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(Journal of Leukocyte Biology. 2003;73:493-501.)
© 2003 by Society for Leukocyte Biology

Fever-like thermal conditions regulate the activation of maturing dendritic cells

Jean-Nicolas Tournier*, Anne Quesnel Hellmann*, Gaëtan Lesca*, Alain Jouan*, Emmanuel Drouet{dagger} and Jacques Mathieu*

* Département de Biologie des agents Transmissibles, Centre de Recherches du Service de Santé des Armées, La Tronche cedex, France; and
{dagger} EA 2939 GDR CNRS, "Virologie moléculaire et structurale," La Tronche, France

Correspondence: Jean-Nicolas Tournier, Centre de Recherches du Service de Santé des Armées, Département de Biologie des agents Transmissibles, BP87; F-38702 La Tronche cedex, France. E-mail: jntournier{at}crssa.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fever is one of the most frequent clinical signs encountered in pathology, especially with respect to infectious diseases. It is currently thought that the role of fever on immunity is limited to activation of innate immunity; however, its relevance to activation of adaptive immunity remains unclear. Dendritic cells (DCs) that behave as sentinels of the immune system provide an important bridge between innate and adaptive immunity. To highlight the role of fever on adaptive immunity, we exposed murine bone marrow-derived lipopolysaccharide (LPS)- or live bacteria-maturing DCs over a 3-h period to 37°C or to fever-like thermal conditions (39°C or 40°C). At these three temperatures, we measured the kinetics of cytokine production and the ability of DCs to induce an allogeneic mixed lymphocyte reaction. Our results show that short exposure of DCs to temperatures of 39°C or 40°C differentially increased the secretion of interleukin (IL)-12p70 and decreased the secretion of IL-10 and tumor necrosis factor {alpha} by maturing DCs. These fever-like conditions induced a regulation of cytokine production at the single-cell level. In addition, short-term exposed LPS-maturing DCs to 39°C induced a stronger reaction with allogeneic CD4+ T cells than maturing DCs incubated at 37°C. These results provide evidence that temperature regulates cytokine secretion and DC functions, both of which are of particular importance in bacterial diseases.

Key Words: hyperthermia • danger signal • cytokine • Salmonella typhimurium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fever is a complex, coordinated autonomic neuroendocrine and behavioral response that is adaptive and is a part of the acute-phase reaction to immune challenges [1 ]. Fever has been associated with improved survival and shortened disease duration in infections [2 3 4 ], but the mechanisms of these beneficial responses are poorly understood. It has been shown that temperature changes induced by fever might activate innate immunity [5 ]; however, its relevance to activation of adaptive immunity is subject to debate [6 ].

We hypothesize that fever may play a role in activation of adaptive immunity through regulation of dendritic cell (DC) functions. Indeed, DCs provide an important bridge between innate and adaptive immunity [7 ], performing two distinct functions at two respective locations [8 ]: in unperturbed tissues in an immature form, where they are adapted for capturing and accumulating antigens, and in lymph nodes in mature form, where after terminal differentiation, DCs acquire potent antigen-presenting capacity and ability to prime T cell response efficiently, thereby initiating the polarization of the emerging T cell response [9 ]. In this manner, DCs are not only key mediators for delivering antigen-specific signals to T cells, but they also convey packets of information to T cells that must be decoded before an appropriate immune response can be mounted [10 ]. During this process, DC maturation is a key step in the initiation of immunity and also has important consequences on the quality of the immune response. The critical process of DC maturation is induced by several danger signals, including microorganisms, necrotic cells, proinflammatory cytokines, and signals from T cells such as CD40 ligand [11 , 12 ]. Until now, the nature of the danger signals known to induce DC maturation and activation is limited to molecular structures.

In this study, we investigated whether lipopolysaccharide (LPS)- or live bacteria-maturing DCs are capable of recognizing physical signals, such as temperature under fever-like conditions. We used Salmonella typhimurium as a paradigm of DC-bacteria interactions to highlight the role of fever on DC functions during an infection. Several aspects of DC-S. typhimurium interactions have been previously studied [13 , 14 ]. It has been shown that S. typhimurium is phagocytosed by murine DCs, but bacteria efficiently survive and replicate in phagosomal compartments in vitro [15 , 16 ]. S. typhimurium triggers the maturation, the secretion of cytokines [15 , 17 ], and the processing of bacterial antigens by major histocompatibility complex (MHC) class I and class II pathways by DCs [18 , 19 ]. Moreover, several reports have shown that DCs play a major role in the control of Salmonella infection in vivo [20 , 21 ].

Here, we present evidence that short exposure of DCs to temperatures of 39°C or 40°C regulates the secretion of cytokines by LPS- and live bacteria-maturing DCs. Our results show that fever-like conditions differentially increased the secretion of interleukin (IL)-12p70 and significantly decreased the secretion of IL-10 and tumor necrosis factor {alpha} (TNF-{alpha}). Moreover, DCs exposed to 39°C induced a stronger reaction with allogeneic CD4+ T cells, as compared with 37°C. By way of these mechanisms, fever may have significant effects on adaptive immunity and elimination of pathogens.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of bone marrow-derived DCs
DCs were generated from proliferating mouse bone marrow progenitors. Briefly, cells were obtained by flushing the bone marrow shafts of femurs and tibias from male BALB/c (H-2d) mice (CERJ, Mayenne, France). Red blood cells were lysed with NH4Cl buffer, and B and T lymphocytes were depleted with anti-pan-T and anti-pan-B beads (Dynal, Oslo, Norway). After washing, bone marrow cells (containing normally less than 2% of CD14-positive cells and more than 3% of Sca-1-positive cells) were seeded in 24-well flat-bottom microtiter plates (Costar-Corning, Cambridge, MA) at 6 x 105 cells/ml in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 5% heat-inactivated fetal calf serum (GibcoTM Invitrogen, Cergy, France), 100 U/ml penicillin–0.1 mg/ml streptomycin (Sigma Chemical Co.), and 2 mM L-glutamine (Sigma Chemical Co.), henceforth referred to as CM. Granulocyte macrophage-colony stimulating factor (GM-CSF; 20 ng/ml; Peprotech, Rocky Hill, NJ) and 10 ng/ml IL-13 (R&D Systems, Oxon, UK) were added to each well. On days 2, 5, and 7, 70% of the medium was replaced by fresh CM containing GM-CSF at 20 ng/ml. Eight-day cultured immature DCs were harvested by vigorously pipetting the cell medium over the adherent stroma. After one wash, DCs were resuspended at 1.5 x 106 cells/ml in CM containing 20 ng/ml GM-CSF for further use.

Fever-like thermal conditions and stimulation of DCs
DCs at 1.5 x 106 cells/ml were seeded in 24-well microplates and were stimulated with LPS from S. typhimurium (Sigma Chemical Co.) at 1 µg/ml or with S. typhimurium at a multiplicity of infection of 1. S. typhimurium (strain C52, Institut Pasteur collection number 103991) were cultured in Luria-Bertani broth at 37°C with shaking until exponential growth; they were quantified spectrophotometrically by determining the optical density at 600 nm.

We sought physiologically relevant conditions under which DC functions are modulated by exposing DCs for several periods of time (from 3 to 24 h) at fever-like conditions (39°C or 40°C). Finally, we focused on short exposures (3 h) to fever-like conditions to be as close as possible to clinical "infectious conditions" characterized by short peaks of fever. Moreover, this strategy offered the opportunity to investigate whether DCs primed in peripheral tissues under fever-like conditions behave in a significantly different manner 24 h later in lymph nodes. DCs were simultaneously stimulated with LPS or live S. typhimurium and were exposed to fever-like conditions (39°C or 40°C) for 3 h and then placed at 37°C until the end of the experiments. Control plates were incubated at 37°C each time. Infection of DCs was allowed to proceed for 1 h, and thereafter, gentamycin (Sigma Chemical Co.) was added at 60 µg/ml in each well to kill any remaining extracellular bacteria. Gentamycin was selected because of its limited uptake by eukaryotic cells. Experiments showed that cell viability was not affected even after long exposure times (24 h) at fever-like conditions (39°C and 40°C). Cell viability was assessed each time by blue trypan exclusion.

Analysis of cell-surface phenotype
Cells were incubated at 106 cells/ml in 0.1% sodium azide–1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with 1 µg/ml 2.4G2 anti-CD16/CD32 antibody (BD Biosciences, Franklin Lakes, NJ) for 10 min at 4°C. Cell suspensions (200 µl) were labeled for 30 min at 4°C in 96-well plates (V-bottom, Nunc, Roskilde, Denmark) with the following antibodies conjugated with fluoroscein isothiocyanate (FITC) or phycoerythrin (PE; all from BD Biosciences): RM4-5 anti-CD4, 53-6.7 anti-CD8{alpha}, M1/70 anti-CD11b, HL3 anti-CD11c, rmC5-3 anti-CD14, 3/23 anti-CD40, RA3-6B2 anti-CD45R/B220, 6-10A1 anti-CD80, GL1 anti-CD86, AMS-32.1 anti-I-Ad, SF1-1.1 anti-H-2 Kd, RB6-8C5 anti-Gr-1. Cells were washed twice with 0.1% sodium azide–1% BSA in PBS and were fixed with 1% formaldehyde (Sigma Chemical Co.) in the washing buffer. Two-color flow cytometry analysis was performed on an EPICS flow cytometer (Beckman Coulter, Fullerton, CA).

Cytokine measurement in supernatants
IL-10, IL-12p70, and TNF-{alpha} secretions were measured in supernatants of DC culture at different times (8, 18, and 24 h) using enzyme-linked immunosorbent assay (ELISA) kits (Quantikine System, R&D Systems). The minimum detectable dose of IL-10, IL-12p70, and TNF-{alpha} was 4, 2.5, and 5.1 pg/ml, respectively.

Intracellular cytokine staining
After incubation for 3 h at 37°C, 39°C, or 40°C, immature LPS- and S. typhimurium-maturing DCs were treated with 10 µg/ml brefeldin A (Sigma Chemical Co.) for intracellular cytokine staining during 16 h at 37°C. Cells were then harvested, stained with FITC-coupled anti-I-Ad monoclonal antibody (mAb; AMS-32.1; BD Biosciences), and fixed in 1% paraformaldehyde. DCs were then permeabilized with 0.1% saponin–1% BSA–PBS and incubated with the following PE-coupled mAb (all from BD Biosciences): anti-mIL10 (JES5-16E3), anti-mIL12p40/p70 (C15.6), anti-mTNF-{alpha} (MP6-XT22). The DCs were then washed and fixed in 1% paraformaldehyde. Two-color flow cytometry analysis was performed on an EPICS flow cytometer (Beckman Coulter).

IL-10 and TNF-{alpha} neutralization
Using the same thermal condition protocol, cells were incubated at 1.5 x 106 cells/ml in CM containing 20 ng/ml GM-CSF and 1 µg/ml LPS in 96-well microplates. Four separate tests were performed by adding 5 µg/ml of the following antibodies at the time of stimulation with LPS: neutralizing anti-IL-10 rat mAb (clone JES052A5, R&D Systems); rat immunoglobulin G (IgG) control (clone R35-95, Pharmingen, San Diego, CA); neutralizing anti-TNF-{alpha} goat IgG polyclonal Ab purified by affinity chromatography (R&D Systems); and normal goat IgG (R&D Systems). IL-12p70 levels were measured after 24 h of culture. Preliminary dose-response experiments indicated that the optimal neutralizing concentration of anti-IL-10 mAb and anti-TNF-{alpha} Ab was 5 µg/ml.

Allogeneic mixed lymphocyte reaction (MLR)
CD4+ T cells were purified from spleens of C57BL/6 (H-2b) mice (CERJ) by magnetic positive cell sorting. After red blood cell lysis with NH4Cl buffer, splenocytes were filtered, and CD4+ T cells were positively selected using magnetic cell sorter CD4 (L3T4) microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of CD4+ T cells was assessed with flow cytometry and was generally >=90%. The DCs that were stimulated with 1 µg/ml LPS or not stimulated were incubated at different temperatures (37°C, 39°C, or 40°C) for 3 h and were then placed at 37°C for the remaining time. After 6 h, the DCs were treated with mitomycin (Sigma Chemical Co.) at 10 µg/106 cells over 20 min, washed twice, and used for MLR. Ten thousand C57BL/6 responder CD4+ T cells per well were cocultured for 5 days at 37°C with different amounts of BALB/c stimulator DCs in 96-well U-bottom microtiter plates (Costar-Corning). During the final 18 h of coculture, 1 µCi [3H]thymidine (ICN Pharmaceuticals, Costa Mesa, CA) was added. Incorporation of [3H]thymidine was measured in a ß-scintillation counter (Microbeta, EG&G Wallac, Turku, Finland).

Statistical analysis
Statistical analyses were done using the two-tailed Student’s t-test. Results are shown as means ± 1 SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fever-like conditions do not change the phenotype of DCs
We assessed the phenotype of the bone marrow-derived cells after 8 days of culture; more than 80% of nonadherent cells generated ex vivo with GM-CSF and IL-13 exhibited CD11c, which is the relatively restricted mouse DC marker. DCs generated with this method were negative for monocyte-macrophage (CD14), B cell (CD45R/B220), granulocyte (Gr-1) markers (granulocyte differentiation was excluded by Giemsa observations, and weak positive staining is a result of cross-reactivity of the antibody with Ly-6C marker) and exhibited a phenotype of myeloid immature DCs (CD11c+, CD11b+hi, CD4-, CD8{alpha}-, MHC-I+, MHC-II+low, CD40-, CD80+, CD86+low; Fig. 1A ).



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  		Figure 1. Phenotypic analysis of immature and maturing DCs at fever-like conditions. The shaded histograms represent isotype fluorescence of DCs; the nonshaded histograms represent the fluorescence of specific markers. (A) Phenotype of bone marrow-derived immature DCs analyzed by flow cytometry after 8 days of culture. (B) Phenotypic analysis of maturing DCs after 18 h of LPS stimulation. (C) Phenotypic analysis of maturing DCs after 18 h of live S. typhimurium stimulation. (B, C) Nonshaded histograms show cells that were incubated at 37°C (light gray line), 39°C (dark gray line), 40°C (solid line); note the superposition of the fluorescence signals at all three temperatures. The isotype controls showed similar levels of fluorescence for cells incubated at 37°C, 39°C, or 40°C. (A–C) Results shown are representative of four different experiments.

 
We first investigated whether temperature elevation within a physiologic range (37–40°C) could modify the expression of cell-surface molecules of immature or LPS- or live bacteria-maturing DCs. Fever-like conditions did not result in the initiation of the final maturation of DCs (data not shown) or in the modification of the maturation-associated, phenotypical changes of LPS (Fig. 1B) or live S. typhimurium-stimulated cells (Fig. 1C) . Three hours of incubation at 39°C or 40°C neither elevated nor inhibited the expression of DC marker CD11c, the costimulatory molecules CD40, CD80, and CD86, and the class II MHC antigen-presenting molecules I-Ad.

Fever-like conditions differentially regulate IL-12p70, IL-10, and TNF-{alpha} secretion
We then investigated whether fever-like conditions could induce LPS-maturing DCs to modulate the level and/or the kinetics of cytokine production. We focused on three major cytokines produced by DCs: IL-12p70, the main cytokine that drives T-helper lymphocyte type 1 polarization [high producers of IL-2 and interferon-{gamma} (IFN-{gamma})] [9 ]; TNF-{alpha}, a pleiotropic cytokine with a diverse range of biological activity in inflammation [22 ]; and IL-10, a cytokine that down-regulates DC functions as well as T cell response [23 , 24 ]. Immature DCs did not produce these cytokines, and temperature did not significantly modulate their level of cytokine production in the supernatants (Fig. 2 ). However, maturing DCs produced the different cytokines with distinct kinetics. High levels of TNF-{alpha} could be detected in DC supernatants 8 h after induction of maturation, whereas IL-10 and IL-12p70 reached a plateau only after 18 h. We observed that fever-like conditions did not modify the kinetics of cytokine secretion, whereas they differentially regulated the amount of cytokines produced. The production of IL-12p70 increased significantly at 39°C and 40°C after 18 h (Fig. 2A) . At 40°C, the secretion was not sustained, and the IL-12p70 concentration after 24 h was not significantly different than that at 37°C. By contrast, the level of IL-10 secreted showed an inverse correlation with temperature after 18 h at 39°C; it is noteworthy that at 40°C, the IL-10 secretion decrease started after 8 h (Fig. 2B) . The level of TNF-{alpha} showed a more marked and earlier (after 8 h) inverse dependence on temperature (Fig. 2C) .



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  		Figure 2. Regulation of cytokine secretions at fever-like thermal conditions after LPS stimulation of DCs. (A) IL-12p70, (B) IL-10, and (C) TNF-{alpha} levels were measured after 8, 18, and 24 h in the supernatants of immature or LPS-stimulated DCs incubated at 37°C (open bars), 39°C (gray bars), or 40°C (solid bars) for 3 h and at 37°C for the remaining time. Data show mean cytokine levels of culture supernatants (±SD) representative of three independent experiments. *, P < 0.05 as compared with 37°C.

 
We next investigated whether the modulations observed with LPS-stimulated DCs were relevant with live S. typhimurium-stimulated DCs. In our experimental conditions, live S. typhimurium induced significantly lower levels of IL-12p70 and TNF-{alpha} at 18 h but higher levels of IL-10 compared with LPS alone (Fig. 3 ). However, the effect of temperature on cytokine secretion patterns was the same; i.e., IL-12p70 was significantly higher at 39°C than 37°C or 40°C, and IL-10 and TNF-{alpha} levels decreased with the increase in temperature. These results suggest that temperature plays an important role during infections by tightly controlling DC cytokine secretions.



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  		Figure 3. Cytokine secretion response as a function of temperature is the same for LPS and S. typhimuriumstimulation. (A) IL-12p70, (B) IL-10, and (C) TNF-{alpha} levels were measured after 18 h in the supernatants of LPS- or of S. typhimurium-stimulated DCs incubated at 37°C (open bars), 39°C (gray bars), or 40°C (solid bars) for 3 h and at 37°C for the remaining time. Data represent mean cytokine levels of culture supernatants (±SD) representative of three independent experiments. *, P < 0.05 as compared with 37°C.

 
To further understand the mechanisms involved in these modifications, cytokine production was assessed at the cell level by intracellular staining, followed by flow cytometry analysis. The detection of cytokine in culture supernatants by ELISA reflects the secretion by a heterogeneous population of cells and is affected by modifications of the half-life of cytokine or its self-consummation by DCs (such as IL-10, IL-12p70, and TNF-{alpha}). Our results showed that LPS and live S. typhimurium induced a significant production of IL-12p40/p70 and TNF-{alpha} by DCs (Fig. 4 ). We did not observe any anti-IL-10 staining, despite having tested several incubation times with brefeldin A (data not shown). We observed a slight increase of the MFI of IL-12p40/p70-positive cells at 39°C compared with 37°C and a decrease of the MFI of TNF-{alpha}-positive cells, which was significantly higher at 40°C than at 39°C. However, the percentage of cytokine-positive cells was not affected by the temperature rise. Taken together, these results suggest that fever regulates cytokine secretion at the single-cell level by modulating the production of cytokine.



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  		Figure 4. Production of IL-12p40/p70 and TNF-{alpha} is regulated at the single-cell level under fever-like conditions. IL-12p40/p70 and TNF-{alpha} productions were followed after LPS (A) or after S. typhimurium stimulation (B) with intracellular staining. The nonshaded, light-gray histograms represent isotype fluorescence of DCs. The nonshaded, black histograms represent the fluorescence of specific markers after incubation at 37°C. The filled gray and black histograms represent, respectively, the fluorescence of DCs incubated at 39°C and 40°C for 3 h and at 37°C for the remaining time. Values of mean fluorescence intensities (MFI) measured over surface indicated by horizontal bars are shown. Results are representative of three different experiments.

 
Role of IL-10 and TNF-{alpha} secretion in IL-12p70 regulation under fever-like conditions
We performed additional tests to see whether the temperature regulation of IL-12p70 secretion depends on an autocrine or paracrine secretion of IL-10 and TNF-{alpha}. It has been shown that IL-12 production after LPS stimulation can be suppressed by IL-10 [23 , 24 ] or TNF-{alpha} [25 , 26 ]. In accordance with a previous report [24 ], we determined that incubation of LPS-maturing DCs with anti-IL-10 mAb induced a higher secretion of IL-12p70 than incubation with an irrelevant mAb (Fig. 5A ). However, the pattern of secretion of IL-12p70 at 37°C, 39°C, or 40°C is unchanged in the presence of anti-IL-10 mAb. These results suggest that even if autocrine IL-10 regulates part of the IL-12p70 secretion, it does not interfere with the increase of IL-12p70 secretion at elevated temperatures. We observed that simultaneous incubation of maturing DCs with anti-TNF-{alpha} antibodies inhibited the secretion of IL-12p70 (Fig. 5B) . However, IL-12p70 secretion was still significantly higher at 39°C than at 37°C. These results suggest that the regulation of IL-12p70 at fever-like thermal conditions did not depend on the TNF-{alpha} decrease. IL-12p70 levels were not controlled by an autocrine or paracrine kinetic cascade dependent on TNF-{alpha} or IL-10. The secretion of IL-10, TNF-{alpha}, and IL-12p70 at elevated temperatures could be controlled independently for each cytokine.



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  		Figure 5. Regulation of IL-12p70 production at fever-like thermal conditions does not depend on autocrine secretion of IL-10 or TNF-{alpha}. IL-12p70 levels were measured in (A) IL-10- or (B) TNF-{alpha}-neutralizing conditions after 24 h of LPS stimulation at 37°C (open bars), 39°C (gray bars), or 40°C (solid bars) for 3 h and at 37°C for the remaining time. Data represent mean cytokine levels of culture supernatants (±SD) representative of three independent experiments. *, P < 0.05 as compared with 37°C.

 
Modulations of immunostimulatory capacity of maturing DCs
To further analyze whether the modulation of cytokine secretion could influence DC functions, we evaluated and compared the immunostimulatory capacity of DCs using an allogeneic MLR. As the modification of cytokine secretion kinetics started already after 8 h, we compared the effects of fever-like thermal conditions on maturing DCs after 6 h of incubation with LPS. Immature DCs exposed at 37°C, 39°C, or 40°C were equally capable of inducing proliferation of MHC-mismatched responder lymphocytes (Fig. 6A ). Conversely, incubation of maturing DCs at 39°C significantly enhanced their ability to stimulate allogeneic T cells, whereas at 40°C, there were no differences (Fig. 6B) . The modification of the ability of DCs to activate MLR is not consistent with modification of molecules expressed at the cell surface but rather with modification of cytokines released in the culture supernatants.



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  		Figure 6. Differential effects of fever-like conditions on stimulatory ability of DCs. (A) Immature DCs and (B) DCs incubated 6 h with LPS were assessed for their ability to induce an allogeneic MLR. [3H] Uptake was measured after 5 days of culture. Data represent mean of cpm of triplicate wells with DCs incubated at 37°C (open squares), 39°C (gray squares), or 40°C (solid squares) for 3 h and at 37°C for the remaining time. Results shown are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The concept of danger signals was postulated as part of an immune-response model [27 ], which suggests that the immune system responds to substances released after damage, rather than to those that are simply foreign [28 ]. In accordance with this model, several recent studies focusing on DCs have reported that molecular structures such as necrotic cells or heat shock proteins (hsp) that induce DC maturation can be perceived as endogenous danger signals [29 30 31 32 ]. Until now, the nature of these danger signals has been limited to molecular structures [12 ]. Our study demonstrates that fever-like conditions do not trigger cytokine secretion of immature DCs nor their phenotypical maturation but regulate the cytokine secretions of maturing DCs. Within the limitations of an in vitro experimental system, our results favor the idea that modifications of the physical environment, such as temperature under fever-like conditions, can be integrated by maturing DCs as an endogenous danger co-signal.

Nonetheless, the effects of fever may be more complex than previously thought. First, the response of DCs to fever-like conditions is not linear, as we report a clear shift in cytokine secretion between 39°C and 40°C. At 39°C, the IL-12p70 secretion is greatly increased, and the IL-10 secretion is decreased. This regulatory mechanism may play a role in controlling the T-helper response toward a type 1 response (high secretion of IFN-{gamma} and IL-2) and in increasing the ability of DCs to induce a MLR. Indeed, IL-10 is known to decrease the overall functions of DCs in humans or in mice [23 , 33 ] by several mechanisms, including the alteration of class II processing of antigens [34 ], the inhibition of IL-12 secretion [24 ], or the generation of functional chemokine decoy receptors, which act in turn as molecular sinks for inflammatory chemokines [35 ]. At 40°C, the increase of IL-12p70 is less important and transient, whereas the decrease of IL-10 and TNF-{alpha} is more pronounced. Above 40°C, hsp are usually induced, and the decrease of the cytokine levels could reflect the well-documented ability of hsp to terminate general protein synthesis in the cell and/or to inhibit transcription of cytokine genes, as has been shown in macrophages for TNF-{alpha} [36 ]. In addition, several previous reports indicated that febrile core temperature is normally below 39°C, and high body temperatures above 40°C can clearly injure the central nervous system as well as other body systems [1 , 37 ]. Temperatures above 40°C may be detrimental to survival of the host; thereby DCs decrease their overall cytokine secretion at 40°C. Furthermore, the marked decrease of TNF-{alpha} secretion may limit the extent and the duration of an inflammatory process and therefore reduce the risk of collateral damage to the host tissues.

A fever signal may have an additional homeostatic role in limiting the release of TNF-{alpha}, which is also known as an endogenous pyrogen. Molecules that are endogenous pyrogens (IL-1ß, TNF-{alpha}, and IFNs) are also endogenous danger signals that directly activate DCs [12 ]. However, apart from their direct stimulatory properties on DCs, indirect pyrogenic effects of TNF-{alpha} on temperature regulation may in turn block the production of TNF-{alpha}. In this way, fever can be understood as a negative feedback process on the release of endogenous pyrogens such as TNF-{alpha}. This can be considered a neuroimmune mechanism for regulation of cytokine production.

Although in vitro studies cannot directly reproduce in vivo events, they may nonetheless provide considerable insight into the impact of temperature elevation on immune defenses. Fever may be a strong adaptive advantage of host survival via rapid selection of the most efficient mechanisms of immunity, thereby contributing to the effectiveness of the response. Nonetheless, the effects of fever-like conditions on DCs triggered by live S. typhimurium highlight an important clue with respect to the role of fever in bacterial infections: Fever may tune adaptive immunity through DC regulation.

Before the advent of antibiotics, "fever therapy" was regarded as a useful treatment of some infectious diseases [38 ]. It can be postulated that the regulatory effects of fever-like conditions on maturing DCs may account for this beneficial effect. In addition, exposure of DCs to fever-like conditions may be used to improve their use in immunotherapy.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from Délégation Générale de l’Armement, Paris, France (PEA 0816). We thank Sandrine Richard and Valérie Blanco for their excellent technical assistance. We also thank Roland Hellmann for improving the English text of the manuscript.

Received October 24, 2002; revised December 20, 2002; accepted January 16, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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