International Immunology, Vol. 4, No.2, pp.
227-232
© 1992 Oxford University Press
Effect of in vivo hyperthermia on thymocyte maturation and selection Shahid Mansoor1, Marcello Span6, Selene Baschieri, Anna Cividalli, Lucia Mosiello, and Gino Doria Division of Physics and Biomedical Sciences, ENEA CRE Casaccia, 00060 Rome, Italy 1 Biomedical Division, National Institute for Biotechnology and Genetic Engineering, 9194 Lahore, Pakistan Key words: hyperthermia, thymocytes, maturation, selection
Two-month-old male mice were exposed to whole-body hyperthermlc treatment in a circulating water bath. After 1 h exposure to 41 °C, mice were kept at room temperature for 24, 48, 72, and 96 h before thymus examination. The total thymocyte number progressively decreased after 24 and 48 h, reached a minimum value at 72 h, and returned to almost normal value after 96 h. Similar changes occurred In the CD4 + CD8 + cell subset. Conversely, the percentage of CD4~CD8~ cells rose to a maximum at 48 h and then declined to normal value at 96 h. The percentage of C D 4 C D 8 + and CD4+CD8~ cell subsets increased to a maximum at 48 h and then declined to normal value at 96 h. These variations in single positive cell subsets correlated well with similar changes in the thymocyte mitotic response to concanavalin A. The observed effects on thymocytes were heat dose dependent, and suggest that hyperthermia induces the development of the most primitive thymocytes and positively selects mature T cells with high mitotic responsiveness. It is proposed that heat shock proteins might be Involved In the hyperthermia-induced alterations of thymocyte maturation and selection. Introduction Fever has been recognized as a sign of pathology since ancient times. High body temperature either during infectious diseases or artificially induced for cancer treatment has been suggested to enhance immune responses and to result in clinically beneficial effects (1,2). Convincing evidence for the beneficial effects of high body temperature also derives from studies on the lizard Dipsosaurus dorsalis which, when placed in a higher than neutral ambient temperature, increases its ability to counteract Aeromonas hydrophila infection and to survive (3). The possibility that the favorable consequences of in vivo hyperthermia are mediated by enhancement of immune responses is not only suggested by results from animals of phylogenetically distant species (2-6) but is also supported by results from in vitro experiments showing that high temperature increases the mitotic and functional activities of T cell subsets (7,8). Nevertheless, the cellular and molecular mechanisms by which hyperthermia affects the immune system are poorly understood. The synthesis of heat shock proteins (hsp) as induced by hyperthermia has been well characterized both in vivo and in vitro. Hsp are a very conserved genetic system in both prokaryotes and eukaryotes. Major hsp share a high degree of homology
(9), and their expression is enhanced in cells reponding to heat, infection, transformation, and other noxious agents (10). Hsp are, therefore, frequently called stress proteins. The immune system recognizes these proteins among the dominant antigens of a broad spectrum of pathogens and as self proteins induced in stressed autologous cells (11,12). The existence of T lymphocytes that recognize self stress proteins suggests that these cells play a role in immune surveillance of injured autologous cells as a defence mechanism against a variety of cellular insults (13). Noteworthily, a large fraction of TCR+ 76 cells recognize self hsp in the skin (14) and TCR 76 cell clones reactive with self and foreign hsp have been found in the neonatal thymus (15). The thymus plays a central role in the development and maintenance of immunity and tolerance, as it provides the microenvironment for T cell maturation and selection (16). Briefly, stem cells located in hemopoietic tissues migrate to the thymus where they undergo an ordered differentiation process. The most primitive thymocytes lack CD4 and CD8 markers (double negative) and, upon gene rearrangement, may develop into cells expressing the TCR yd which then seed to the periphery, mostly to the epithelial layers of skin and intestine. If the rearrangements
Correspondence to: G. Doria, Immunology Laboratory, ENEA CRE Casaccia, CP 2400, 00100 Rome AD, Italy Transmitting editor- L Moretta.
Received 24 April 1991, accepted 1 November 1991
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
Abstract
228
Effect of hyperthermia on thymocytes
of 7 and 8 genes are unproductive, double negative thymocytes may develop into CD4 + CD8 + (double positive) cells that initially express a low level of TCR a/3. Double positive cells undergo positive and negative selection mediated by interactions with MHC molecules expressed on epithelial and dendritic cells, and develop into CD4~CD8 + and CD4 + CD8~ (single positive) cells which then seed to the peripheral lymphoid tissues. It is possible that exogenous stressful stimuli, such as hyperthermia, induce hsp that affect the differentiation process of T cell maturation and selection. We have, therefore, examined the effect of hyperthermia on the thymus of adult mice, in terms of cellulanty, thymccyte phenotype, and mitotic responsiveness. We report herein that hyperthermia induces profound changes in the thymocyte population, which affect the generation of normal T cells.
color staining, thymocytes were incubated with anti-CD4 PE-conjugated mAb and anti-CD8 FITC-conjugated mAb for 30 mm on ice, washed, and analyzed by flow cytometry. Samples
Animals (C3H/RI/CnexDBA/2J/Cne)F, male mice were raised in our animal facilities and used at the age of 8 - 9 weeks. Each control and experimental group was composed of three animals.
Fig. 1. Effect of treatment at 41 or 37°C on thymus cell count. After treatment mice were kept at room temperature for 24 - 96 h before thymus examination. Control values from untreated mice are also shown. 37 °C
41 °C
Hyperthermia Hyperthermic treatment was delivered without anesthesia in a circulating-water bath, using a specially designed jig (17). For the delivery of whole-body hyperthermia, the jig was lowered into the bath in such a way that the water reached the mouse neck level. The temperature used during the hyperthermic treatment was 41, 42, or 43°C, as monitored by temperature probes (Omega Engineering Inc., CN). Control animals were either untreated or exposed to 37°C under identical conditions. The mean temperature of mice (legs, flank, and neck regions) was 41.3 ± 0.2 and 37.6 ± 0.3°C for mice exposed to 41 and 37°C, respectively. No variation was determined for 42 and 43°C. No mouse died during the experiments. After exposure to 37 or 41 °C for 1 h, mice were kept at room temperature (24-25°C) for 24, 48, 72, or 96 h. Mice exposed to 41, 42, or 43°C for different times to determine dose-dependent effects were kept at room temperature for a period of 72 h.
CD
a o a o
Flow cytometry Reagents to detect CD4 and CD8 antigens were anti-L3T4 phycoerythrin (PE)-conjugated mAb (clone GK 1.5) and anti-Lyt 2.2 fluorescein isothiocyanate (FITQ-conjugated mAb (clone 53-6.7) respectively. The reagents were purchased from Beckton - Dickinson (Mountain View, CA) and optimal concentrations were determined in preliminary experiments. For two-
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Cell preparations Thymuses were aseptically removed from three mice of each group, pooled, and single thymccyte suspensions were prepared by tapping thymuses on a 100 /un, wire mesh gauze. Cells were centrifuged at 1200r.p.m. (200 g) in a Beckman refrigerated centrifuge for 10min and resuspended in complete medium, consisting of RPMI 1640 (Sigma, St Louis, MO), supplemented with 10% FCS (Seromed, Berlin, West Germany), 2 mM L-glutamine (Gibco, Grand Island, NY), 5 x 10~5 M 2-mercaptoethanol, and 10 /ig/ml gentamicin (Shering, Kenilworth, NY).
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Fig. 2. Effect of treatment at 41 or 37 °C on the distribution erf thymocyte subsets defined by CD4 and CD8 markers. After treatment mice were kept at room temperature for 24 - 96 h before thymus examination. Control values from untreated mice are also shown
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
41-C
Methods
Effect of hyperthermia on thymocytes 229 of 10,000-20,000 viable cells were analyzed for double-color fluorescence in a FACStar Plus machine. All fluorescence signals were collected in log mode. Data were evaluated as contour or dot plots generated by using Consort 30 or FACStar Plus software (Beckton - Dickinson) respectively. Mitotic response Thymocytes suspended in complete medium with or without concanavalm A (Con A; Miles-Yeda Laboratory, Rehovot, Israel) were distributed in flat bottom 96-well microtiter plates (Falcon Plastics 3040, Oxnard, CA) and cultured at 37°C in 5% CO2 humidified incubator for 48 h. Each group of triplicate cultures contained 106 cells in 0.2 ml medium alone, or with 0 5, 1, or
37 °C
Results Mice were exposed to 41 °C for 1 h, and then kept at room temperature for 24, 48, 72, and 96 h before thymus examination. The total thymocyte number progressively decreased after 24 and 48 h, reached a minimum value at 72 h and returned to almost normal values after 96 h Exposure to 37°C for 1 h under identical conditions and then to room temperature for 24 - 9 6 h caused a small decrease in thymus cellularity (Fig. 1). Figures 2 and 3 show the distribution of the four thymocyte
24 hr
48 hr
•
96hr
Fig. 4. Mitotic response of thymocytes from mice treated at 41 or 37°C. After treatment mice were kept at room temperature for 24 - 96 h before thymocytes were harvested and cultured with 1 fig Con A for 48 h. Control values from untreated mice are also shown.
. 0
72 hr
cfl-
96 hr
20 -
10 "•••
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Time of treatment ( rrin)
Fig. 5. Heat dose dependence of the effect of hyperthermia on thymocyte count Mice were treated at 41, 42, or 43°C and kept at room temperature for 72 h before thymus examination. Control values from untreated mice are also shown.
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
41 °C
2 ng Con A. At 4 h before harvesting, cultures received 0.5 ^Ci of tritiated thymidine (specific activity 1.739 GBq/mmol) (The Radiochemical Centre, Amersham, UK) in 20 /J. Cultures were then sacrificed with an automated cell harvester (Skatron, Lier, Norway). Radioactivity was measured in a Miniaxi Tri-Carb 4000 Scintillation counter (Packard) and expressed as counts per minute (c.p.m).
230
Effect of hyperthermia on thymocytes 43 °C
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Time of treatment ( min ) Fig. 6. Effect of treatment at 41, 42, or 43°C on the distribution of thymocyte subsets defined by CD4 and CD8 markers. Treated mice were kept at room temperature for 72 h before thymocyte analysis Control values from untreated mice are also shown.
subsets defined by the CD4 and CD8 markers and evaluated by flow cytometry. After 1 h treatment at 41 °C, the percent of double negative cells increased to a maximum at 48 h and then declined to normal value at 96 h, while the double positive cell subset displayed similar variations as observed for the total thymocyte population. The percentage of single positive cells increased to a maximum at 48 h and then declined to normal value at 96 h. These changes in the thymocyte phenotype suggest that hyperthermia induced the development of the most primitive CD4~CD8~ thymocytes, selection of mature CD4-CD8 + and CD4 + CD8~ cells, and depletion in the CD4 + CD8 + and total thymocytes. Although exposure to 37°C for 1 h resulted in a small decrease in thymus cellularity, it did not affect the phenotype distribution of the four cell subsets. Thymocytes from mice sacrificed at 2 4 - 9 6 h after 1 h exposure to 41 °C were stimulated in vitro with different doses of Con A. The mitotic response to 1 HQ Con A/well was found highest in all groups. Figure 4 shows that the maximum response to Con A is increased by hyperthermia, reaches a peak value
at 48 h, and remains higher than in untreated controls at 96 h. One hour exposure to 37°C did not affect the thymocyte mitotic response to Con A. The observed effects of hyperthermia were found to be heat dose dependent. Mice were exposed to 41, 42, or 43°C for different times, and then sacrificed after 72 h at room temperature. Thymus cellularity was progressively decreased with increasing temperature and time of exposure (Fig. 5). Flow cytometry analysis of the four thymocyte subsets revealed that the increase in the percentage of double negative and single positive cells, as well as the decrease in the percentage of double positive cells, are both heat dose dependent (Fig. 6). Discussion The present findings indicate that hyperthermia induces profound changes in the thymus. The total number was decreased to - 30% of the untreated control value at 72 h after 1 h exposure to 41 °C. This result is consistent with the decrease in thymus
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
10
Effect of hyperthermia on thymocytes 231
The double negative cell subset exhibited the most significant changes after hyperthermia as it increased up to 2- to 3-fold at 48 h This subset comprises (i) very primitive thymocytes expressing the stem cell markers Thy-1to, Lin", Sca-1+ (21), and lacking TCR, CD4, and CD8, and (li) differentiated thymocytes expressing TCR76 or TCRa/3 (16). Thus, hyperthermia may have favored stem cell migration from bone marrow to the thymus and/or development of CD4-CD8" TCR76"1" and TCRa/3+ cells which seed to the periphery (14,22). Row cytometry analysis with an appropriate mAb will resolve this issue. Preliminary results showed a 3-fold increase in the frequency of TCR^S* cells in the CD4-CD8- cell population. It has been shown that hyperthermia of lymph node cells in vitro induces enrichment of TCR75+ cells (19). Under the present conditions of hyperthermic treatment in a circulating water bath, TCRy6+ cells have probably been positively selected in the skin. Since hyperthermia induces the synthesis of hsp (9) and these molecules are recognized by TCR76"1" cells (13), self hspinduced in heat-stressed keratinocytes are likely to be involved in the activation of peripheral TCR75+ cells displaying a limited antigen receptor repertoire. It has been proposed that these cells recognize hsp associated with MHC molecules on the stressed keratinocytes and, upon stimulation by IL-1, will kill the damaged cells. By this surveillance mechanism, TCR76+ cells could act as protection against a wide variety of cellular insults, including heat, organic solvents, irradiation, infection, and transformation, by detecting a common antigen expressed on the damaged cell without the need to recognize the diverse antigens of myriad of external agents (14). The presence of TCR76+ cells in the thymus at birth, when skin, gut, lung, and other body surfaces are exposed for the first time to many noxious stimuli, may be developmentally programmed. Large numbers of cells with the same or similar specificities may provide a pod for rapid immune responses to certain critical antigens without the need for prior clonal expansion (23). Hsp might play a role also in thymocyte maturation and selection. Induction of hsp in the epithelial and dendritic cells may interfere with cell recognition of MHC class I and II molecules involved in thymocyte maturation and, therefore, with the positive
and negative selection of the antigen-specific T cell repertoire (24). Furthermore, it is possible that hyperthermia affects the synthesis of MHC molecules, as mediated by the induction of IFN-7 (6), and modulates the expression of adhesion molecules, such as lymphocyte function antigen-3 and intercellular adhesion molecular-1, which are critical for normal T cell development (25). Some of these possibilities are currently under test in our laboratory. Acknowledgements Work supported by ENEA-Euratom Contract Publication no. 2615 of the Euratom Biology Division
Abbreviations Con A FITC hsp PE
concanavalm A fluorescein isothiocyanate heat shock proteins phycoerythnn
References 1 Mondovi, B., Santoro, A S., Strom, R , Faiola, R , and Fanelli, A R. 1972 Increased immunogenicity of Ehriich ascite cells after heat treatment. Cancer 30885. 2 Duff, G. W and Durum, S K. 1983. The pyrogenic and mitogenic actions of interieukin-1 are related Nature 304449 3 Kluger, M. J , Ringler, D. H., and Anver, M R. 1975. Fever and survival Science 188:166. 4 Covert, J. B and Reynolds, W. W. 1977. Survival value of fever in fish. Nature 267.43. 5 Kluger, M J and Vaughn, L. K. 1978. Fever and survival in rabbits infected with Pasteurella multocida J. Physiol. 282.243 6 Downing, J. F , Martinez-Valdez, H., Elizondo, R. S , Walker, E B , and Taylor, M. W. 1988 Hyperthermia in humans enhances interferon7 synthesis and alters the peripheral lymphocyte population J. Interferon Res. 8:143 7 Jampel, H. D., Duff, G. W., Gershon, R. K , Atkino, E., and Durum, S. K. 1983. Fever and unmunoregulation. III. Hyperthermia augments the primary in vitro humoral immune response J. Exp. Med 157.1229 8 Ciavarra, R. P , Silvester, S , and Brody, T. 1987 Analysis of T-cell subset proliferation at afebrile and febrile temperatures differential response of Lyt-1 + 23~ lymphocytes to hyperthermia following mitogen and antigen stimulation and its functional consequences on development of cytotoxic lymphocytes. Cell Immunol 107:293. 9 Lindquist, S. and Craig, E. A 1988. The heat shock proteins Annu. Rev. Genet 22:631 10 Polla, B. S. 1988. A role of heat shock proteins in inflammation. Immunol. Today 9:134 11 Kaufmann, S. H. E. 1990. Heat shock proteins and the immune response. Immunol. Today 11 129. 12 Young, R. A. 1990. Stress proteins and immunology. Annu. Rev. Immunol 8:401. 13 Young, R. A. and Eltott, T. 1989. Stress proteins, infection and immune surveillance. Cell 59:5. 14 Asarnow, D M., Kuziel, W. A., Bonyhadi, M., Tigelaar, R. E., Tucker, P. W., and Allison, P. 1988. Limited diversity of 76 antigen receptor genes of thy-1 + dendritic epidermal cells Cell 55.837. 15 O'Brien, R. L., Happ, M P., Dallas, A., Palmer, E., Kubo, R., and Born, W. 1989. Stimulation of a major subset of lymphocytes expressing T cell receptor 75 by an antigen derived from Mycobacterium tuberculosis. Cell 57:667. 16 Fowlkes, B. J. and Pardoll, D. M 1989. Molecular and cellular events of T cell development Adv. Immunol. 44.207. 17 Cividalli, A., Parmigiani, P., and Moscati, M. 1989. A multi-jig system for radiation and hyperthermia treatment for mice. RT/PAS, 20 ENEA 18 Liburdy, R. P. 1979. Radiofrequency radiation alters the immune system: modulation of T- and B-lymphocyte levels and cell mediated immunocompetence by hyperthermic radiation. Radial. Res. 77:34.
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
weight as observed after radiofrequency hyperthermic treatment (18). The fall in thymus cellularity was heat dose dependent but the duration and temperature of the hyperthermic treatments are not likely to cause thermal cell death (19,20). Since in the normal differentiation process a great majority of the double positive cells die within the thymus (16), the decrease in thymus cellularity suggests that hyperthermia has accelerated the normal process of cellular differentiation. This interpretation is consistent with the observation that the decrease in the percentage of CD4 + CD8 + cells was concomitant with the parallel increase in the percentage of CD4"CD8 + and CD4+CD8" cells, and in the thymocyte mitotic response to Con A. Thus, not only the accelerated fall of double positive cells but also the enhanced positive selection and migration of mature single positive cells to the periphery may contribute to thymus depletion. Note that the maximum variation in thymocyte subsets occurred at 48 h, thus preceding the maximum fall in total cell number which was observed at 72 h. This view is also supported by the finding (18) that hyperthermia induces an increase in the percentage of splenic T cells, possibly as a result of enhanced cell migration from the thymus to the periphery.
232
Effect of hyperthermia on thymocytes
19 Rajasekar, Ft., Sim, G., and Augustin, A. 1990. Self heat shock and y6 T-cell reactivity. Proc. Nail Acad. Sd. USA, 87.1767. 20 Ciavarra, R. P. and Simeone, A. 1990. T lymphocyte stress response. I. Induction of heat shock protein synthesis at febrile temperature is correlated with heat enhanced resistance to hyperttiermic stress but not to heavy metal toxicrty or dexamethasone-induced immunosuppression. Cell Immunol 129:363 21 Spangrude, G. J., Heimfeld, S , and Weissman, I. L 1988. Purification and characterization of mouse hematopcnetic stem cells Science 24158. 22 Guidos, C. J., Weissman, I. L, and Adkins, B. 1989. Developmental
potential of CD4 CD8 thymocytes. Periphery progeny include mature CD4~CD8~ T cells bearing ap T cell receptor J. Immunol 142:3773. 23 Born, W., Happ, M. P., Dallas, A., Reardon, C , Kubo, R., Shinnick, T , Brennan, P , and O'Brien, R 1990. Recognition of heat shock proteins and y& cell function. Immunol Today 11:40. 24 Sargent, C. A., Dunham, I., Trowsdale, J., and Campbell, R D. 1989. Human major hstocompatibtlity complex contains genes for the major heat shock protein HSP 70. Proc. Natl Acad. Sci. USA, 86:1968. 25 Singer, K. H. 1990. Interactions between epithelial cells and T lymphocytes- role of adhesion molecules. J. Leukocyte Bid. 48:367.
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227-232
© 1992 Oxford University Press
Effect of in vivo hyperthermia on thymocyte maturation and selection Shahid Mansoor1, Marcello Span6, Selene Baschieri, Anna Cividalli, Lucia Mosiello, and Gino Doria Division of Physics and Biomedical Sciences, ENEA CRE Casaccia, 00060 Rome, Italy 1 Biomedical Division, National Institute for Biotechnology and Genetic Engineering, 9194 Lahore, Pakistan Key words: hyperthermia, thymocytes, maturation, selection
Two-month-old male mice were exposed to whole-body hyperthermlc treatment in a circulating water bath. After 1 h exposure to 41 °C, mice were kept at room temperature for 24, 48, 72, and 96 h before thymus examination. The total thymocyte number progressively decreased after 24 and 48 h, reached a minimum value at 72 h, and returned to almost normal value after 96 h. Similar changes occurred In the CD4 + CD8 + cell subset. Conversely, the percentage of CD4~CD8~ cells rose to a maximum at 48 h and then declined to normal value at 96 h. The percentage of C D 4 C D 8 + and CD4+CD8~ cell subsets increased to a maximum at 48 h and then declined to normal value at 96 h. These variations in single positive cell subsets correlated well with similar changes in the thymocyte mitotic response to concanavalin A. The observed effects on thymocytes were heat dose dependent, and suggest that hyperthermia induces the development of the most primitive thymocytes and positively selects mature T cells with high mitotic responsiveness. It is proposed that heat shock proteins might be Involved In the hyperthermia-induced alterations of thymocyte maturation and selection. Introduction Fever has been recognized as a sign of pathology since ancient times. High body temperature either during infectious diseases or artificially induced for cancer treatment has been suggested to enhance immune responses and to result in clinically beneficial effects (1,2). Convincing evidence for the beneficial effects of high body temperature also derives from studies on the lizard Dipsosaurus dorsalis which, when placed in a higher than neutral ambient temperature, increases its ability to counteract Aeromonas hydrophila infection and to survive (3). The possibility that the favorable consequences of in vivo hyperthermia are mediated by enhancement of immune responses is not only suggested by results from animals of phylogenetically distant species (2-6) but is also supported by results from in vitro experiments showing that high temperature increases the mitotic and functional activities of T cell subsets (7,8). Nevertheless, the cellular and molecular mechanisms by which hyperthermia affects the immune system are poorly understood. The synthesis of heat shock proteins (hsp) as induced by hyperthermia has been well characterized both in vivo and in vitro. Hsp are a very conserved genetic system in both prokaryotes and eukaryotes. Major hsp share a high degree of homology
(9), and their expression is enhanced in cells reponding to heat, infection, transformation, and other noxious agents (10). Hsp are, therefore, frequently called stress proteins. The immune system recognizes these proteins among the dominant antigens of a broad spectrum of pathogens and as self proteins induced in stressed autologous cells (11,12). The existence of T lymphocytes that recognize self stress proteins suggests that these cells play a role in immune surveillance of injured autologous cells as a defence mechanism against a variety of cellular insults (13). Noteworthily, a large fraction of TCR+ 76 cells recognize self hsp in the skin (14) and TCR 76 cell clones reactive with self and foreign hsp have been found in the neonatal thymus (15). The thymus plays a central role in the development and maintenance of immunity and tolerance, as it provides the microenvironment for T cell maturation and selection (16). Briefly, stem cells located in hemopoietic tissues migrate to the thymus where they undergo an ordered differentiation process. The most primitive thymocytes lack CD4 and CD8 markers (double negative) and, upon gene rearrangement, may develop into cells expressing the TCR yd which then seed to the periphery, mostly to the epithelial layers of skin and intestine. If the rearrangements
Correspondence to: G. Doria, Immunology Laboratory, ENEA CRE Casaccia, CP 2400, 00100 Rome AD, Italy Transmitting editor- L Moretta.
Received 24 April 1991, accepted 1 November 1991
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
Abstract
228
Effect of hyperthermia on thymocytes
of 7 and 8 genes are unproductive, double negative thymocytes may develop into CD4 + CD8 + (double positive) cells that initially express a low level of TCR a/3. Double positive cells undergo positive and negative selection mediated by interactions with MHC molecules expressed on epithelial and dendritic cells, and develop into CD4~CD8 + and CD4 + CD8~ (single positive) cells which then seed to the peripheral lymphoid tissues. It is possible that exogenous stressful stimuli, such as hyperthermia, induce hsp that affect the differentiation process of T cell maturation and selection. We have, therefore, examined the effect of hyperthermia on the thymus of adult mice, in terms of cellulanty, thymccyte phenotype, and mitotic responsiveness. We report herein that hyperthermia induces profound changes in the thymocyte population, which affect the generation of normal T cells.
color staining, thymocytes were incubated with anti-CD4 PE-conjugated mAb and anti-CD8 FITC-conjugated mAb for 30 mm on ice, washed, and analyzed by flow cytometry. Samples
Animals (C3H/RI/CnexDBA/2J/Cne)F, male mice were raised in our animal facilities and used at the age of 8 - 9 weeks. Each control and experimental group was composed of three animals.
Fig. 1. Effect of treatment at 41 or 37°C on thymus cell count. After treatment mice were kept at room temperature for 24 - 96 h before thymus examination. Control values from untreated mice are also shown. 37 °C
41 °C
Hyperthermia Hyperthermic treatment was delivered without anesthesia in a circulating-water bath, using a specially designed jig (17). For the delivery of whole-body hyperthermia, the jig was lowered into the bath in such a way that the water reached the mouse neck level. The temperature used during the hyperthermic treatment was 41, 42, or 43°C, as monitored by temperature probes (Omega Engineering Inc., CN). Control animals were either untreated or exposed to 37°C under identical conditions. The mean temperature of mice (legs, flank, and neck regions) was 41.3 ± 0.2 and 37.6 ± 0.3°C for mice exposed to 41 and 37°C, respectively. No variation was determined for 42 and 43°C. No mouse died during the experiments. After exposure to 37 or 41 °C for 1 h, mice were kept at room temperature (24-25°C) for 24, 48, 72, or 96 h. Mice exposed to 41, 42, or 43°C for different times to determine dose-dependent effects were kept at room temperature for a period of 72 h.
CD
a o a o
Flow cytometry Reagents to detect CD4 and CD8 antigens were anti-L3T4 phycoerythrin (PE)-conjugated mAb (clone GK 1.5) and anti-Lyt 2.2 fluorescein isothiocyanate (FITQ-conjugated mAb (clone 53-6.7) respectively. The reagents were purchased from Beckton - Dickinson (Mountain View, CA) and optimal concentrations were determined in preliminary experiments. For two-
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14
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71
• •III
••
Mill Hill Ccanl
f*
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71
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Cell preparations Thymuses were aseptically removed from three mice of each group, pooled, and single thymccyte suspensions were prepared by tapping thymuses on a 100 /un, wire mesh gauze. Cells were centrifuged at 1200r.p.m. (200 g) in a Beckman refrigerated centrifuge for 10min and resuspended in complete medium, consisting of RPMI 1640 (Sigma, St Louis, MO), supplemented with 10% FCS (Seromed, Berlin, West Germany), 2 mM L-glutamine (Gibco, Grand Island, NY), 5 x 10~5 M 2-mercaptoethanol, and 10 /ig/ml gentamicin (Shering, Kenilworth, NY).
«
cm
o
2 o
•III, 14
41
It
ti
Time after treatment
CMM
•'
14
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41
it
71
••
••
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Fig. 2. Effect of treatment at 41 or 37 °C on the distribution erf thymocyte subsets defined by CD4 and CD8 markers. After treatment mice were kept at room temperature for 24 - 96 h before thymus examination. Control values from untreated mice are also shown
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
41-C
Methods
Effect of hyperthermia on thymocytes 229 of 10,000-20,000 viable cells were analyzed for double-color fluorescence in a FACStar Plus machine. All fluorescence signals were collected in log mode. Data were evaluated as contour or dot plots generated by using Consort 30 or FACStar Plus software (Beckton - Dickinson) respectively. Mitotic response Thymocytes suspended in complete medium with or without concanavalm A (Con A; Miles-Yeda Laboratory, Rehovot, Israel) were distributed in flat bottom 96-well microtiter plates (Falcon Plastics 3040, Oxnard, CA) and cultured at 37°C in 5% CO2 humidified incubator for 48 h. Each group of triplicate cultures contained 106 cells in 0.2 ml medium alone, or with 0 5, 1, or
37 °C
Results Mice were exposed to 41 °C for 1 h, and then kept at room temperature for 24, 48, 72, and 96 h before thymus examination. The total thymocyte number progressively decreased after 24 and 48 h, reached a minimum value at 72 h and returned to almost normal values after 96 h Exposure to 37°C for 1 h under identical conditions and then to room temperature for 24 - 9 6 h caused a small decrease in thymus cellularity (Fig. 1). Figures 2 and 3 show the distribution of the four thymocyte
24 hr
48 hr
•
96hr
Fig. 4. Mitotic response of thymocytes from mice treated at 41 or 37°C. After treatment mice were kept at room temperature for 24 - 96 h before thymocytes were harvested and cultured with 1 fig Con A for 48 h. Control values from untreated mice are also shown.
. 0
72 hr
cfl-
96 hr
20 -
10 "•••
••'
ID*
!•
CD8 Fig. 3. Two color immunofluorescence analysis of CD4 and CD8 expression on thymocytes. Mice were treated at 41 or 37°C and kept at room temperature for 24 - 96 h before thymocyte analysis.
Time of treatment ( rrin)
Fig. 5. Heat dose dependence of the effect of hyperthermia on thymocyte count Mice were treated at 41, 42, or 43°C and kept at room temperature for 72 h before thymus examination. Control values from untreated mice are also shown.
Downloaded from http://intimm.oxfordjournals.org/ at New York University on April 11, 2015
41 °C
2 ng Con A. At 4 h before harvesting, cultures received 0.5 ^Ci of tritiated thymidine (specific activity 1.739 GBq/mmol) (The Radiochemical Centre, Amersham, UK) in 20 /J. Cultures were then sacrificed with an automated cell harvester (Skatron, Lier, Norway). Radioactivity was measured in a Miniaxi Tri-Carb 4000 Scintillation counter (Packard) and expressed as counts per minute (c.p.m).
230
Effect of hyperthermia on thymocytes 43 °C
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41 °C
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Time of treatment ( min ) Fig. 6. Effect of treatment at 41, 42, or 43°C on the distribution of thymocyte subsets defined by CD4 and CD8 markers. Treated mice were kept at room temperature for 72 h before thymocyte analysis Control values from untreated mice are also shown.
subsets defined by the CD4 and CD8 markers and evaluated by flow cytometry. After 1 h treatment at 41 °C, the percent of double negative cells increased to a maximum at 48 h and then declined to normal value at 96 h, while the double positive cell subset displayed similar variations as observed for the total thymocyte population. The percentage of single positive cells increased to a maximum at 48 h and then declined to normal value at 96 h. These changes in the thymocyte phenotype suggest that hyperthermia induced the development of the most primitive CD4~CD8~ thymocytes, selection of mature CD4-CD8 + and CD4 + CD8~ cells, and depletion in the CD4 + CD8 + and total thymocytes. Although exposure to 37°C for 1 h resulted in a small decrease in thymus cellularity, it did not affect the phenotype distribution of the four cell subsets. Thymocytes from mice sacrificed at 2 4 - 9 6 h after 1 h exposure to 41 °C were stimulated in vitro with different doses of Con A. The mitotic response to 1 HQ Con A/well was found highest in all groups. Figure 4 shows that the maximum response to Con A is increased by hyperthermia, reaches a peak value
at 48 h, and remains higher than in untreated controls at 96 h. One hour exposure to 37°C did not affect the thymocyte mitotic response to Con A. The observed effects of hyperthermia were found to be heat dose dependent. Mice were exposed to 41, 42, or 43°C for different times, and then sacrificed after 72 h at room temperature. Thymus cellularity was progressively decreased with increasing temperature and time of exposure (Fig. 5). Flow cytometry analysis of the four thymocyte subsets revealed that the increase in the percentage of double negative and single positive cells, as well as the decrease in the percentage of double positive cells, are both heat dose dependent (Fig. 6). Discussion The present findings indicate that hyperthermia induces profound changes in the thymus. The total number was decreased to - 30% of the untreated control value at 72 h after 1 h exposure to 41 °C. This result is consistent with the decrease in thymus
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Effect of hyperthermia on thymocytes 231
The double negative cell subset exhibited the most significant changes after hyperthermia as it increased up to 2- to 3-fold at 48 h This subset comprises (i) very primitive thymocytes expressing the stem cell markers Thy-1to, Lin", Sca-1+ (21), and lacking TCR, CD4, and CD8, and (li) differentiated thymocytes expressing TCR76 or TCRa/3 (16). Thus, hyperthermia may have favored stem cell migration from bone marrow to the thymus and/or development of CD4-CD8" TCR76"1" and TCRa/3+ cells which seed to the periphery (14,22). Row cytometry analysis with an appropriate mAb will resolve this issue. Preliminary results showed a 3-fold increase in the frequency of TCR^S* cells in the CD4-CD8- cell population. It has been shown that hyperthermia of lymph node cells in vitro induces enrichment of TCR75+ cells (19). Under the present conditions of hyperthermic treatment in a circulating water bath, TCRy6+ cells have probably been positively selected in the skin. Since hyperthermia induces the synthesis of hsp (9) and these molecules are recognized by TCR76"1" cells (13), self hspinduced in heat-stressed keratinocytes are likely to be involved in the activation of peripheral TCR75+ cells displaying a limited antigen receptor repertoire. It has been proposed that these cells recognize hsp associated with MHC molecules on the stressed keratinocytes and, upon stimulation by IL-1, will kill the damaged cells. By this surveillance mechanism, TCR76+ cells could act as protection against a wide variety of cellular insults, including heat, organic solvents, irradiation, infection, and transformation, by detecting a common antigen expressed on the damaged cell without the need to recognize the diverse antigens of myriad of external agents (14). The presence of TCR76+ cells in the thymus at birth, when skin, gut, lung, and other body surfaces are exposed for the first time to many noxious stimuli, may be developmentally programmed. Large numbers of cells with the same or similar specificities may provide a pod for rapid immune responses to certain critical antigens without the need for prior clonal expansion (23). Hsp might play a role also in thymocyte maturation and selection. Induction of hsp in the epithelial and dendritic cells may interfere with cell recognition of MHC class I and II molecules involved in thymocyte maturation and, therefore, with the positive
and negative selection of the antigen-specific T cell repertoire (24). Furthermore, it is possible that hyperthermia affects the synthesis of MHC molecules, as mediated by the induction of IFN-7 (6), and modulates the expression of adhesion molecules, such as lymphocyte function antigen-3 and intercellular adhesion molecular-1, which are critical for normal T cell development (25). Some of these possibilities are currently under test in our laboratory. Acknowledgements Work supported by ENEA-Euratom Contract Publication no. 2615 of the Euratom Biology Division
Abbreviations Con A FITC hsp PE
concanavalm A fluorescein isothiocyanate heat shock proteins phycoerythnn
References 1 Mondovi, B., Santoro, A S., Strom, R , Faiola, R , and Fanelli, A R. 1972 Increased immunogenicity of Ehriich ascite cells after heat treatment. Cancer 30885. 2 Duff, G. W and Durum, S K. 1983. The pyrogenic and mitogenic actions of interieukin-1 are related Nature 304449 3 Kluger, M. J , Ringler, D. H., and Anver, M R. 1975. Fever and survival Science 188:166. 4 Covert, J. B and Reynolds, W. W. 1977. Survival value of fever in fish. Nature 267.43. 5 Kluger, M J and Vaughn, L. K. 1978. Fever and survival in rabbits infected with Pasteurella multocida J. Physiol. 282.243 6 Downing, J. F , Martinez-Valdez, H., Elizondo, R. S , Walker, E B , and Taylor, M. W. 1988 Hyperthermia in humans enhances interferon7 synthesis and alters the peripheral lymphocyte population J. Interferon Res. 8:143 7 Jampel, H. D., Duff, G. W., Gershon, R. K , Atkino, E., and Durum, S. K. 1983. Fever and unmunoregulation. III. Hyperthermia augments the primary in vitro humoral immune response J. Exp. Med 157.1229 8 Ciavarra, R. P , Silvester, S , and Brody, T. 1987 Analysis of T-cell subset proliferation at afebrile and febrile temperatures differential response of Lyt-1 + 23~ lymphocytes to hyperthermia following mitogen and antigen stimulation and its functional consequences on development of cytotoxic lymphocytes. Cell Immunol 107:293. 9 Lindquist, S. and Craig, E. A 1988. The heat shock proteins Annu. Rev. Genet 22:631 10 Polla, B. S. 1988. A role of heat shock proteins in inflammation. Immunol. Today 9:134 11 Kaufmann, S. H. E. 1990. Heat shock proteins and the immune response. Immunol. Today 11 129. 12 Young, R. A. 1990. Stress proteins and immunology. Annu. Rev. Immunol 8:401. 13 Young, R. A. and Eltott, T. 1989. Stress proteins, infection and immune surveillance. Cell 59:5. 14 Asarnow, D M., Kuziel, W. A., Bonyhadi, M., Tigelaar, R. E., Tucker, P. W., and Allison, P. 1988. Limited diversity of 76 antigen receptor genes of thy-1 + dendritic epidermal cells Cell 55.837. 15 O'Brien, R. L., Happ, M P., Dallas, A., Palmer, E., Kubo, R., and Born, W. 1989. Stimulation of a major subset of lymphocytes expressing T cell receptor 75 by an antigen derived from Mycobacterium tuberculosis. Cell 57:667. 16 Fowlkes, B. J. and Pardoll, D. M 1989. Molecular and cellular events of T cell development Adv. Immunol. 44.207. 17 Cividalli, A., Parmigiani, P., and Moscati, M. 1989. A multi-jig system for radiation and hyperthermia treatment for mice. RT/PAS, 20 ENEA 18 Liburdy, R. P. 1979. Radiofrequency radiation alters the immune system: modulation of T- and B-lymphocyte levels and cell mediated immunocompetence by hyperthermic radiation. Radial. Res. 77:34.
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weight as observed after radiofrequency hyperthermic treatment (18). The fall in thymus cellularity was heat dose dependent but the duration and temperature of the hyperthermic treatments are not likely to cause thermal cell death (19,20). Since in the normal differentiation process a great majority of the double positive cells die within the thymus (16), the decrease in thymus cellularity suggests that hyperthermia has accelerated the normal process of cellular differentiation. This interpretation is consistent with the observation that the decrease in the percentage of CD4 + CD8 + cells was concomitant with the parallel increase in the percentage of CD4"CD8 + and CD4+CD8" cells, and in the thymocyte mitotic response to Con A. Thus, not only the accelerated fall of double positive cells but also the enhanced positive selection and migration of mature single positive cells to the periphery may contribute to thymus depletion. Note that the maximum variation in thymocyte subsets occurred at 48 h, thus preceding the maximum fall in total cell number which was observed at 72 h. This view is also supported by the finding (18) that hyperthermia induces an increase in the percentage of splenic T cells, possibly as a result of enhanced cell migration from the thymus to the periphery.
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19 Rajasekar, Ft., Sim, G., and Augustin, A. 1990. Self heat shock and y6 T-cell reactivity. Proc. Nail Acad. Sd. USA, 87.1767. 20 Ciavarra, R. P. and Simeone, A. 1990. T lymphocyte stress response. I. Induction of heat shock protein synthesis at febrile temperature is correlated with heat enhanced resistance to hyperttiermic stress but not to heavy metal toxicrty or dexamethasone-induced immunosuppression. Cell Immunol 129:363 21 Spangrude, G. J., Heimfeld, S , and Weissman, I. L 1988. Purification and characterization of mouse hematopcnetic stem cells Science 24158. 22 Guidos, C. J., Weissman, I. L, and Adkins, B. 1989. Developmental
potential of CD4 CD8 thymocytes. Periphery progeny include mature CD4~CD8~ T cells bearing ap T cell receptor J. Immunol 142:3773. 23 Born, W., Happ, M. P., Dallas, A., Reardon, C , Kubo, R., Shinnick, T , Brennan, P , and O'Brien, R 1990. Recognition of heat shock proteins and y& cell function. Immunol Today 11:40. 24 Sargent, C. A., Dunham, I., Trowsdale, J., and Campbell, R D. 1989. Human major hstocompatibtlity complex contains genes for the major heat shock protein HSP 70. Proc. Natl Acad. Sci. USA, 86:1968. 25 Singer, K. H. 1990. Interactions between epithelial cells and T lymphocytes- role of adhesion molecules. J. Leukocyte Bid. 48:367.
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