INFECTION AND IMMUNrrY, Aug. 1992, p. 3105-3110
Vol. 60, No. 8
0019-9567/92/083105-06$02.00/0 Copyright X 1992, American Society for Microbiology
hsp7O Synthesis in Schwann Cells in Response to Heat Shock and Infection with Mycobactenium leprae YASMIN MISTRY,1 DOUGLAS B. YOUNG,2 AxND3 RAMA MUKHERJEEl* Microbiology Division, National Institute of Immunology, Shahid Jeet Singh Marg, New Delhi-10 067,
India, 1 and Medical Research Council Tuberculosis and Related Infections Unit, Hammersmith Hospital, London W12 OHS, United Kingdom2 Received 22 April 1992/Accepted
5
May 1992
Induction of heat shock protein synthesis was monitored in murine and monkey Schwann cells exposed to elevated temperatures. Synthesis of the stress-inducible 70-kDa heat shock protein (hsp7O) was detected in both murine and primate Schwann cells by metabolic labelling and by immunoblotting with a specific monoclonal antibody. hsp70 synthesis was also induced in Schwann cells after infection with Mycobacterium leprae and was detected from 24 h to 1 week postinfection. These results are discussed with respect to the possible role of heat shock proteins in immunopathological events associated with the clinical manifestations of leprosy.
Leprosy is a chronic infectious disease caused by Mycobacterium leprae and currently affects an estimated 10 to 11 million individuals worldwide (25). Neuritis and nerve damage are the most important features of leprosy, and invasion of the nerve is a unique characteristic of M. leprae (24). Histopathological observations and in vitro studies have demonstrated that Schwann cells, the glial cells of the peripheral nervous system, are the preferred hosts, harboring large numbers of intracellular bacilli (12, 20, 34). Leprosy bacilli do not have a direct cytotoxic effect on Schwann cells, and it has been suggested that the nerve damage is a result of cell-mediated immune responses to mycobacterial and/or host antigens (6, 13). Factors which may play a role in such cell-mediated immunopathology include the release of cytokines (such as tumor necrosis factor and gamma interferon) from locally activated macrophages (3) and a direct attack on infected cells by cytotoxic T lymphocytes (23, 28, 29). T-cell-mediated lysis of Schwann cells infected in vitro with M. leprae has been described previously (28, 30). In the murine system, a role was shown for CD8+ alpha/beta cytotoxic T cells (29), with unrestricted killer cells fulfilling a similar function in a human Schwann cell model (30). In each case, a central role was proposed for Schwann cell heat shock proteins. Evidence was presented to indicate that the murine CD8+ cytotoxic T cells recognize antigenic determinants shared by bacterial and Schwann cell heat shock proteins (29), while in human Schwann cells, the induction of heat shock proteins was suggested to play a role in allowing infected cells to resist killing (30). In all of these studies, a role for hsp65 has been implicated. The role of hsp70, which is equally important, however, remains to be established. Heat shock proteins were initially identified by their elevated synthesis after exposure of cultured cells or organisms to a sudden temperature increase (for a review, see reference 15). Subsequently it was found that structurally related proteins are present constitutively in all cells and that heat shock could be replaced by a variety of other environmental stresses as inducers of the response (16). The heat shock response is generally considered to be ubiquitous, but there is evidence to suggest that the response may not be
identical in all cell types. In the rabbit brain, for example, in situ hybridization analysis revealed marked differences in heat shock gene expression of hsp70 in different cells both constitutively and after hyperthermia (27). A further indication of a specialized aspect to the heat shock response within the nervous system is provided by the observation of a rapid transfer of heat shock-like proteins with molecular sizes of 70 and 95 kDa from adaxonal glial cells into axons in the giant squid at elevated temperatures (32). Induction of a heat shock response after exposure to physical trauma has been demonstrated in the central nervous system (4), and detection of heat shock proteins has been suggested as a useful general marker for nerve damage (9). In this study, we have characterized the heat shock response of murine and monkey Schwann cells in an in vitro culture system and have analyzed the effect of infection with M. leprae on the expression of Schwann cell hsp70. MATERIALS AND METHODS
Schwann cell cultures from neonatal mice. The sciatic and brachial plexus from 2- to 3-day-old pups of BALB/c mice were dissected under sterile conditions by using a dissecting microscope (model SZ40; Olympus, Tokyo, Japan). These nerves were chopped into fine pieces and incubated in 0.05% collagenase (Sigma Chemical Co., St. Louis, Mo.) for 30 min at 37°C, and cells were dispersed by passing twice through 21- and 23-gauge needles. The cell suspension was collected after centrifugation at 500 x g for 10 min, washed with Dulbecco's minimal essential medium (DMEM; Flow Laboratories) and plated onto 25-mm2 sterile tissue culture flasks. Cultures were maintained in growth medium consisting of DMEM supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 IU/ml; streptomycin, 100 ,ug/ml; gentamicin, 100 p.g/ml) for a period of 7 to 10 days, with replenishment of the medium twice a week. Schwann cell cultures from adult monkeys. Peripheral nerves, mainly the sciatic and the ulnar, were dissected from adult rhesus monkeys anesthetized with ketamine (5 mg/kg of body weight). A modification of the technique described by Askanas et al. (2) was used to set up Schwann cell cultures from these nerves. Dissected nerves were immedinerves
ately transferred to DMEM containing gentamicin (100 ,ug/ *
ml). Under sterile conditions, the
Corresponding author. 3105
nerves were
cleaned of
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epineural tissue by use of a dissecting microscope and cut into 1-mm3 explants. Explants were treated with 0.25% trypsin (Difco Laboratories, Detroit, Mich.) for 5 min and then explanted onto collagen gel-coated six-well plates (Coming Glass Works, Coming, N.Y.). Collagen extraction and coating were done as previously described (20). The explants were maintained in growth medium which was replenished twice a week. After 7 to 10 days, when the cells had emerged sufficiently from the explants, the original explants were removed and reexplanted on fresh collagencoated culture plates. The glial cell outgrowth from these reexplants was greatly enriched in the Schwann cell population. The reexplanted cultures enriched for Schwann cells were subcultured to obtain dissociated monolayer cultures which were used for heat shock protein induction experiments. For this, explants were removed and the bed of Schwann cells embedded in collagen gel was first treated with 0.05% collagenase for a total of 20 min at 37°C and with 0.25% trypsin for 10 min to obtain a single cell suspension. The cells were obtained by centrifugation at 500 x g for 10 min, washed once with fresh DMEM, and then plated onto 25-mm2 culture flasks. Immunofluorescence staining. Schwann cells were identified by indirect immunofluorescence staining by using antibodies to S100 protein (26). Cells plated on coverslips were fixed with 4% formal saline for 10 min at 4°C and then permeabilized with 0.005% Nonidet P-40 (Sigma). The cells were stained with a 1:50 dilution of S100 antiserum (Dakopatts, Copenhagen, Denmark) for 30 min at room temperature and then labelled with a 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (from the reagent bank, National Institute of Immunology, New Delhi, India). Fluorescence was observed under UV light on a Microphot-FX microscope (Nikon, Tokyo, Japan). Infection with M. leprae. Leprosy bacilli were isolated from frozen armadillo tissue by using the method described by Ambrose et al. (1). The bacillary count was determined by using the method described by Hart and Rees (11). The absence of other bacterial contaminants was confirmed by lack of growth on nutrient agar and Lowenstein-Jensen media. To prepare heat-killed bacilli, bacterial suspensions were autoclaved at 15 lb/in2 for 15 min. Schwann cells were infected by incubation with 107 bacilli per ml of growth medium for 24 h. Unphagocytosed bacilli were removed by repeated washing with DMEM, and cultures were then maintained for periods of up to 1 week postinfection. The presence of acid-fast bacilli in infected cultures was monitored by Ziehl-Neelsen staining (10). Heat shock treatment. Schwann cells growing on 25-mm2 culture flasks were washed twice with methionine-free DMEM (GIBCO Laboratories, Grand Island, N.Y.) and allowed to remain in the same medium for 30 min. Cells were heat shocked by incubating flasks in a water bath for 30 min at 42 or 45°C, and then the temperature was returned to 37°C for recovery and radiolabelling of the cells. Metabolic labelling. Schwann cell cultures washed and incubated with 2 ml of methionine-free DMEM in 25-mm2 flasks as described above were labelled with [35S]methionine (5 ,Ci/ml; specific activity, >800 Ci/mmol; New England Nuclear, Boston, Mass.) for 1 h at 37°C. The adherent cells were then washed three times with complete DMEM and detached from the culture surface by mechanical scraping with a rubber policeman. The cell pellet was washed twice with phosphate-buffered saline, and the total protein content was estimated by the method of Lowry et al. (17). PAGE and Western blotting (immunoblotting). Radiola-
IN?FECT. IMMUN.
belled cell extracts were prepared for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) by boiling for 5 min in Laemmli sample buffer containing SDS and 2-mercaptoethanol (14). Samples containing equal amounts of protein were separated by electrophoresis in gels containing 10% polyacrylamide as described by Laemmli (14). For two-dimensional PAGE (2D-PAGE), labelled cell extracts were prepared in sample buffer containing urea, Nonidet P-40, 2-mercaptoethanol, and ampholytes (Pharmacia LKB Biotechnology, Bromma, Sweden) as described by O'Farrell (22). Proteins were separated in the first dimension by using tube gels containing ampholytes in the pH range of 3.5 to 9.5 at a concentration of 0.4% and ampholytes in the pH range of 4 to 6 at a concentration of 1.6% (22). In the second dimension, proteins were separated by SDS-PAGE as described above. Electrophoresis was carried out by using a mini-gel system (Hoefer Scientific Instruments, San Francisco, Calif.). Proteins separated by SDS-PAGE and 2D-PAGE were transferred to nitrocellulose membranes by electroblotting as described by Towbin et al. (31). Autoradiographs were prepared from blots by exposure to X-ray film (Hyperfilm-MP; Amersham) for 3 days at room temperature. For quantitation, autoradiographs were scanned by using a scanning densitometer and GS 365 Data System Software (Hoefer Scientific). To localize heat shock proteins, blots were incubated with the following monoclonal antibodies: C92, specific for the heat-inducible form of hsp7o (35) (SPA801; StressGen Biotechnologies Corp., Sidney, Canada); N27, which recognizes both heat-inducible and constitutive forms of hsp70 (33) (SPA802; StressGen Biotechnologies); and cosII, raised against the purified hsp7o of Mycobactenium tuberculosis (18). After incubation with monoclonal antibody, blots were washed and further incubated with peroxidase-conjugated anti-mouse immunoglobulin (1:2,000; Dakopatts) as a secondary antibody. Finally, bound antibody was visualized by incubation with 3,3'-diaminobenzidine dihydrochloride and hydrogen peroxide. RESULTS Schwann cell cultures. Schwann cells were cultured in vitro from nerves of newborn mice and adult monkeys. Dissociated single-cell cultures were readily obtained from the neonatal mice, but a technique of explantation-reexplantation had to be used for nerves from adult monkeys. Highly enriched Schwann cell cultures were obtained from nerves from adult monkeys by reexplantation of the original explants onto a fresh collagen-coated surface. A subsequent subculture of these yielded a monolayer of dissociated Schwann cells of approximately 95% purity. The cultured Schwann cells were identified by their typical bipolar morphology (Fig. la) and by their positive staining with antibodies to S100 protein, a specific marker which differentiates Schwann cells from neurofibroblasts in vitro (Fig. lb). Heat shock response. Dissociated murine and monkey Schwann cell cultures were heat shocked at 42 or 45°C for 30 min, and synthesis of heat shock proteins was then monitored after returning the cells to a temperature of 37°C. Synthesis of the heat-inducible form of hsp7o, recognized by monoclonal antibody C92, was detectable after heat shock at 42°C (data not shown), but the effect was more pronounced after a 45°C heat shock. Figure 2a shows a Western blot of cell extracts prepared from cultures at various times after a 45°C heat shock and developed with monoclonal antibody C92. Heat-inducible hsp70 was not detected in extracts from
VOL. 60, 1992
M. LEPRAE INDUCES hsp70 IN SCHWANN CELLS
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FIG. 1. Characterization of Schwann cell cultures. Schwann cells from nerves from adult monkeys were cultured as described in the text. (a) Viewed under a phase-contrast microscope, the Schwann cells have a characteristic bipolar morphology. Bar, 100 Fm. (b) Immunofluorescence staining with antibody to S100 indicated that Schwann cells accounted for approximately 95% of the cells in culture. Bar, 10 ,um.
murine Schwann cells maintained at 37°C (Fig. 2a, lane 1). After heat shock and then a recovery period of 3 h or more, however, a prominent C92-positive band was seen (Fig. 2a, lanes 4 to 6). A weak C92-positive band was observed in unstressed monkey Schwann cells (Fig. 2a, lane 7), but again heat shock resulted in a marked enhancement of hsp70 synthesis (Fig. 2a, lanes 8 to 12). Staining of the same samples with monoclonal antibody N27 (which recognizes constitutive as well as heat-inducible hsp70) showed no differences between control and heat-shocked samples on SDS-PAGE (data not shown). Further analysis of murine Schwann cell extracts by 2D-PAGE did, however, reveal a difference in the N27 staining pattern between control and heat-shocked cells (Fig. 3). In addition to the constitutive forms of the protein (Fig. 3, arrow labelled "c") stained by N27 in control cells (Fig. 3a), a new protein (Fig. 3, arrow labelled "1") was seen in the heat-shocked cells with a more acidic pI (Fig. 3b), corresponding to the heat-inducible hsp70 detected by C92 (Fig. 3d). Increased synthesis of hsp70 after heat shock could also be seen by SDS-PAGE analysis of cell extracts labelled with
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FIG. 2. Analysis of the heat shock response in cultured Schwann cells. Murine and monkey Schwann cell cultures were exposed to heat shock for 30 min at 45°C and then allowed to recover at 37°C as described in the text. [35S]methionine was added at the times indicated, and cell extracts prepared after a 1-h labelling period were analyzed by SDS-PAGE. Lanes 1 to 6 show extracts from murine Schwann cells. Lane 1, control cells maintained at 37°C; lanes 2 to 6, cells labelled immediately after heat shock (lane 2) and after 1 (lane 3), 3 (lane 4), 6 (lane 5), and 10 (lane 6) h of recovery. Lanes 7 to 12 show extracts from monkey Schwann cells treated as described for lanes 1 to 6, respectively. (a) hsp70 synthesis was monitored by Western blot with monoclonal antibody C92 (specific for the heat-inducible hsp70). Induction of hsp70 synthesis during the recovery period is clearly seen in lanes 4 to 6 (murine Schwann cells) and in lanes 8 to 12 (monkey Schwann cells). A low-level expression of the inducible hsp70 was seen in the control monkey cells (lane 7). (b) Autoradiograph showing metabolic labelling of the same cultures shown in panel A. Increased incorporation into a 70-kDa protein was observed after heat shock (indicated by arrowhead). This band precisely overlapped that recognized by the C92 monoclonal antibody.
[35Slmethionine (Fig. 2b). In murine Schwann cells, a reduction in total protein synthesis was clearly evident during the first hour after heat shock (Fig. 2b, lane 2), with enhanced hsp70 synthesis being seen during the subsequent recovery period. Quantitative densitometric analysis showed no incorporation of [35Slmethionine into the 70-kDa band in control murine Schwann cells; however, incorporation increased with recovery, reaching a maximum of 12.5% after 3 h, and subsequently dropped to 6.6% after 10 h. Inhibition of
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protein synthesis immediately after heat shock was not seen with the monkey Schwann cells, but increased hsp70 synthesis was again evident during the recovery phase (Fig. 2b, lanes 7 to 12). Maximum incorporation (9.7% of total [35S]methionine incorporated) was obtained after 6 h of recovery in monkey cell cultures. Effect of infection with M. leprae. Monkey Schwann cells readily phagocytosed M. leprae during 24 h of incubation in culture, with more than 90% of the cells harboring acid-fast bacilli (Fig. 4). Murine Schwann cells were less active in phagocytosis, and the monkey cells were therefore chosen as a model for detailed analysis of the effect of infection on hsp70 synthesis. As reported previously for Schwann cells in culture (21), phagocytosis was relatively specific for M.
leprae, with only a very limited uptake of other mycobacteria occurring under these conditions (while 90% of Schwann cells phagocytosed M. leprae, both live and killed, less than 15% of cells phagocytosed either H37Rv or H37Ra). Synthesis of hsp70 in infected cells was monitored by immunoblot and autoradiography by using the protocols established for the heat shock response. C92 immunoblot analysis of extracts from cells infected for periods of up to 1 week clearly showed an increase in synthesis of the heatinducible hsp7O (Fig. 5a). The increased synthesis was apparent as early as the first time point analyzed, i.e., 24 h after the addition of bacteria to the culture (Fig. 5a, lane 2). Bacterial viability was not essential for hsp70 induction, since phagocytosis of heat-killed organisms elicited a comparable effect (Fig. Sa, lanes 6 and 7). To discount the possibility that increased staining with C92 was due to cross-reactive recognition of M. leprae hsp70, Western blots with M. leprae extracts were probed with C92 and cosII (data not shown). The antimycobacterial antibody (cosII) bound strongly to M. leprae hsp70, but C92 was completely negative. No difference in N27 staining was detectable between the control and infected cultures (Fig. Sb). While the continued presence of the inducible hsp70, as detected by immunoblotting with C92 antibody, was evident in Schwann cells up to 1 week postinfection, it was of interest to determine whether or not increased synthesis was maintained throughout this period. Analysis of protein synthesis profiles by [35S]methionine labelling throughout the period of infection showed that increased incorporation into the 70-kDa band could in fact still be seen 1 week after the initial infection (Fig. Sc). Comparable control cultures maintained without infection over the same period did not show any increase in hsp70 synthesis. It is unlikely that the change in the [35S]methionine labelling profile represents incorporation into M. leprae proteins since M. leprae cells have an extremely slow growth rate (doubling time of 12 to 14 days) and are unlikely to synthesize detectable amounts of protein during the 1-h labelling period, the procedure for preparation of cell extracts will give only very inefficient lysis of M. leprae, and the phenomenon was also observed with dead bacilli.
FIG. 4. Infection of Schwann cells with M. leprae. About 90% of the Schwann cells cultured in vitro from adult monkeys phagocytosed M. leprae in 24 h. The cultures were stained for acid-fast bacilli by using the Ziehl-Neelsen method. Bar, 2.5 pLm.
DISCUSSION Exposure of Schwann cell cultures to heat shock in vitro induced a heat shock response similar to that of other mammalian cells, with induction of a major 70-kDa heat shock protein. Although the responses were similar in murine and monkey Schwann cells, staining with the monoclonal antibody C92 showed a continuous increase in the response up to 10 h after recovery in murine Schwann cells (Fig. 2a), unlike the results in the metabolic labeling experiment (Fig. 2b), where induction appeared to be a transient phenomenon. This could be explained if the degradation of this protein was slow, leading to an accumulation within the cell which would be detected by the monoclonal antibody. In monkey Schwann cells, however, the amount of protein detected by Western blot correlated with its synthesis as estimated by incorporation of radiolabel. The two species also differed in that a small amount of the heat-inducible hsp70 could be detected in unstressed monkey cells but not in the comparable mouse cells. This result may simply reflect a higher affinity of monoclonal antibody C92 for the monkey hsp70, but the observation that protein synthesis in the monkey cells was more resistant to inhibition by heat shock
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FIG. 3. 2D-PAGE analysis of constitutive and inducible hsp70. Control (a and c) and heat-shocked (b and d) murine Schwann cells were compared by 2D-PAGE and immunoblot. (a) Constitutive expression of hsp70 (arrow labelled "c") was seen in blots from control cells developed with monoclonal antibody N27. (b) Additional N27 staining was seen in the heat-shocked cells migrating with a more acidic position during isoelectric focusing (arrow labelled "1"). (c) No staining was obtained with C92 staining of control cells. (d) A faint spot corresponding to that labelled "I" in the N27 stained heat-shocked cells was stained with the C92 antibody in heatshocked cells.
M. LEPRAE INDUCES hsp70 IN SCHWANN CELLS
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am01W -WO -0 FIG. 5. Detection of hsp70 in Schwann cells infected with M. leprae. Monkey Schwann cells were infected with 20 x 106 live or heat-killed M. leprae per flask for 24 h. Cultures were labelled with
[35S]methionine for 1 h and then processed for SDS-PAGE at various time intervals after infection. Lanes: 1, control uninfected Schwann cells; 2 to 5, cells labelled immediately after washing off unphagocytosed bacilli (lane 1) and at 12 h (lane 3), 48 h (lane 4), and 1 week (lane 5) postinfection; 6, Schwann cells infected with heat-killed M. leprae terminated immediately after infection; 7, heat-killed M. leprae-infected cultures at 1 week postinfection. (a) Induction of hsp70 synthesis was detected with monoclonal antibody C92 (specific for inducible hsp7o). Positive staining was observed at all time intervals in cultures infected with either live or heat-killed M. leprae (lanes 2 to 7). A low level of hsp7o was seen in control uninfected Schwann cells. (b) Staining with N27 monoclonal
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would also be consistent with an elevated resting level of hsp7O in these cells. It will be of interest to extend this approach to look at hsp7O synthesis by Schwann cells in vivo or in organized nerve cell cultures. It has been reported that the glial cells of the giant squid may be able to transfer hsp7O into the axon during stress (32). It has been reported that the equivalent glial cells constitutively synthesize hsp7O (27), which is further increased after stress conditions such as ischemia (33) or physical trauma (4), in the central nervous system. It is possible that Schwann cells can fulfill a similar function in the peripheral nervous system. Heat shock proteins are known to be induced by a variety of stimuli in addition to heat shock itself, and the results reported here demonstrate that bacterial infection can induce hsp70 synthesis in Schwann cells. It has previously been reported that, upon infection with viruses in vitro, eukaryotic cells respond by synthesis of stress proteins (8). Phagocytosis of erythrocytes also induces heat shock protein synthesis in macrophages (7). In this case it was shown that phagocytosis per se did not provide a signal for heat shock protein induction, with iron-dependent activation of reactive oxygen metabolism playing an essential role in the induction phenomenon (7). In the Schwann cell studies described here, since phagocytosis is relatively specific for M. leprae, we were unable to determine whether the phagocytic process itself was the trigger for hsp70 induction or whether additional events dependent on the presence of the intracellular bacilli were required. hsp70 induction is not dependent on metabolic activity of the bacteria since autoclaved M. leprae cells, which were readily phagocytosed by monkey Schwann cells, also induced hsp70 synthesis. In contrast to the classical heat shock response, which is a transient phenomenon (19), induction of hsp7O after uptake of live or killed M. leprae was seen to persist over a 1-week period. Entry of Salmonella typhimurium into the hostile environment of a host macrophage has previously been shown to result in induction of bacterial heat shock proteins (5). It is possible that an analogous induction of M. leprae heat shock proteins occurs after entry into Schwann cells, but the extremely low rate of M. leprae metabolism would make this hypothesis difficult to test experimentally. Alternatively, since the microbicidal functions characteristic of macrophages have not been demonstrated in Schwann cells, it is possible that the intracellular environment for M. leprae in this case is less hostile than in a macrophage and that elevated bacterial heat shock protein synthesis may not be necessary for survival of the pathogen. Our results lend support to the inferred role of heat shock proteins in T-cell-mediated responses to infected Schwann cells (28, 30) where the focus was on hsp65. Steinhoff et al. (29) have also proposed a role for gamma interferon-induced Schwann cell hsp65 as a target antigen recognized by selfreactive cytotoxic T cells. The possibility of hsp70 in the infected Schwann cells playing a similar role has yet to be established. In summary, we have demonstrated that Schwann cells do
antibody (which detects both constitutive and inducible hsp70) showed no difference between uninfected control (lane 1) and M. leprae-infected (lanes 2 to 7) Schwann cells. (c) Autoradiograph of the same cultures shown in panels a and b, labelled with [35S]methionine. Increased incorporation was observed in a 70-kDa protein corresponding to the band recognized by C92 in all infected cultures.
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respond to stress by induction of hsp70 and that infection with M. leprae provides an appropriate stimulus to trigger this response. It is reasonable to suggest, therefore, that a local accumulation of both endogenous Schwann cell and bacterial heat shock proteins may occur at the site of M. leprae infection. In light of the frequent identification of stress proteins as immune targets in a wide range of infectious and autoimmune diseases (for a review, see reference 36), the results of this study provide support for the hypothesis that heat shock proteins could play a role in the immunopathology of leprosy. ACKNOWLEDGMENT This investigation received financial support from the UNDP/ World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). REFERENCES 1. Ambrose, E. J., S. R. Khanolkar, and R. G. Chulawalla. 1978. A rapid test for bacillary resistance to dapsone. Lepr. India 50:131-145. 2. Askanas, V., W. K. Engel, M. C. Dalakas, J. V. Lawrence, and L. S. Carter. 1980. Human Schwann cells in tissue culture. Histochemical and ultrastructural studies. Arch. Neurol. 37: 329-331. 3. Barnes, P. F., S. J. Fong, P. J. Brennan, P. E. Twomey, A. Mazumdar, and R. L. Modlin. 1990. Local production of tumor necrosis factor and IFN-gamma in tuberculosis pleuritis. J. Immunol. 145:149-154. 4. Brown, I. R., S. Rush, and G. 0. Ivy. 1989. Induction of heat shock gene at the site of tissue injury in the rat brain. Neuron 2:1559-1564. 5. Buchmeier, N. A., and F. Heffron. 1990. Induction of Salmonella stress proteins upon infection of macrophages. Science 248:730732. 6. Cammer, W., B. R. Bloom, W. T. Norton, and S. Gordin. 1978. Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: a possible mechanism of inflammatory demyelination. Proc. Natl. Acad. Sci. USA 75: 1554-1558. 7. Clerget, M., and B. S. Polla. 1990. Erythrophagocytosis induces heat shock protein synthesis by human monocytes-macrophages. Proc. Natl. Acad. Sci. USA 87:1081-1085. 8. Collins, P. L., and L. E. Hightower. 1982. Newcastle disease virus stimulates the cellular accumulation of stress (heat shock) mRNAs and proteins. J. Virol. 44:703-707. 9. Gonzalez, M. F., K. Shiraishi, K. Hisanaga, S. M. Sagar, M. Mandabach, and F. R. Sharp. 1989. Heat shock proteins as markers of neural injury. Mol. Brain Res. 6:93-100. 10. Harada, K. 1976. The nature of mycobacterial acid-fastness. Stain Technol. 51:255-260. 11. Hart, P. D., and R. J. W. Rees. 1960. Effect of macrocyclon in acute and chronic pulmonary tuberculosis infection in mice as shown by viable and total bacterial counts. Br. J. Exp. Pathol. 41:414-421. 12. Iyer, C. G. S. 1965. Predilection of M. leprae for nerves. Neurohistopathologic observations. Int. J. Lepr. 33:634 645. 13. Kaplan, G., and Z. A. Cohn. 1986. The immunobiology of leprosy. Int. Rev. Exp. Pathol. 28:45-78. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
227:680-685. 15. Lindquist, S. 1986. The heat shock response. Annu. Rev. Biochem. 55:1151-1191. 16. Lindquist, S., and E. A. Craig. 1988. The heat-shock proteins. Annu. Rev. Genet. 22:631-677. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J.
INFECT. IMMUN. Biol. Chem. 193:265-275. 18. Mehiert, A., and D. B. Young. 1989. Biochemical and antigenic characterization of the Mycobacterium tuberculosis 71 kD antigen, a member of the 70 kD heat-shock protein family. Mol. Microbiol. 3:125-130. 19. Mizzen, L. A., and W. J. Welch. 1988. Characterization of the thermotolerant cell. 1. Effect on protein synthesis activity and the regulation of heat-shock protein 70 expression. J. Cell Biol. 106:1105-1116. 20. Mukherjee, R., P. R. Mahadevan, and N. H. Antia. 1980. Organized nerve culture. I. A technique to study the effect of M. leprae infection. Int. J. Lepr. 48:183-188. 21. Mukherjee, R., and N. H. Antia. 1986. Adherence of M. leprae to Schwann cells in vitro-a specific phenomenon. IRCS Med. Sci. 13:853-854. 22. O'Farrell, P. H. 1975. High resolution two-demensional electrophoresis of proteins. J. Biol. Chem. 250:4007-4021. 23. Ottenhoff, T. H. M., B. K. Ab, J. D. A. Van Embden, J. E. R. Thole, and R. Kiessling. 1988. The recombinant 65-kd heat shock protein of Mycobacterium bovis Bacillus CalmetteGuerin M. tuberculosis is a target molecule for CD4+ cytotoxic T lymphocytes that lyse human monocytes. J. Exp. Med. 168:1947-1952. 24. Pearson, J. M. H., and W. F. Ross. 1975. Nerve involvement in leprosy-pathology, differential diagnosis and principles of management. Lepr. Rev. 46:199-212. 25. Sansarricq, H. 1983. Recent changes in leprosy control. Lepr. Rev. 54(Special Issue):7S-16S. 26. Scarpini, E., G. Meola, P. Baron, S. Beretta, M. Velicogna, and G. Scarlato. 1986. S100 protein and laminin: immunocytochemical markers for human Schwann cells in vitro. Exp. Neurol. 93:77-83. 27. Sprang, G. K., and I. R. Brown. 1987. Selective induction of heat shock gene in fibre tracts and cerebellar neurons of the rabbit brain detected by in situ hybridization. Mol. Brain Res. 3:89-93. 28. Steinhoff, U., and S. H. E. Kaufmann. 1988. Specific lysis by CD8+ T cells of Schwann cells expressing Mycobacterium leprae antigens. Eur. J. Immunol. 18:969-972. 29. Steinhoff, U., B. Schoel, and S. H. E. Kaufmnann. 1990. Lysis of interferon-gamma activated Schwann cell by cross-reactive CD8+ alpha/beta T cells with specificity for the mycobacterial 65 kD heat shock protein. Int. Immunol. 2:279-284. 30. Steinhoff, U., A. Wand-Wfirttenberger, A. Bremerich, and S. H. E. Kaufmann. 1991. Mycobacterium leprae renders Schwann cells and mononuclear phagocytes susceptible or resistant to killer cells. Infect. Immun. 59:684-688. 31. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350-4354. 32. Tyteli, M., G. S. Greenberg, and R. J. Laselk 1986. Heat shock-like protein is transferred from glia to axon. Brain Res. 363:161-164. 33. Vass, K., W. J. Welch, and T. S. Nowak, Jr. 1988. Localization of 70-kDa stress protein induction in gerbil brain after ischemia. Acta Neuropathol. 77:128-135. 34. Weddell, G., D. Jamison, and E. Palmer. 1959. Recent investigations into sensory and neurohistological changes in leprosy, p. 96-113. In R. G. Cochrane (ed.), Leprosy in theory and practice. John Wright & Sons, Ltd., Bristol, United Kingdom. 35. Welch, W. J., and J. P. Suhan. 1986. Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. J. Cell Biol. 103:2035-2052. 36. Young, D. B., A. Mehlert, and D. F. Smith. 1990. Stress proteins and infectious diseases, p. 131-165. In R. Morimoto, A. Tissieres, and C. Georgopoulos (ed.), Stress proteins in biology and medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Vol. 60, No. 8
0019-9567/92/083105-06$02.00/0 Copyright X 1992, American Society for Microbiology
hsp7O Synthesis in Schwann Cells in Response to Heat Shock and Infection with Mycobactenium leprae YASMIN MISTRY,1 DOUGLAS B. YOUNG,2 AxND3 RAMA MUKHERJEEl* Microbiology Division, National Institute of Immunology, Shahid Jeet Singh Marg, New Delhi-10 067,
India, 1 and Medical Research Council Tuberculosis and Related Infections Unit, Hammersmith Hospital, London W12 OHS, United Kingdom2 Received 22 April 1992/Accepted
5
May 1992
Induction of heat shock protein synthesis was monitored in murine and monkey Schwann cells exposed to elevated temperatures. Synthesis of the stress-inducible 70-kDa heat shock protein (hsp7O) was detected in both murine and primate Schwann cells by metabolic labelling and by immunoblotting with a specific monoclonal antibody. hsp70 synthesis was also induced in Schwann cells after infection with Mycobacterium leprae and was detected from 24 h to 1 week postinfection. These results are discussed with respect to the possible role of heat shock proteins in immunopathological events associated with the clinical manifestations of leprosy.
Leprosy is a chronic infectious disease caused by Mycobacterium leprae and currently affects an estimated 10 to 11 million individuals worldwide (25). Neuritis and nerve damage are the most important features of leprosy, and invasion of the nerve is a unique characteristic of M. leprae (24). Histopathological observations and in vitro studies have demonstrated that Schwann cells, the glial cells of the peripheral nervous system, are the preferred hosts, harboring large numbers of intracellular bacilli (12, 20, 34). Leprosy bacilli do not have a direct cytotoxic effect on Schwann cells, and it has been suggested that the nerve damage is a result of cell-mediated immune responses to mycobacterial and/or host antigens (6, 13). Factors which may play a role in such cell-mediated immunopathology include the release of cytokines (such as tumor necrosis factor and gamma interferon) from locally activated macrophages (3) and a direct attack on infected cells by cytotoxic T lymphocytes (23, 28, 29). T-cell-mediated lysis of Schwann cells infected in vitro with M. leprae has been described previously (28, 30). In the murine system, a role was shown for CD8+ alpha/beta cytotoxic T cells (29), with unrestricted killer cells fulfilling a similar function in a human Schwann cell model (30). In each case, a central role was proposed for Schwann cell heat shock proteins. Evidence was presented to indicate that the murine CD8+ cytotoxic T cells recognize antigenic determinants shared by bacterial and Schwann cell heat shock proteins (29), while in human Schwann cells, the induction of heat shock proteins was suggested to play a role in allowing infected cells to resist killing (30). In all of these studies, a role for hsp65 has been implicated. The role of hsp70, which is equally important, however, remains to be established. Heat shock proteins were initially identified by their elevated synthesis after exposure of cultured cells or organisms to a sudden temperature increase (for a review, see reference 15). Subsequently it was found that structurally related proteins are present constitutively in all cells and that heat shock could be replaced by a variety of other environmental stresses as inducers of the response (16). The heat shock response is generally considered to be ubiquitous, but there is evidence to suggest that the response may not be
identical in all cell types. In the rabbit brain, for example, in situ hybridization analysis revealed marked differences in heat shock gene expression of hsp70 in different cells both constitutively and after hyperthermia (27). A further indication of a specialized aspect to the heat shock response within the nervous system is provided by the observation of a rapid transfer of heat shock-like proteins with molecular sizes of 70 and 95 kDa from adaxonal glial cells into axons in the giant squid at elevated temperatures (32). Induction of a heat shock response after exposure to physical trauma has been demonstrated in the central nervous system (4), and detection of heat shock proteins has been suggested as a useful general marker for nerve damage (9). In this study, we have characterized the heat shock response of murine and monkey Schwann cells in an in vitro culture system and have analyzed the effect of infection with M. leprae on the expression of Schwann cell hsp70. MATERIALS AND METHODS
Schwann cell cultures from neonatal mice. The sciatic and brachial plexus from 2- to 3-day-old pups of BALB/c mice were dissected under sterile conditions by using a dissecting microscope (model SZ40; Olympus, Tokyo, Japan). These nerves were chopped into fine pieces and incubated in 0.05% collagenase (Sigma Chemical Co., St. Louis, Mo.) for 30 min at 37°C, and cells were dispersed by passing twice through 21- and 23-gauge needles. The cell suspension was collected after centrifugation at 500 x g for 10 min, washed with Dulbecco's minimal essential medium (DMEM; Flow Laboratories) and plated onto 25-mm2 sterile tissue culture flasks. Cultures were maintained in growth medium consisting of DMEM supplemented with 10% fetal calf serum and antibiotics (penicillin, 100 IU/ml; streptomycin, 100 ,ug/ml; gentamicin, 100 p.g/ml) for a period of 7 to 10 days, with replenishment of the medium twice a week. Schwann cell cultures from adult monkeys. Peripheral nerves, mainly the sciatic and the ulnar, were dissected from adult rhesus monkeys anesthetized with ketamine (5 mg/kg of body weight). A modification of the technique described by Askanas et al. (2) was used to set up Schwann cell cultures from these nerves. Dissected nerves were immedinerves
ately transferred to DMEM containing gentamicin (100 ,ug/ *
ml). Under sterile conditions, the
Corresponding author. 3105
nerves were
cleaned of
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epineural tissue by use of a dissecting microscope and cut into 1-mm3 explants. Explants were treated with 0.25% trypsin (Difco Laboratories, Detroit, Mich.) for 5 min and then explanted onto collagen gel-coated six-well plates (Coming Glass Works, Coming, N.Y.). Collagen extraction and coating were done as previously described (20). The explants were maintained in growth medium which was replenished twice a week. After 7 to 10 days, when the cells had emerged sufficiently from the explants, the original explants were removed and reexplanted on fresh collagencoated culture plates. The glial cell outgrowth from these reexplants was greatly enriched in the Schwann cell population. The reexplanted cultures enriched for Schwann cells were subcultured to obtain dissociated monolayer cultures which were used for heat shock protein induction experiments. For this, explants were removed and the bed of Schwann cells embedded in collagen gel was first treated with 0.05% collagenase for a total of 20 min at 37°C and with 0.25% trypsin for 10 min to obtain a single cell suspension. The cells were obtained by centrifugation at 500 x g for 10 min, washed once with fresh DMEM, and then plated onto 25-mm2 culture flasks. Immunofluorescence staining. Schwann cells were identified by indirect immunofluorescence staining by using antibodies to S100 protein (26). Cells plated on coverslips were fixed with 4% formal saline for 10 min at 4°C and then permeabilized with 0.005% Nonidet P-40 (Sigma). The cells were stained with a 1:50 dilution of S100 antiserum (Dakopatts, Copenhagen, Denmark) for 30 min at room temperature and then labelled with a 1:100 dilution of fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (from the reagent bank, National Institute of Immunology, New Delhi, India). Fluorescence was observed under UV light on a Microphot-FX microscope (Nikon, Tokyo, Japan). Infection with M. leprae. Leprosy bacilli were isolated from frozen armadillo tissue by using the method described by Ambrose et al. (1). The bacillary count was determined by using the method described by Hart and Rees (11). The absence of other bacterial contaminants was confirmed by lack of growth on nutrient agar and Lowenstein-Jensen media. To prepare heat-killed bacilli, bacterial suspensions were autoclaved at 15 lb/in2 for 15 min. Schwann cells were infected by incubation with 107 bacilli per ml of growth medium for 24 h. Unphagocytosed bacilli were removed by repeated washing with DMEM, and cultures were then maintained for periods of up to 1 week postinfection. The presence of acid-fast bacilli in infected cultures was monitored by Ziehl-Neelsen staining (10). Heat shock treatment. Schwann cells growing on 25-mm2 culture flasks were washed twice with methionine-free DMEM (GIBCO Laboratories, Grand Island, N.Y.) and allowed to remain in the same medium for 30 min. Cells were heat shocked by incubating flasks in a water bath for 30 min at 42 or 45°C, and then the temperature was returned to 37°C for recovery and radiolabelling of the cells. Metabolic labelling. Schwann cell cultures washed and incubated with 2 ml of methionine-free DMEM in 25-mm2 flasks as described above were labelled with [35S]methionine (5 ,Ci/ml; specific activity, >800 Ci/mmol; New England Nuclear, Boston, Mass.) for 1 h at 37°C. The adherent cells were then washed three times with complete DMEM and detached from the culture surface by mechanical scraping with a rubber policeman. The cell pellet was washed twice with phosphate-buffered saline, and the total protein content was estimated by the method of Lowry et al. (17). PAGE and Western blotting (immunoblotting). Radiola-
IN?FECT. IMMUN.
belled cell extracts were prepared for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) by boiling for 5 min in Laemmli sample buffer containing SDS and 2-mercaptoethanol (14). Samples containing equal amounts of protein were separated by electrophoresis in gels containing 10% polyacrylamide as described by Laemmli (14). For two-dimensional PAGE (2D-PAGE), labelled cell extracts were prepared in sample buffer containing urea, Nonidet P-40, 2-mercaptoethanol, and ampholytes (Pharmacia LKB Biotechnology, Bromma, Sweden) as described by O'Farrell (22). Proteins were separated in the first dimension by using tube gels containing ampholytes in the pH range of 3.5 to 9.5 at a concentration of 0.4% and ampholytes in the pH range of 4 to 6 at a concentration of 1.6% (22). In the second dimension, proteins were separated by SDS-PAGE as described above. Electrophoresis was carried out by using a mini-gel system (Hoefer Scientific Instruments, San Francisco, Calif.). Proteins separated by SDS-PAGE and 2D-PAGE were transferred to nitrocellulose membranes by electroblotting as described by Towbin et al. (31). Autoradiographs were prepared from blots by exposure to X-ray film (Hyperfilm-MP; Amersham) for 3 days at room temperature. For quantitation, autoradiographs were scanned by using a scanning densitometer and GS 365 Data System Software (Hoefer Scientific). To localize heat shock proteins, blots were incubated with the following monoclonal antibodies: C92, specific for the heat-inducible form of hsp7o (35) (SPA801; StressGen Biotechnologies Corp., Sidney, Canada); N27, which recognizes both heat-inducible and constitutive forms of hsp70 (33) (SPA802; StressGen Biotechnologies); and cosII, raised against the purified hsp7o of Mycobactenium tuberculosis (18). After incubation with monoclonal antibody, blots were washed and further incubated with peroxidase-conjugated anti-mouse immunoglobulin (1:2,000; Dakopatts) as a secondary antibody. Finally, bound antibody was visualized by incubation with 3,3'-diaminobenzidine dihydrochloride and hydrogen peroxide. RESULTS Schwann cell cultures. Schwann cells were cultured in vitro from nerves of newborn mice and adult monkeys. Dissociated single-cell cultures were readily obtained from the neonatal mice, but a technique of explantation-reexplantation had to be used for nerves from adult monkeys. Highly enriched Schwann cell cultures were obtained from nerves from adult monkeys by reexplantation of the original explants onto a fresh collagen-coated surface. A subsequent subculture of these yielded a monolayer of dissociated Schwann cells of approximately 95% purity. The cultured Schwann cells were identified by their typical bipolar morphology (Fig. la) and by their positive staining with antibodies to S100 protein, a specific marker which differentiates Schwann cells from neurofibroblasts in vitro (Fig. lb). Heat shock response. Dissociated murine and monkey Schwann cell cultures were heat shocked at 42 or 45°C for 30 min, and synthesis of heat shock proteins was then monitored after returning the cells to a temperature of 37°C. Synthesis of the heat-inducible form of hsp7o, recognized by monoclonal antibody C92, was detectable after heat shock at 42°C (data not shown), but the effect was more pronounced after a 45°C heat shock. Figure 2a shows a Western blot of cell extracts prepared from cultures at various times after a 45°C heat shock and developed with monoclonal antibody C92. Heat-inducible hsp70 was not detected in extracts from
VOL. 60, 1992
M. LEPRAE INDUCES hsp70 IN SCHWANN CELLS
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FIG. 1. Characterization of Schwann cell cultures. Schwann cells from nerves from adult monkeys were cultured as described in the text. (a) Viewed under a phase-contrast microscope, the Schwann cells have a characteristic bipolar morphology. Bar, 100 Fm. (b) Immunofluorescence staining with antibody to S100 indicated that Schwann cells accounted for approximately 95% of the cells in culture. Bar, 10 ,um.
murine Schwann cells maintained at 37°C (Fig. 2a, lane 1). After heat shock and then a recovery period of 3 h or more, however, a prominent C92-positive band was seen (Fig. 2a, lanes 4 to 6). A weak C92-positive band was observed in unstressed monkey Schwann cells (Fig. 2a, lane 7), but again heat shock resulted in a marked enhancement of hsp70 synthesis (Fig. 2a, lanes 8 to 12). Staining of the same samples with monoclonal antibody N27 (which recognizes constitutive as well as heat-inducible hsp70) showed no differences between control and heat-shocked samples on SDS-PAGE (data not shown). Further analysis of murine Schwann cell extracts by 2D-PAGE did, however, reveal a difference in the N27 staining pattern between control and heat-shocked cells (Fig. 3). In addition to the constitutive forms of the protein (Fig. 3, arrow labelled "c") stained by N27 in control cells (Fig. 3a), a new protein (Fig. 3, arrow labelled "1") was seen in the heat-shocked cells with a more acidic pI (Fig. 3b), corresponding to the heat-inducible hsp70 detected by C92 (Fig. 3d). Increased synthesis of hsp70 after heat shock could also be seen by SDS-PAGE analysis of cell extracts labelled with
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FIG. 2. Analysis of the heat shock response in cultured Schwann cells. Murine and monkey Schwann cell cultures were exposed to heat shock for 30 min at 45°C and then allowed to recover at 37°C as described in the text. [35S]methionine was added at the times indicated, and cell extracts prepared after a 1-h labelling period were analyzed by SDS-PAGE. Lanes 1 to 6 show extracts from murine Schwann cells. Lane 1, control cells maintained at 37°C; lanes 2 to 6, cells labelled immediately after heat shock (lane 2) and after 1 (lane 3), 3 (lane 4), 6 (lane 5), and 10 (lane 6) h of recovery. Lanes 7 to 12 show extracts from monkey Schwann cells treated as described for lanes 1 to 6, respectively. (a) hsp70 synthesis was monitored by Western blot with monoclonal antibody C92 (specific for the heat-inducible hsp70). Induction of hsp70 synthesis during the recovery period is clearly seen in lanes 4 to 6 (murine Schwann cells) and in lanes 8 to 12 (monkey Schwann cells). A low-level expression of the inducible hsp70 was seen in the control monkey cells (lane 7). (b) Autoradiograph showing metabolic labelling of the same cultures shown in panel A. Increased incorporation into a 70-kDa protein was observed after heat shock (indicated by arrowhead). This band precisely overlapped that recognized by the C92 monoclonal antibody.
[35Slmethionine (Fig. 2b). In murine Schwann cells, a reduction in total protein synthesis was clearly evident during the first hour after heat shock (Fig. 2b, lane 2), with enhanced hsp70 synthesis being seen during the subsequent recovery period. Quantitative densitometric analysis showed no incorporation of [35Slmethionine into the 70-kDa band in control murine Schwann cells; however, incorporation increased with recovery, reaching a maximum of 12.5% after 3 h, and subsequently dropped to 6.6% after 10 h. Inhibition of
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protein synthesis immediately after heat shock was not seen with the monkey Schwann cells, but increased hsp70 synthesis was again evident during the recovery phase (Fig. 2b, lanes 7 to 12). Maximum incorporation (9.7% of total [35S]methionine incorporated) was obtained after 6 h of recovery in monkey cell cultures. Effect of infection with M. leprae. Monkey Schwann cells readily phagocytosed M. leprae during 24 h of incubation in culture, with more than 90% of the cells harboring acid-fast bacilli (Fig. 4). Murine Schwann cells were less active in phagocytosis, and the monkey cells were therefore chosen as a model for detailed analysis of the effect of infection on hsp70 synthesis. As reported previously for Schwann cells in culture (21), phagocytosis was relatively specific for M.
leprae, with only a very limited uptake of other mycobacteria occurring under these conditions (while 90% of Schwann cells phagocytosed M. leprae, both live and killed, less than 15% of cells phagocytosed either H37Rv or H37Ra). Synthesis of hsp70 in infected cells was monitored by immunoblot and autoradiography by using the protocols established for the heat shock response. C92 immunoblot analysis of extracts from cells infected for periods of up to 1 week clearly showed an increase in synthesis of the heatinducible hsp7O (Fig. 5a). The increased synthesis was apparent as early as the first time point analyzed, i.e., 24 h after the addition of bacteria to the culture (Fig. 5a, lane 2). Bacterial viability was not essential for hsp70 induction, since phagocytosis of heat-killed organisms elicited a comparable effect (Fig. Sa, lanes 6 and 7). To discount the possibility that increased staining with C92 was due to cross-reactive recognition of M. leprae hsp70, Western blots with M. leprae extracts were probed with C92 and cosII (data not shown). The antimycobacterial antibody (cosII) bound strongly to M. leprae hsp70, but C92 was completely negative. No difference in N27 staining was detectable between the control and infected cultures (Fig. Sb). While the continued presence of the inducible hsp70, as detected by immunoblotting with C92 antibody, was evident in Schwann cells up to 1 week postinfection, it was of interest to determine whether or not increased synthesis was maintained throughout this period. Analysis of protein synthesis profiles by [35S]methionine labelling throughout the period of infection showed that increased incorporation into the 70-kDa band could in fact still be seen 1 week after the initial infection (Fig. Sc). Comparable control cultures maintained without infection over the same period did not show any increase in hsp70 synthesis. It is unlikely that the change in the [35S]methionine labelling profile represents incorporation into M. leprae proteins since M. leprae cells have an extremely slow growth rate (doubling time of 12 to 14 days) and are unlikely to synthesize detectable amounts of protein during the 1-h labelling period, the procedure for preparation of cell extracts will give only very inefficient lysis of M. leprae, and the phenomenon was also observed with dead bacilli.
FIG. 4. Infection of Schwann cells with M. leprae. About 90% of the Schwann cells cultured in vitro from adult monkeys phagocytosed M. leprae in 24 h. The cultures were stained for acid-fast bacilli by using the Ziehl-Neelsen method. Bar, 2.5 pLm.
DISCUSSION Exposure of Schwann cell cultures to heat shock in vitro induced a heat shock response similar to that of other mammalian cells, with induction of a major 70-kDa heat shock protein. Although the responses were similar in murine and monkey Schwann cells, staining with the monoclonal antibody C92 showed a continuous increase in the response up to 10 h after recovery in murine Schwann cells (Fig. 2a), unlike the results in the metabolic labeling experiment (Fig. 2b), where induction appeared to be a transient phenomenon. This could be explained if the degradation of this protein was slow, leading to an accumulation within the cell which would be detected by the monoclonal antibody. In monkey Schwann cells, however, the amount of protein detected by Western blot correlated with its synthesis as estimated by incorporation of radiolabel. The two species also differed in that a small amount of the heat-inducible hsp70 could be detected in unstressed monkey cells but not in the comparable mouse cells. This result may simply reflect a higher affinity of monoclonal antibody C92 for the monkey hsp70, but the observation that protein synthesis in the monkey cells was more resistant to inhibition by heat shock
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FIG. 3. 2D-PAGE analysis of constitutive and inducible hsp70. Control (a and c) and heat-shocked (b and d) murine Schwann cells were compared by 2D-PAGE and immunoblot. (a) Constitutive expression of hsp70 (arrow labelled "c") was seen in blots from control cells developed with monoclonal antibody N27. (b) Additional N27 staining was seen in the heat-shocked cells migrating with a more acidic position during isoelectric focusing (arrow labelled "1"). (c) No staining was obtained with C92 staining of control cells. (d) A faint spot corresponding to that labelled "I" in the N27 stained heat-shocked cells was stained with the C92 antibody in heatshocked cells.
M. LEPRAE INDUCES hsp70 IN SCHWANN CELLS
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[35S]methionine for 1 h and then processed for SDS-PAGE at various time intervals after infection. Lanes: 1, control uninfected Schwann cells; 2 to 5, cells labelled immediately after washing off unphagocytosed bacilli (lane 1) and at 12 h (lane 3), 48 h (lane 4), and 1 week (lane 5) postinfection; 6, Schwann cells infected with heat-killed M. leprae terminated immediately after infection; 7, heat-killed M. leprae-infected cultures at 1 week postinfection. (a) Induction of hsp70 synthesis was detected with monoclonal antibody C92 (specific for inducible hsp7o). Positive staining was observed at all time intervals in cultures infected with either live or heat-killed M. leprae (lanes 2 to 7). A low level of hsp7o was seen in control uninfected Schwann cells. (b) Staining with N27 monoclonal
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would also be consistent with an elevated resting level of hsp7O in these cells. It will be of interest to extend this approach to look at hsp7O synthesis by Schwann cells in vivo or in organized nerve cell cultures. It has been reported that the glial cells of the giant squid may be able to transfer hsp7O into the axon during stress (32). It has been reported that the equivalent glial cells constitutively synthesize hsp7O (27), which is further increased after stress conditions such as ischemia (33) or physical trauma (4), in the central nervous system. It is possible that Schwann cells can fulfill a similar function in the peripheral nervous system. Heat shock proteins are known to be induced by a variety of stimuli in addition to heat shock itself, and the results reported here demonstrate that bacterial infection can induce hsp70 synthesis in Schwann cells. It has previously been reported that, upon infection with viruses in vitro, eukaryotic cells respond by synthesis of stress proteins (8). Phagocytosis of erythrocytes also induces heat shock protein synthesis in macrophages (7). In this case it was shown that phagocytosis per se did not provide a signal for heat shock protein induction, with iron-dependent activation of reactive oxygen metabolism playing an essential role in the induction phenomenon (7). In the Schwann cell studies described here, since phagocytosis is relatively specific for M. leprae, we were unable to determine whether the phagocytic process itself was the trigger for hsp70 induction or whether additional events dependent on the presence of the intracellular bacilli were required. hsp70 induction is not dependent on metabolic activity of the bacteria since autoclaved M. leprae cells, which were readily phagocytosed by monkey Schwann cells, also induced hsp70 synthesis. In contrast to the classical heat shock response, which is a transient phenomenon (19), induction of hsp7O after uptake of live or killed M. leprae was seen to persist over a 1-week period. Entry of Salmonella typhimurium into the hostile environment of a host macrophage has previously been shown to result in induction of bacterial heat shock proteins (5). It is possible that an analogous induction of M. leprae heat shock proteins occurs after entry into Schwann cells, but the extremely low rate of M. leprae metabolism would make this hypothesis difficult to test experimentally. Alternatively, since the microbicidal functions characteristic of macrophages have not been demonstrated in Schwann cells, it is possible that the intracellular environment for M. leprae in this case is less hostile than in a macrophage and that elevated bacterial heat shock protein synthesis may not be necessary for survival of the pathogen. Our results lend support to the inferred role of heat shock proteins in T-cell-mediated responses to infected Schwann cells (28, 30) where the focus was on hsp65. Steinhoff et al. (29) have also proposed a role for gamma interferon-induced Schwann cell hsp65 as a target antigen recognized by selfreactive cytotoxic T cells. The possibility of hsp70 in the infected Schwann cells playing a similar role has yet to be established. In summary, we have demonstrated that Schwann cells do
antibody (which detects both constitutive and inducible hsp70) showed no difference between uninfected control (lane 1) and M. leprae-infected (lanes 2 to 7) Schwann cells. (c) Autoradiograph of the same cultures shown in panels a and b, labelled with [35S]methionine. Increased incorporation was observed in a 70-kDa protein corresponding to the band recognized by C92 in all infected cultures.
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respond to stress by induction of hsp70 and that infection with M. leprae provides an appropriate stimulus to trigger this response. It is reasonable to suggest, therefore, that a local accumulation of both endogenous Schwann cell and bacterial heat shock proteins may occur at the site of M. leprae infection. In light of the frequent identification of stress proteins as immune targets in a wide range of infectious and autoimmune diseases (for a review, see reference 36), the results of this study provide support for the hypothesis that heat shock proteins could play a role in the immunopathology of leprosy. ACKNOWLEDGMENT This investigation received financial support from the UNDP/ World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR). REFERENCES 1. Ambrose, E. J., S. R. Khanolkar, and R. G. Chulawalla. 1978. A rapid test for bacillary resistance to dapsone. Lepr. India 50:131-145. 2. Askanas, V., W. K. Engel, M. C. Dalakas, J. V. Lawrence, and L. S. Carter. 1980. Human Schwann cells in tissue culture. Histochemical and ultrastructural studies. Arch. Neurol. 37: 329-331. 3. Barnes, P. F., S. J. Fong, P. J. Brennan, P. E. Twomey, A. Mazumdar, and R. L. Modlin. 1990. Local production of tumor necrosis factor and IFN-gamma in tuberculosis pleuritis. J. Immunol. 145:149-154. 4. Brown, I. R., S. Rush, and G. 0. Ivy. 1989. Induction of heat shock gene at the site of tissue injury in the rat brain. Neuron 2:1559-1564. 5. Buchmeier, N. A., and F. Heffron. 1990. Induction of Salmonella stress proteins upon infection of macrophages. Science 248:730732. 6. Cammer, W., B. R. Bloom, W. T. Norton, and S. Gordin. 1978. Degradation of basic protein in myelin by neutral proteases secreted by stimulated macrophages: a possible mechanism of inflammatory demyelination. Proc. Natl. Acad. Sci. USA 75: 1554-1558. 7. Clerget, M., and B. S. Polla. 1990. Erythrophagocytosis induces heat shock protein synthesis by human monocytes-macrophages. Proc. Natl. Acad. Sci. USA 87:1081-1085. 8. Collins, P. L., and L. E. Hightower. 1982. Newcastle disease virus stimulates the cellular accumulation of stress (heat shock) mRNAs and proteins. J. Virol. 44:703-707. 9. Gonzalez, M. F., K. Shiraishi, K. Hisanaga, S. M. Sagar, M. Mandabach, and F. R. Sharp. 1989. Heat shock proteins as markers of neural injury. Mol. Brain Res. 6:93-100. 10. Harada, K. 1976. The nature of mycobacterial acid-fastness. Stain Technol. 51:255-260. 11. Hart, P. D., and R. J. W. Rees. 1960. Effect of macrocyclon in acute and chronic pulmonary tuberculosis infection in mice as shown by viable and total bacterial counts. Br. J. Exp. Pathol. 41:414-421. 12. Iyer, C. G. S. 1965. Predilection of M. leprae for nerves. Neurohistopathologic observations. Int. J. Lepr. 33:634 645. 13. Kaplan, G., and Z. A. Cohn. 1986. The immunobiology of leprosy. Int. Rev. Exp. Pathol. 28:45-78. 14. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
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