Fever-like temperature induces maturation of dendritic cells through induction of hsp90
Sreyashi Basu1 and
Pramod K. Srivastava1
1 Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, MC1601, Farmington, CT 06030-1601, USA
Correspondence to: P. Srivastava; E-mail: srivastava{at}nso2.uchc.edu
Transmitting editor: P. S. Ohashi
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Abstract
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Fever is a phylogenetically conserved biological phenomenon and a common consequence of infection. Here, we examine in vitro and in vivo the effect of febrile temperature on dendritic cells (DC), a key antigen-presenting cell in the immune system. Elevated temperatures are observed to cause immature DC to mature, specifically through elevation of intracellular levels of hsp90. Surprisingly, even brief exposure to elevated temperatures has a powerful effect on the immunostimulatory capacity of DC. These results bear on the mechanisms of the salutary effects of fever as well as of behavioral elevations of temperature such as saunas and warm blankets.
Keywords: dendritic cell, fever, hsp90
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Introduction
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Fever is a conserved physiological process, reported in bony fish (1), amphibians (2), reptiles (3,4) and mammals (5). It imparts a definite survival value to the affected organism (6). Organisms that are unable to increase their body temperature physiologically resort to behaviors that increase body temperature (4). Fever imposes a significant metabolic cost in humans (7) and other species (8). The phylogenetic conservation of this metabolically expensive mechanism is a strong argument for its survival value (9). Artificial elevation of body temperature has been used for therapy of afflictions as early as 400 BCE (10) through Hippocrates (11) to modern times and may well be the most widely used method of self-therapy among humans (saunas, blankets, soup, etc.). Despite the overwhelming evidence for the beneficial effects of fever for the host, little is known regarding the mechanisms that mediate them. This anomaly derives partly from the concomitance of elevated temperatures and elevated cytokine levels during fever, and from the abundant information available on the mechanisms of action of cytokines. In this regard, Repasky et al. have shown previously that hyperthermia (without infection, i.e. without elevated cytokines) regulates lymphocyte delivery to high endothelial venules (12) and plays a role in migration of Langerhans cells to lymph nodes (13,14). In light of the increasing realization that dendritic cells (DC) play a key role in several aspects of the adaptive and innate immune response (15), we have focused our attention here on the interaction between elevated temperatures and DC in vitro as well as in vivo.
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Methods
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Mice, cells and reagents
C57Bl/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Bone marrow-derived DC were generated from the femurs and tibia of C57BL/6 mice. The bone marrow was flushed out, and the leukocytes obtained and cultured as described (16) in complete RPMI 1640 with 10% heat-inactivated FCS and 20 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF; Endogen, Woburn, MA) for 6 days. Fresh medium with GM-CSF was added to the plates on day 3. Antibodies against CD86 (B7-2), CD40, CD11c and MHC II for FACS analysis were purchased from PharMingen (San Diego, CA). Antibodies to hsp70 and hsc70 were purchased from Stressgen (Victoria, Canada), and antibodies to hsp84 and hsp86 were purchased from Neomarkers (Freemont, CA). NiCl2 was purchased from Sigma (St Louis, MO). hsp70 (a mixture of hsc70 and hsp70) from a mouse tumor line (heat-shocked for 1 h at 42°C and allowed to recover for 16 h at 37°C) and hsp90 were purified from mouse tumors as previously described (17).
Heat-shocking DC cultures
The DC were cultured in Petri dishes for 6 days. In some experiments (as indicated) the Petri dishes were moved to 39.5 or 41°C for heat shock without splitting or re-plating to avoid physical manipulation of DC cultures. In other experiments, the DC (non-adherent cells) were harvested from Petri dishes (20 ml cultures) after day 6, and split in two separate 24-well plates or culture tubes in smaller volumes and then either incubated at higher temperature or at 37°C. In all cases, parallel cultures were handled identically.
Cellular loading of proteins or geldanamycin
hsp70, hsp90, ovalbumin (OVA; Sigma) or geldanamycin (GIBCO, Grand Island, NY) at indicated amounts in 100 µl volume were incubated with 100 µl DOTAP {N-[-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (C43H83NO8S); Roche Molecular Biochemicals} for 15 min at room temperature. In all loading experiments, 1 x 106 DC were washed 3 times with OPTI-MEM I medium (GIBCO) and then incubated in 1 ml of OPTI-MEM I media with a protein/DOTAP mixture for 30 min at 37°C. Control cells were either loaded with medium and DOTAP or were incubated with protein alone in the absence of DOTAP. After loading, cells were thoroughly washed 3 times with DC medium and then heat-shocked at 41°C for 6 h in 1 ml. After heat shock the cells were allowed to recover for 16 h for testing the expression of maturation markers or used directly in 16-h T cell stimulation assays.
Immunoblotting for I
B
Heat-shocked DC extracts were assayed by western blot analysis using anti-I
B
(rabbit polyclonal) as described in the manufacturers handbook (Cell Signaling, Beverly, MA).
Preparation of nuclear extracts and electrophoretic mobility shift assay
Heat-shocked DC were washed with PBS [lipopolysaccharide (LPS) free] and re-suspended in cold lysis buffer [buffer A: 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 1 µM aprotinin, 1 µM pepstatin and 14 µM leupeptin] with 0.1% NP-40 and incubated on ice for 30 min. Nuclei were pelleted at 14,000 r.p.m. for 2 min at 4°C. Proteins were extracted from the nuclei in a hypertonic buffer [buffer C: 20 mM HEPES (pH 7.9), 0.4M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1 µM aprotinin, 1 µM pepstatin and 14 µM leupeptin] on ice for 30 min (mixing frequently by vortexing). Nuclear debris was pelleted by centrifugation at 14,000 r.p.m. for 5 min at 4°C. Supernatant was collected and protein concentration was measured by the Bradford assay. The standard DNA-binding reaction was performed using a
B DNA probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') as described (18).
Confocal and fluorescence microscopy
For surface staining, heat-shocked or non-heat-shocked DC were labeled with FITC-labeled anti-CD11c and phycoerythrin (PE)-labeled anti-I-Ab. After staining cells were fixed by paraformaldehyde and visualized using a Zeiss LSM confocal microscope. For intracellular staining of heat-shocked DC, cells were fixed with 4% paraformaldehyde for 20 min on ice and then permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Cells were then washed in PBS and blocked with 4% BSA for 30 min. The cells were then incubated with the primary anti-p50 (H-119; Santa Cruz Biotechnology, Santa Cruz, CA) in the blocking buffer for 1 h at room temperature, washed and stained with Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 1 h at room temperature in the dark. After staining and washing, the cell pellet was mixed with DAPI (1:1000) for nuclear staining, then an equal volume of SlowFade (Molecular Probes) and placed onto a glass slide; the cover slip was sealed with nail polish. Fluorescence distribution was analyzed using a Carl Zeiss Axioplan 2 IE imaging system.
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Results
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Maturation of bone marrow DC under fever-like temperatures
CD11c+ DC constitute
75% of bone marrow-derived cultures on day 6 in GM-CSF-containing medium (Fig. 1A). Such cultures from C57BL/6 mice were exposed to temperatures of 37, 39.5 or 41°C for 0, 6, 12 or 24 h followed by recovery at 37°C overnight. The CD11c+ populations of these cultures were monitored for expression of cell-surface markers of maturation, i.e. MHC II, CD86 and CD40, at the end of recovery. The DC did not lose viability upon exposure to 39.5 or 41°C for up to 12 h. Cultures exposed to 39.5°C for 24 h also did not show loss of viability. However, exposure to 41°C for 24 h did cause loss of viability (data not shown), and therefore that specific time and temperature combination was not tested. Analysis of cell-surface markers showed that exposure to either of the elevated temperatures caused maturation of DC as demonstrated by higher mean fluorescence intensity values of the maturation markers as well as a higher proportion of cells expressing these markers (Fig. 1B). Exposure to 41°C caused a more pronounced maturation than did exposure to 39.5°C. However, exposure of DC to 39.5°C for 24 h generally showed the same level of expression of CD86 and CD40 as observed upon exposure to 41°C for 12 h (Fig. 1B). The phenomenon of heat-induced maturation was also examined by confocal microscopy of cells unexposed or exposed to 41°C, and stained with antibodies to MHC II (I-Ab, coupled to PE) and CD11c (coupled to FITC). Cells not exposed to elevated temperature stained positive for CD11c, but not for MHC II, by virtue of being immature. In contrast, cells exposed to elevated temperature showed a dramatically enhanced expression of MHC II. An overlay of these images shows that CD11c+ cells now express dramatically enhanced levels of MHC II (Fig. 1C).

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Fig. 1. Kinetics of maturation of DC after heat shock. (A) Day 6 bone marrow cultures were analyzed for the expression of CD14+, Gr1+ and CD11c+ by FACS. Gr1 and CD14 were used as granulocyte- and monocyte-specific markers in order to test the purity of the DC preparations. (B) Bone marrow cultures were incubated at 37, 39.5 or 41°C for different time points as indicated and allowed to recover at 37°C overnight. The cells were stained with FITC-conjugated anti-CD11c antibody and for MHC II, CD40 or B7-2 (by respective antibodies coupled to PE), and analyzed by two-color staining using FACS. The mean fluorescence intensity values of the activation markers on total CD11c+ cells and percentages of double-positive cells (CD11c+ and positive for the indicated markers) are plotted. (C) Confocal microscopy of non-heat-shocked or heat-shocked (at 41°C for 6 h) DC stained with anti-CD11cFITC antibody and anti-I-AbPE antibody. (D) Dot-plots of DC cultures incubated at 37 or 41°C for 6 h, followed by overnight recovery and staining with FITC-labeled anti-CD11c antibody and PE-labeled anti-I-Ab antibody.
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Expression of heat shock proteins is cell-type specific under fever-like temperatures
The bone marrow cultures consisting predominantly of DC (Fig. 1A) were exposed to 39.5 or 41°C (6, 12 or 24 h followed by recovery at 37°C overnight) and were analyzed for expression of steady-state levels of the heat shock proteins hsc70, hsp70 and hsp90. Hsc70 was expressed constitutively and either temperature caused induction of hsp90, but surprisingly not of hsp70 (Fig. 2A). The heat treatment regimen used here is expected to cause induction of hsp70 as well; as a control for the regimen, we therefore tested the effect of this regimen of heat shock on non-DC. We observe that exposure of CT26 colon carcinoma cells to 41°C results in clear induction of hsp70; exposure of DC to the same regimen in the same experiment did not result in such induction (Fig. 2B). In view of the well-known conservation of the heat-shock response (19), cell-type specificity observed in Fig. 2(B) is surprising and the lack of induction of hsp70 in DC under these conditions may be a peculiarity of DC.

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Fig. 2. Hyperthermia induces hsp90, but not hsp70, in DC. (A) The bone marrow cultures were heat-shocked at 39.5 or 41°C for 4 h and allowed to recover at 37°C overnight. Cell lysates were resolved by SDSPAGE and immunoblotted using antibodies to hsp86, hsc73 or hsp72. (B) CT26 tumor cells or bone marrow-derived DC were heat-shocked at 41°C for the indicated time and allowed to recover at 37°C overnight. Cell lysates were resolved by SDSPAGE and immunoblotted using antibodies to hsp72.
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Response of DC to chemical agents that mimic heat shock
Elevation of cellular temperature results in a classical heat-shock response characterized by elevated expression of a number of heat shock proteins (19). Certain chemical signals, such as exposure to nickel or stannous chloride, are known to mimic this response (19). In order to determine if common components of the heat-shock response were responsible for the induced maturation of DC as observed in Fig. 1, immature DC were exposed to 300 µM NiCl2 for 20 h at 37°C instead of heat shock. The DC were analyzed for cell-surface markers of maturation as in Fig. 1, and indeed it was observed that exposure to 300 µM NiCl2 mediates up-regulation of MHC II, CD40 and CD86 on immature DC, thus converting them into mature DC (Fig. 3A). The DC also showed up-regulation of hsp90 upon exposure to NiCl2 (Fig. 3B). Interestingly, induction of hsp72 was not observed in nickel-treated DC (data not shown).

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Fig. 3. Effect of NiCl2 on DC mimics heat shock response in both maturation and expression of heat shock proteins. (A) Day 6 bone marrow cells were cultured with 300 µM of NiCl2 as indicated for 20 h at 37°C. Cells were analyzed for expression of surface markers by three-color staining using FACS as in Fig. 1. The mean fluorescence intensity values for I-Ab, B7-2 and CD40 on CD11c+ cells are plotted. (B) Day 6 bone marrow cells were cultured with NiCl2 or medium for 4 h at 37°C, and washed thoroughly and cultured overnight at 37°C for recovery. Cell lysates were resolved by SDSPAGE and immunoblotted using antibody to hsp86. The blots were also probed with antibody to hsp72; no induction of hsp72 was observed (not shown).
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Geldanamycin, an inhibitor of hsp90, inhibits DC maturation under fever-like temperature
The preferential induction of hsp90 in heat-exposed DC led us to ask if hsp90 was actually playing a role in maturation or if it was a side event. The antibiotic geldanamycin, which binds hsp90, but not hsp70, family members specifically (20), was used for this analysis. Immature DC were chemically micro-injected (using the lipid DOTAP) with geldanamycin (or saline as a negative control) followed by heat shock and monitored for maturation as measured by expression of MHC II. Sequestration of hsp90 was observed to abrogate completely the maturation of DC by heat. When DC were also micro-injected with hsp70 or hsp90 in order to determine if the abrogation of maturation could be reversed, introduction of additional hsp90, but not hsp70, was found to be effective. The reversal of geldanamycin-induced inhibition by adding exogenous hsp90 was substantial, but not total, because of the difficulty in getting hsp90 to a high enough concentration in the DOTAP micelles. This is entirely consistent with the fact that hsp90 is expressed intracellularly at a very high concentration. Interestingly, endotoxin-induced maturation of immature DC was also observed to be completely inhibited by experimental sequestration of intracellular hsp90 by geldanamycin (Fig. 4).

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Fig. 4. Maturation of DC in response to fever-like temperature is abrogated by introduction of geldanamycin into the cytosol. Bone marrow cultures were loaded with medium alone, geldanamycin (10 µM) alone, geldanamycin with hsp90 (100 µg) or geldanamycin with hsp70 (70 µg) using DOTAP. After extensive washing, cells were heat-shocked for 6 h at 41°C and allowed to recover at 37°C overnight or treated with 100 ng/ml LPS at 37°C. The cells were analyzed for expression of I-Ab by FACS. The percentages shown are CD11c+ cells that are also positive for I-Ab.
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Fever-like temperature activates nuclear translocation of NF
B in bone marrow-derived DC
One of the early events in maturation of DC through other agents such as endotoxin or exogenous application of heat shock proteins involves degradation of the cytosolic factor I
B and consequent translocation of the I
B-bound NF
B to the nucleus where it acts as a transcription factor for a number of genes of immunological significance (21,22). The heat-treated immature DC cultures were analyzed for these phenomena. Lysates of DC were probed for I
B content by immunoblotting and for translocation of NF
B into the nucleus by an electrophoretic mobility shift assay. I
B content was observed to gradually diminish in a time-dependent manner, beginning 4 h after heat shock and reaching a minimum at 10 h, after which it recovered rapidly to a level even higher than the initial level. Concomitantly, NF
B levels in the nucleus began to rise at 4 h and reached a peak at 10 h, after which they began to diminish (Fig. 5A). These events were also observed microscopically. Non-heat-shocked and heat-shocked DC were stained with an antibody to the p50 component of NF
B. p50 was detected clearly in the cytosol in non-heat-shocked cells and it could be observed to migrate into the nucleus in heat-shocked DC (Fig. 5B). These studies demonstrate that the heat-induced maturation of DC appears to follow the canonical path of maturation of DC as known to be elicited by other agents.
Heat-shocked bone marrow-derived DC are more potent stimulators of T cells than untreated cells
The heat-shocked DC were tested for their ability to stimulate T cells in several independent assays in vitro and in vivo. In studies in vitro, non-heat-shocked or heat-shocked (41°C, 6 h) cultures of DC (b haplotype) were used to stimulate purified T cells obtained from naive spleen cells of BALB/c (d haplotype) mice. The heat-shocked DC were significantly better stimulators of the allogeneic cells in the entire range of DC:T ratios tested. The stimulation of T cells by non-heat-shocked DC at the DC:T ratio of 1:1600 was practically undetectable; the heat-shocked DC cultures were detectably able to stimulate T cells under these conditions. At the highest DC:T ratio tested (1:200), the heat-shocked DC were 3-fold as effective as non-heat-shocked DC (Fig. 6A). This phenomenon was then tested in an antigen-specific system. DC cultures were micro-injected (using DOTAP) with OVA protein, introducing it into the endogenous pathway of antigen presentation leading to presentation of the SIINFEKL epitope by Kb of the DC. The antigen-loaded DC were not or were exposed to heat shock (41°C, 6 h) and were cultured with an MHC I-restricted T cell line, Moja, specific for the Kb/SIINFEKL epitope. Stimulation of T cells was monitored by release of IFN-
. DC micro-injected with medium were unable to stimulate Moja regardless of whether or not they were heat-shocked. The non-heat-shocked DC micro-injected with OVA stimulated Moja specifically. However, the same DC, upon heat shock, stimulated Moja nearly 3-fold as compared to their non-heat-shocked counterparts (Fig. 6B). Thus, in antigen-dependent and -independent assays in vitro, heat-shocked DC were significantly superior to non-heat-shocked DC in stimulating T cells.

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Fig. 6. Effect of fever-like hyperthermia on the antigen presentation by DC in vitro and in vivo. (A) Non-heat-shocked or heat-shocked DC (C57BL/6J) were irradiated (2500 rad) and cultured with 2 x 105 T cells purified from naive BALB/cJ spleen. After a 3-day co-culture, 2.5 µCi/ml [3H]thymidine was added to each well for 16 h and thymidine uptake measured. (B) Day 6 DC (C57BL/6J) were loaded with OVA (1 mg) using DOTAP for 30 min in serum-free medium. Cells were washed and heat-shocked at 41°C for 6 h. The loaded heat-shocked or non-heat-shocked DC were used to stimulate a syngeneic OVA-specific CD8+ T cell line (Moja) at 37°C for 20 h. Culture supernatants were tested for the presence of IFN- (pg/ml) by ELISA as a marker for cytotoxic T lymphocyte stimulation. (C) Experimental design for (D) and (E). (D and E) C57BL/6J mice were injected intradermally with PBS or 1 mg OVA in 100 µl. Purified CD11c+ cells from the lymph nodes of injected mice were co-cultured with B3Z cells in a ratio of 1:2 (DC:T) for 16 h at 37°C. The cells were washed and fixed, and X-gal added to them for 16 h at 37°C as described (20). The total number of blue cells in each well was also counted (E).
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DC from heat-shocked mice are more potent antigen presenters
The ability of DC exposed to elevated temperatures in vivo to stimulate antigen-specific T cells was tested (see Fig. 6C for experimental design). Elevation of temperatures in vivo is nearly always linked to infection (and hence elevated cytokine levels), but it was our goal to examine the effect of elevated temperatures alone. The alternative usually involves placing mice at elevated air temperatures for several hours, at which time the hypothalamic mechanisms that set the body temperature are overwhelmed and the core temperature increases (13). We injected C57BL/6 mice intradermally at a mid-ventral location with OVA (1 mg). Immediately following injection, the mice were not or were exposed to 41°C for 1 h by placing them in an air incubator. Such treatment results in elevation of the core temperature. The mice were allowed to recover at ambient temperature overnight. The inguinal and axillary lymph nodes of the mice were isolated. There were no notable differences in the numbers of CD11c+ cells in the lymph nodes of non-heat-shocked and heat-shocked mice. CD11c+ cells from both groups of mice were used to stimulate cognate T cells and, in this parameter, they were profoundly different. The following assay was used for this experiment. CD11c+ cells were incubated with B3Z cells which express the TCR recognizing Kb/SIINFEKL whose engagement leads to activation of a transfected ß-galactosidase gene (23). The incubation of cultures with X-gal, a substrate of ß-galactosidase, results in deposition of an insoluble blue product of reaction within the cells, leading to visualization of activated T cells. This system thus permits quantitative determination of activated T cells. Incubation of CD11c+ cells from PBS-immunized mice with B3Z cells resulted in no detectable activation of B3Z cells, regardless of whether or not the mice were heat-shocked. CD11c+ cells from immunized, non-heat-shocked mice were able to stimulate significant numbers of B3Z cells. However, similar CD11c+ cells from heat-shocked mice stimulated at least 3-fold more B3Z cells (Fig. 6D and E). Thus, a transient exposure of the mouse to elevated temperatures, under non-febrile conditions, leads to a powerful difference in the immunostimulatory capability of the skin DC.
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Discussion
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The results shown here provide an outline of the specific mechanism through which fever may exert a beneficial effect on the host immune response. The following series of convergent events can be seen to happen upon infection and fever. Elevation of body temperature can cause maturation of DC in skin and other tissues such that having received the antigen from infectious (or other) pathological agents in the immature stage, the DC mature and migrate to the draining lymph nodes. The mature DC are better presenters of antigen to the naive T cells in the lymph nodes (15). Clearly, this series of events is not operating in isolation of other events during infection and fever. The elevation of levels of cytokines such as tumor necrosis factor (TNF)-
, frequently concomitant with fever, also contributes to DC maturation (24). At the same time, the infected cells must undergo lytic cell death in many, but not all, conditions of infection. The heat shock proteins released from cells as a result of necrotic death (22,25) have been shown to cause translocation of NF
B into the nucleus and maturation of DC (22). The mammalian DNA released under these conditions also mediates the same effect (26). In addition to mediating maturation, the non-covalent heat shock proteinspeptide complexes formed during infection in the infected cells and released from cells as a result of necrotic death are taken up by DC through the CD91 receptor and the peptides re-presented (or cross-presented) by the MHC I molecules of the DC to the cognate T cells (27,28).
Elevated temperatures and cytokine levels during infection, although happening in concert, are independent of each other. While elevated temperature and TNF both cause maturation of DC, one does not require the other in order to mediate that effect. Elevated temperature alone or TNF alone can mediate DC maturation. As an example, fever range hyperthermia has been shown to enhance contact hypersensitivity, presumably through enhanced migration of Langerhans cells (13), and elevated temperatures enhance adhesion of lymphocytes to the endothelium (12). However, such elevations of body temperatures do not necessarily mediate elevation of cytokine levels in the blood (13,29). Similarly, the effects of elevated temperature or of TNF are independent of the maturation of DC elicited by the heat shock proteins (12) or DNA (26) released as a result of cell lysis during infection. The selection of redundant, but independent, pathways to key biological objectives is a time-tested evolutionary strategy.
The observation that hsp90, but not hsp70, is involved in mediating the fever-induced maturation of DC is surprising. hsp90 is the most abundant chaperone in the cytosol of all cells, but hsp70 is the most highly heat-inducible. Both heat shock proteins have been shown to be present in the activation cluster of molecules that associate with each other during the action of LPS on antigen-presenting cells (30). Further, both heat shock proteins have been shown to be targets of action of taxol on macrophages (31). From the perspective of the studies shown here, hsp90 (but not hsp70) has specifically been shown to be involved in nuclear translocation of NF
B and expression of TNF in macrophages treated with taxol or with LPS. Thus, elevated temperature mediates DC maturation following the same pathways (through hsp90 and NF
B) as utilized by LPS or taxol in activating macrophage.
Our observation that even a short exposure to elevated temperature can profoundly affect the immunostimulatory capacity of skin DC is surprising to us, although in hindsight it is consistent with the data in vitro. DC exposed in vitro for 6 h to elevated temperature followed by ambient temperature show distinctly different maturation characteristics. Similarly, DC of mice exposed as briefly as 1 h to elevated temperatures followed by ambient temperature overnight become profoundly more immunostimulatory. This observation requires further exploration in terms of possible anti-viral and anti-tumor resistance of animals exposed to elevated temperatures for brief periods. Considerable anecdotal data point towards the salutary effects of saunas and exposure to warmer climates for relief from infections. The observations reported here provide a possible and testable mechanism for those data.
How do the DC sense heat? One would imagine that such sensory perceptions are in the domain of sensory neurons. Our studies show that the sensory neurons and DC may have common sensory mechanisms. VR1/VRL3 receptors, identified as heat receptors on sensory neurons by Julius and colleagues (3237), are also expressed on DC and are likely involved in heat sensing by them (data not shown). This observation suggests that the immune and nervous systems sense heat through the same receptors, but through divergent pathways and for distinct purposes. This idea is consonant with the considerable and growing evidence of the common phylogenetic origins of the immune and nervous systems, and of the central role of the phagocyte (or related cells) as a common progenitor for both these systems (38).
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Acknowledgements
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We thank Jaqueline Beltran for technical assistance and Jason Kirk (CBIT, UCHC) for assisting in confocal microscopy. The work was supported by NIH grant CA64394 and by a research agreement with Antigenics, in which P. K. S. has a significant financial interest.
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Abbreviations
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DCdendritic cell
GM-CSFgranulocyte macrophage colony stimulating factor
LPSlipopolysaccharide
OVAovalbumin
PEphycoerythrin
TNFtumor necrosis factor
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