitized erythrocytes is one of the host’s defence tures similar to those observed in malaria. In the nervous mechanisms. Phagocytic cells release soluble factors, system, HSPs are transported by slow axonal transincluding oxygen free radicals, other reactive oxygen port but are also transferred from glia to axon, allowing species, such as hydrogen peroxide, and tumor necrosis rapid supply of HSPs to any part of an injured axon31. factor cx(TNF-a), that may cause intraerythrocytic death During cerebral malaria, host HSPs may also be induced, of the parasites. Reactive oxygen species and TNF pro- and play a significant role in protection from brain duced by monocytes-macrophages are involved in intradamage. erythrocytic killing of the malarial parasites but both also cause tissue injury. HSPs, free radicals and parasites It has been shown that phagocytosis of sheep Toxic oxygen species probably induce HSP production erythrocytes by human macrophages induces synthesis of both in parasites and in host cells where they may serve the classical HSPs and of a 32 kDa oxidation-specific similar protective functions. Log phase, avirulent, prostress protein, heme oxygenase (Ref. 24 and Fig. 1) in the mastigotes of L. donovani chugusi appeared to be more macrophages. Whether or not phagocytosis of para- sensitive to HzOz than stationary, virulent, prositized red blood cells induces a distinct, specific stress mastigotes32. Pre-exposure of avirulent promastigotes to response in the phagocytes is currently being examined in heat shock induces a similar level of resistance to HzOz as our laboratory. In any event, stress proteins synthesized that found in stationary promastigotes; both the heat by macrophages during phagocytosis of parasitized red shocked and stationary promastigotes showed increased blood cells or in association with the host’s fever induced HSP70 transcriptiod2, and transcriptional activity was a by the infection, provides a second link between malaria prerequisite for H202 resistance induced by heat shock. and the stress response. These data suggest a role for parasite HSPs in protection The role of TNF-a in malaria has been well docu- from oxidative damage. Likewise, a major antigen of S. mented both in the mouse model and in humans25 and is mansoni is a glutathione transferase, which may also be discussed in detail by Titus and colleagues (this issue). part of parasite defences against nonspecific, oxidationWhereas TNF-a does induce the production of super- mediated, immune attack, since it both inhibits lipid oxide dismutase (Table 1 and Ref. 26) and perhaps an peroxidation and scavenges hydroxyalkenalsss.
changes to resemble that of intracellular amastigotes, but even before the changes in morphology are observed, the infectivity of heat-shocked L. brusiliensis panamensis promastigotes increases 17.This finding suggests a role for HSPs in virulence and in the adaptation of these organisms to a change in environment when they invade mammalian tissues or the insect vector. In bacteria, a similar role for HSPs has recently been established. Macrophagesensitive mutant Salmonellu fail to synthesize a subset of stress proteins normally induced during the infection of macrophages, indicating that these proteins contribute to bacterial survival within the macrophagest8. HSPs may thus have contributed to the evolutionary selection of pathogens, which may in part explain the conservation and immunodominance of these proteins.
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key molecules Work mentioned here was supported by the Swiss National Research Foundation (Grant 3.960-0.87). The author is grateful to S. Kantengwa for helpful discussions and critical review of the manuscript.
Another peculiarity of oxidative injury to parasites is that it is also mediated by eosinophils, which besides their unique, prolonged release of superoxide and eosinophil peroxidase, characteristically secrete toxic granule proteins such as major basic protein, which are involved in parasite killing. This may be of particular relevance with respect to the stress response. Indeed, selective spontaneous HSP synthesis by human alveolar mactophages recovered by bronchoalveolar lavage from patients with interstitial lung diseases and alveolar eosinophilia has been observed (B. Polla et al., unpublished). It thus appears that when macrophages are infected by parasites, both host and pathogen HSPs may be induced. The net effect (if any) of HSP synthesis on host-parasite interactions (for example pathogen clearance, immunogenicity or virulence) may result from a subtle balance between the amount, type and availability for immune recognition of the HSPs produced by the host and the pathogen. Similarly, HSP synthesis by parasites during anti-parasite therapy, as described for Giurdiu fumbliu in response to mettonidazole and quinacrine3” may reflect either the stress imposed on the pathogens by the drugs ot a putative escape mechanism. However, if HSPs are assumed to be essentially good for the host, drugs would be expected to induce HSPs in host cells, as seen when gold compounds and penicillamine are used for the treatment of rheumatoid atthritis3’. Chloroquine may have a similar effect as gold on host cell HSP synthesis but via another mechanism, in this case alterations of the cytoskeleton (inhibition of phagosome-lysosome fusion), which ate known to play a role in the stress response. Parasite HSPs as immunogens Finally, HSPs have to be discussed with respect to their antigenicity. The possibility has been raised that molecular mimicry between host and pathogen HSPs could be involved in protective immunity and/or autoimmunityG6,9J9. The potential role of HSPs, mainly HSP65, in autoimmunity has been particularly developed with tespect to mycobactetia and rheumatoid arthritis. However, in parasites, although HSPs ate prominent immunogens, there appears to be a sufficient lack of homology between parasite and host HSPs to permit a specific host immune response to attack the parasite HSPs selectively3b38. HSP70 is a major antigen in chronic T. crwzi infection (Chagas disease, a condition in which autoimmune processes may be involved in pathogenesis), but despite a 73% similarity at the amino acid level between human and T. crtrzi HSP70, antibodies to T. crtrzi do not teact with human HSP70 (Ref. 39). f. fulcipurum HSP70 is expressed on infected hepatocytes and becomes a target for antibody-dependent cellmediated cytotoxicity 23.The 1Cll epitope preferentially recognized by sera from immune monkeys is located in a poorly conserved region of the HSP family. Thus the potential of HSP-like antigens for the development of anti-malaria vaccines remains13. Finally, the role of HSPs as selective targets for y6 T cells40 remains even more speculative than the role of HSPs in immunology and parasitology. We shall thus await more data in this rapidly moving field, perhaps discussing them in a future combined issue of Purusitology Today and Immunology Today.
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