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Am J Physiol Regul Integr Comp Physiol 278: R157-R165, 2000;
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Vol. 278, Issue 1, R157-R165, January 2000

Role of hypothermia induced by tumor necrosis factor on apoptosis and function of inflammatory neutrophils in mice

Toshiko Mizuno1, Yukiko Kannan1, Midori Tokunaga1, Mitsuaki Moriyama1, Yasuo Kiso2,5, Ken Kusakabe2, Jyoji Yamate3, Kenichi Kiyomiya4, and Tsukasa Sugano1

1 Department of Veterinary Physiology, 2 Department of Veterinary Anatomy, 3 Department of Veterinary Pathology, and 4 Department of Toxicology, College of Agriculture, Osaka Prefecture University, Sakai 599-8531; and 5 Department of Veterinary Anatomy, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in body temperature and cell infiltration, mediated by cytokines including tumor necrosis factor-alpha (TNF-alpha ), occur during inflammation, but a role of body temperature on inflammatory responses remains obscure. Intraperitoneal injection of 10% casein to mice resulted in transient hypothermia followed by neutrophil accumulation in peritoneal cavities. Peritoneal TNF-alpha was rapidly raised, and pretreatment of mice with an anti-TNF-alpha antibody promoted temperature restoration and partially inhibited neutrophil accumulation. To investigate direct effects of body temperature on neutrophils, peritoneal or peripheral blood neutrophils were cultured at 35°C or 37°C with or without recombinant murine TNF-alpha (100 ng/ml) or a protein synthesis inhibitor cycloheximide (1 µg/ml). Significant inhibition of spontaneous and TNF-alpha -induced apoptosis was obtained at 35°C compared with 37°C, an effect that was not altered by the addition of cycloheximide. Moreover, phagocytic ability of peritoneal neutrophils was significantly enhanced by incubating them at the lower temperature. These results indicate that mild hypothermia induced by endogenous TNF-alpha has enhancing roles on neutrophil survival and function during peritoneal inflammation.

inflammation; body temperature; tumor necrosis factor-alpha


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INFLAMMATION IS A COMPLEX cellular and biochemical response to injurious stimuli and is regulated by an extensive network of cytokines. Above all, tumor necrosis factor-alpha (TNF-alpha ) is well known to be released immediately after an injury to local tissue and to play multiple roles in acute phase responses including recruitment of neutrophils and fever. Neutrophils primarily accumulate and play a pivotal role in host defense by phagocytosing and killing microbes or scavenging irritants but are relatively short-lived and die by apoptosis. TNF-alpha is known to stimulate neutrophil adhesion to endothelial cells (11, 19) and to enhance phagocytosis and superoxide production of neutrophils (1, 28). TNF-alpha , on the other hand, has been reported to promote neutrophil apoptosis (31, 39, 41, 42, 46).

Fever is currently thought to be caused by endogenous pyrogens released by macrophages and accessory cells during injury and inflammation, and TNF-alpha as well as interleukin (IL)-1 and -6 are regarded as pyrogens (22). Actually, there are several reports in which the injection of TNF-alpha produced fever in human and experimental animals (30, 37). In addition, polyclonal and monoclonal antibodies against TNF-alpha (anti-TNF) have been found to attenuate lipopolysaccharide (LPS)- and turpentine-induced fever (4, 18, 29). However, Long et al. (26) have shown opposite results, that is, anti-TNF further enhanced LPS-induced fever, and Klir et al. (21) have reported that intraperitoneal injection of human recombinant TNF-alpha attenuated LPS-induced fever in rats. Moreover, hypothermia was observed immediately following injection of relatively high concentrations of LPS in mice and rats, and anti-TNF and TNF-alpha -soluble receptor prevented this (6, 23). Furthermore, Leon et al. (24) just recently reported that TNF p55/p75-receptor (TNFR) knockout mice attenuated hypothermia induced by cecal ligation and puncture (CLP). These results suggest a possible role for TNF-alpha not only as a pyrogen but also as an antipyretic factor (cryogen) to limit the magnitude of fever or more actively lower body temperature.

As to a physiological role of body temperature on neutrophil function, it has been generally reported that mild hyperthermia (up to 40°C) activates neutrophil function, but severe hyperthermia (more than 42°C) or hypothermia inhibits it (15, 44, 45, 47, 48). However, most of the results were obtained from in vivo experiments in which body temperature was changed physically or chemically or in vitro experiments using peripheral blood neutrophils, some of whose physiological characteristics are known to be different from those of inflammatory or tissue neutrophils (2, 31, 49). Thus the present study was performed to determine whether a physiological correlation between neutrophil accumulation and changes in body temperature exists and the role of endogenous TNF-alpha in this correlation during acute inflammation by using mice injected intraperitoneally with casein. Moreover, to investigate a possible role of changes in body temperature on neutrophil function at an inflammatory site, we studied the effect of different incubation temperatures within a physiological range on apoptosis of neutrophils isolated from the casein-injected peritoneal cavity and from normal peripheral blood and on phagocytic activity of peritoneal neutrophils. Most of all, this is the first report demonstrating that physiological temperature changes directly alter the progress of neutrophil apoptosis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. Albino ICR mice (from Japan SLC, Hamamatsu, Japan) were raised in our laboratory, and male mice aged 2-3 mo were used in this study.

Measurement of body temperature. Rectal temperature was measured using an electric thermometer with a probe (Muromachi Kikai, Tokyo, Japan) every 1 h after mice were injected intraperitoneally with 2 ml of various doses of LPS-free sodium caseinate (casein; Wako Pure Chemical Industries, Osaka, Japan) in saline warmed to 37°C. A mouse was put on a metal cover of a cage, and the tail was gently held, then the probe was inserted 1.7 cm into the rectum. Experiments were conducted after mice were trained for several days. In some experiments, mice were preinjected with 1 ml polyclonal rabbit anti-mouse TNF-alpha (Genzyme, Cambridge, MA) diluted 50-fold in saline and warmed to 37°C 2 h before the injection of casein.

Isolation of peritoneal exudate cells. Isolation of peritoneal exudate cells (PEC) after casein injection was carried out by techniques previously described (16, 17). Briefly, mice were killed by decapitation 5 h after intraperitoneal injection of 10% casein, and PECs were collected by washing the peritoneum with MEM (Gibco BRL Life Technologies, Rockville, MD) containing 1% heparin. PECs, in which the population of neutrophils was greater than 80%, estimated by Giemsa staining, were used for the cultures.

Isolation of peripheral blood neutrophils. Heparinized peripheral blood was obtained from intact mice by cardiac puncture under pentobarbital sodium anesthesia (0.1 mg/100 g body wt). Neutrophils were purified by a method described by Tsuchida et al. (42). Briefly, the heparinized blood was mixed with an equal volume of 37°C plasma gel that had been prepared as follows. Three grams of gelatin, 0.7 g of NaCl, and 0.2 g of CaCl2 were dissolved in 100 ml distilled water at 60°C and stored at 4°C. The mixture of blood and plasma gel was allowed to stand until erythrocytes were settled. The leukocyte-rich supernatant was transferred to an another tube and washed with MEM. The pellet was suspended in 5 ml MEM and overlayered onto the same volume of Ficoll-Paque (sp. gr. 1.077; Amersham Pharmacia Biotech, Uppsala, Sweden), then centrifuged at 800 g for 10 min at 4°C. The pellet containing neutrophils and erythrocytes was allowed to lyse the erythrocytes with hypotonic NaCl and was washed with MEM twice. The purity of neutrophils was more than 75% (the remaining cells were mostly lymphocytes).

Cell culture. PECs were cultured in 24-well tissue culture plates (Becton Dickinson, Tokyo, Japan). Each well contained 106 PECs in 1 ml alpha MEM (GIBCO) supplemented with 1% newborn calf serum (NCS; GIBCO) with or without 100 ng/ml recombinant mouse TNF-alpha (rmTNF-alpha ; Pepro Tech, Rocky Hill, NJ) or 1 µg/ml cycloheximide (CHX; Wako). Peripheral blood neutrophils were cultured at 105 cells/100 µl in each well of 96-well plates (Becton Dickinson). Plates were incubated at 35°C or 37°C in a fully humidified atmosphere of 5% CO2 in air.

Examination of apoptosis. Morphological examination of apoptosis was performed by using Giemsa staining. Briefly, cells were spun in a cytocentrifuge (Cytospin; Shandon Scientific, Cheshire, Wales, UK) and were fixed in methanol followed by staining for 30 min with 10-times diluted Giemsa solution. Giemsa-stained preparations were observed under light microscopy (×400), and percentages of apoptotic neutrophils were estimated from 200 Giemsa-stained neutrophils per slide (16). Neutrophil apoptosis was also confirmed by staining with 1 µg/ml DNA-specific fluorochrome Hoechst 33258 under fluorescent microscopy.

Electrophoresis of DNA. Isolation and electrophoresis of PEC DNA were carried out by a modification of a technique described by Ishizawa et al. (14). Briefly, 5 × 105 PECs, cultured with or without rmTNF-alpha at 35°C or 37°C, were suspended in 200 µl PBS in a 1.5-ml microfuge tube. Ten microliters proteinase K (10 mg/ml; Sigma Chemical, St. Louis, MO), 10 µl RNase (10 mg/ml ribonuclease A; Sigma), and 50 µl 5% SDS were added and incubated at 37°C for 30 min. After addition of 300 µl NaI solution and incubation at 60°C for 15 min, 500 µl 100% isopropanol (Wako) were added, then vigorously shaken, and allowed to stand for 15 min at room temperature. The NaI solution contained 6 M sodium iodide (Wako), 13 mM EDTA (EDTA-Na, Wako), 0.5% sodium-N-lauroylsarcosinate (Wako), 10 mg/ml glycogen (Wako), and 26 mM Tris-HCl (pH 8, Wako). A tube was centrifuged at 15,000 g for 15 min to precipitate DNA, the supernatant was discarded, 1 ml 50% isopropanol was added, and the mixture was centrifuged at 15,000 g for 15 min. After the supernatant was discarded, 1 ml 100% isopropanol was added and centrifuged at 15,000 g for 15 min. The DNA precipitate was vacuum-dried and dissolved in 50 µl Tris-EDTA (TE) buffer at 4°C. TE buffer contained 100 µl 40 mM Tris-HCl, 10 µl 1 mM EDTA, and 890 µl ultrapurified water. Twenty five microliters of each sample were mixed with 2 µl loading buffer (0.25% bromophenol blue and 40% sucrose) and loaded into each well of a 2% agarose gel containing 1 µg/ml ethidium bromide. As a molecular size standard of DNA, 1 µl marker solution (Maker 4, phi ×174/Hae digest; Wako) was mixed with 24 µl TE buffer. Electrophoresis was carried out at 100 V, 80 mA, until the marker dye had migrated 3-4 cm.

TNF-alpha bioassay. TNF-alpha in a peritoneal exudate fluid (PEF) was measured using a modification of a bioassay technique previously described (9). PEFs were frozen at -80°C until used. Assays were performed using TNF-alpha -sensitive L929 tumorigenic murine fibroblast cell line. L929 cells were seeded at 104 cells/well in 100 µl DMEM (GIBCO) containing 10% NCS in a 96-well flat-bottom microtiter plate (Becton Dickinson) and incubated for 12 h in 5% CO2 atmosphere at 37°C. The medium was discarded and replaced with 100 µl PEF solution diluted 40- to 80-fold by MEM containing 5% NCS. To construct a dose-response curve, 100 µl of the serial dilutions of rmTNF-alpha were added. One hundred microliters actinomycin D (final concentration 1 mg/ml; Wako) were also added, and a plate was incubated for 18 h. Supernatant was removed, and cells were treated with 40 µl 2% crystal violet (in 25 mM methanol) for 20 min. After 100 µl 0.5% SDS were added, absorbance was measured at 570 nm on Immuno Reader NJ-2001 (Nippon InterMed, Tokyo, Japan). TNF-alpha values for PEF samples were determined from a standard curve generated using rmTNF-alpha .

Phagocytosis assay. Phagocytosis was assessed using a modified technique previously described (17). PECs were suspended at a concentration of 2 × 105 cells/ml in alpha MEM containing 1% NCS. Hydrophilic microsphere lumispheres (2-µm diameter; Toray Research Center, Tokyo, Japan) were suspended at 2 × 108 microspheres/ml in PBS. Ten microliters rmTNF-alpha (100 ng/ml) or MEM were mixed with 90 µl cell suspension and 10 µl lumisphere solution. The mixture was incubated for 20 min at 35°C or 37°C. To stop the phagocytic response, 1 ml cold PBS was added. After centrifugation at 1,500 rpm for 5 min, 10 µl fuchsin solution diluted 10-fold were added to the pellet. Extracellular lumispheres were stained red with fuchsin, but they were transparent when phagocytosed. Under light microscopy (×400), the percentages of neutrophils phagocytosing more than one lumisphere were estimated from a total of 200 neutrophils.

Statistical analysis. ANOVA followed by Fisher's post hoc test (protected least-squares difference) was performed for multiple comparison.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell influx and body temperature changes induced by intraperitoneal injection of casein. Figure 1A shows the time course of the values of total PECs and peritoneal neutrophils after injection of a 10% casein solution. To eliminate the influence of circadian rhythms on leukocyte function (13, 40), PECs were collected between 1200 and 1500. PEC values increased rapidly, and a maximum response was observed 15 h after injection. At 5 h after the injection, PECs contained mostly neutrophils (81.8 ± 0.3%) but contained some of both neutrophils (63.5 ± 3.5%) and monocytes (33.8 ± 2.3%) at 15 h.


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  		Fig. 1.   Peritoneal exudate cell (PEC) and neutrophil numbers (A) and body temperatures (B) after intraperitoneal injection of 10% casein into mice. Collection of PECs and measurement of rectal temperature were performed just before injection (0 h) and 1, 2, 5, 10, 15, 24, and 48 h after the injection. Each value is a mean ± SE (n = 2-7). * P < 0.05, ** P < 0.01 compared with 0 h, and # P < 0.01 compared with saline by ANOVA/Fisher's protected least-squares difference (PLSD).

Body temperature fell suddenly after injection of 10% casein (Fig. 1B). Just before the casein injection, the average rectal temperature of mice was 38.0 ± 0.1°C and fell to 34.8 ± 0.6°C 1 h after the injection. On the other hand, saline injection did not cause rectal temperature to fall after 1 h (37.5 ± 0.4°C and 38.2 ± 0.5°C, before and 1 h after the injection, respectively). Until 3 h, temperature was still significantly lower in the casein-injected mice compared with saline-injected mice or with 0 h, but it recovered afterwards.

We examined the effects of 2% and 5% casein in addition to 10% casein, but no significant differences of body temperature from saline injection were observed nor was there neutrophil accumulation until 5 h after the smaller doses of casein injection (data not shown).

From these results, we deduced that intraperitoneal injection of 10% casein caused a rapid decrease in mouse body temperature and neutrophil accumulation during the restoration of body temperature.

TNF-alpha levels in PEFs after casein injection. PEFs were drawn 1 and 5 h after injection of saline or 2, 5, and 10% casein, and TNF-alpha bioactivity was assessed (Table 1). A dramatic elevation of TNF-alpha concentration was observed 1 h after 10% casein injection (632.83 ± 257.90 ng/ml). At 5 h, the TNF-alpha concentration had decreased but was still relatively high (92.53 ± 34.69 ng/ml). Casein at 2% and 5% did not cause significant release of TNF-alpha at 1 and 5 h after injection.

                              
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  		Table 1.   TNF-alpha activity in PEF after injection of various doses of casein

To investigate the roles of internally elevated TNF-alpha on the changes in body temperature and neutrophil infiltration, anti-TNF was applied 2 h before injection of 10% casein (Fig. 2A). Complete blockade of the TNF-alpha activity in the PEF sample withdrawn 1 h after 10% casein injection was confirmed by anti-TNF treatment using the above-mentioned bioassay (data not shown). Anti-TNF itself had no effect on body temperature before injection of casein. Also, body temperature decreased 1 h after the injection of casein in the presence of anti-TNF to the same extent as in the presence of saline (-2.82 ± 0.50°C vs. -2.85 ± 0.76°C from 0 h). However, anti-TNF promoted restoration of body temperature, that is, significantly elevated body temperature 2 h after casein injection, whereas it takes 4 h to significantly recover temperature without anti-TNF pretreatment.


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  		Fig. 2.   Effects of pretreatment of anti-tumor necrosis factor (TNF) antibody on changes in body temperature (A) and peritoneal neutrophil numbers (B) after injection of 10% casein. Anti-TNF diluted 50-fold in saline or only saline was intraperitoneally injected 2 h before injection of 10% casein or saline. Changes in body temperature indicate difference of temperatures based on 0 h. Each value is a mean ± SE (n = 4-15). * P < 0.01 compared with 1 h and # P < 0.01 compared with saline + casein by ANOVA/Fisher's PLSD. PECs were collected 5 h after injection of casein (or saline as a control), and number of neutrophils was counted. * P < 0.05 and ** P < 0.01 compared with saline + casein by ANOVA/Fisher's PLSD.

Anti-TNF pretreatment also partially inhibited neutrophil infiltration caused by injection of 10% casein (Fig. 2B). The number of peritoneal neutrophils was 9.24 (±1.76) × 106 cells in anti-TNF pretreatment and 19.81 (±3.09) × 106 cells in saline pretreatment 5 h after casein injection. In the case of anti-TNF pretreatment followed by saline injection, the number of peritoneal neutrophils was 2.03 (±0.29) × 106 cells.

It was therefore found that TNF-alpha , which was rapidly released into the peritoneal cavity by casein injection, not only enhanced neutrophil infiltration but also delayed recovery of decreased body temperature.

Effect of incubating temperature on apoptosis of neutrophils in vitro. We next examined the direct effect of changes in temperature within a physiological range on apoptosis of neutrophils. PECs collected 5 h after injection of 10% casein were incubated at 37°C or 35°C for up to 24 h in medium with or without the addition of 100 ng/ml rmTNF-alpha and stained with Giemsa. Apoptosis was determined by typical morphological features such as condensed nuclei, vacuolated cytoplasm, and some apoptotic bodies as described previously (16). Morphological features of apoptotic nuclei were also confirmed by Hoechst 33258 staining (data not shown). As shown in Fig. 3A, the population of apoptotic neutrophils was increased more rapidly at 37°C (on and after 6 h) than at 35°C (on and after 18 h) in both the presence and the absence of TNF-alpha . TNF-alpha accelerated ongoing apoptosis at 37°C after 18 h, but not at 35°C until 24 h. We also cultured PECs at 39°C, and a significant increase of apoptotic neutrophils was obtained during 3-12 h in culture compared with 37°C (data not shown).


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  		Fig. 3.   Effect of incubation temperature on apoptosis of peritoneal neutrophils (A) and peripheral blood neutrophils (B) in vitro. A: PECs were collected 5 h after injection of 10% casein and were cultured for 6, 12, 18, or 24 h in presence (closed symbols) or absence (open symbols) of 100 ng/ml recombinant mouse (rm) TNF-alpha at 37°C or 35°C. Proportion of apoptotic neutrophils was assessed by morphological observation of Giemsa-stained preparations under light microscopy. Each value is a mean ± SE (n = 13-21). * P < 0.01 compared with 0 h, # P < 0.01 compared with 37°C control and 35°C TNF-alpha at 18 h, and § P < 0.01 compared with 35°C control by ANOVA/Fisher's PLSD. B: blood neutrophils were cultured for 18 h. * P < 0.05 and ** P < 0.01 by ANOVA/Fisher's PLSD.

For biochemical estimation of apoptosis, DNA was isolated from PECs cultured for 3 and 10 h at 37°C or 35°C and electrophoresed by agarose gel. As shown in Fig. 4, the ladder with ~200-bp typical steps of endonuclease activation did not appear in DNA of PEC cultured for 3 h at both 37°C and 35°C (lanes 1-4), regardless of the presence or the absence of rmTNF-alpha . However, typical ladders were observed in DNA of PEC cultured for 10 h at 37°C in which the density of the ladder was greater in DNA of PEC cultured with TNF-alpha (lane 6) than without TNF-alpha (lane 5). On the other hand, at 35°C, distinct ladders were barely visible both in culture with or without TNF-alpha (lanes 7 and 8).


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  		Fig. 4.   Agarose gel electrophoresis of DNA isolated from 5 × 105 PECs cultured at 35°C and 37°C. Lanes 1-4, DNA cultured for 3 h; lanes 5-8, DNA cultured for 10 h. Lanes 1 and 5, DNA cultured at 37°C in control medium; lanes 2 and 6, DNA cultured at 37°C in medium containing 100 ng/ml rmTNF-alpha ; lanes 3 and 7, DNA cultured at 35°C in control medium; lanes 4 and 8, DNA cultured at 35°C in medium containing rmTNF-alpha . Lane M, a molecular size standard (phi ×174/Hae digest, Maker 4). Molecular sizes in bp are indicated on left for gel. Similar results were obtained from 3 experiments.

These results indicate that apoptosis of peritoneal neutrophils was delayed as temperature of culture was lowered in a physiological range and that the lower temperature also disturbed TNF-alpha -promoted neutrophil apoptosis.

To further examine whether lower temperature inhibition of apoptosis is restricted to inflammatory neutrophils or not, we next assessed apoptosis of neutrophils isolated from peripheral blood of intact mice. Equal to the peritoneal neutophils, incubation for 18 h at 35°C decreased both spontaneous and TNF-induced apoptosis of blood neutrophils compared with 37°C (Fig. 3B), thus suggesting that lower temperature suppresses neutrophil apoptosis in a neutrophil activation- or localization-independent manner.

Role of protein synthesis in temperature-dependent inhibition of neutrophil apoptosis. To understand the mechanism by which a lower temperature suppresses neutrophil apoptosis, 1 µg/ml CHX, a protein synthesis inhibitor, was added to the PEC culture medium, which was then cultured for 18 h at 37°C or 35°C (Fig. 5). Significant promotion of neutrophil apoptosis was observed by the addition of CHX at 37°C (36.40 ± 3.46% vs. 23.85 ± 2.73% with or without CHX), but not at 35°C. This suggests that the neutrophils synthesized apoptosis inhibitory protein during culture at 37°C, but the lowered temperature (35°C) suppressed apoptosis in a protein synthesis-independent manner.


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  		Fig. 5.   Effect of protein synthesis inhibitor on temperature-dependent apoptosis of peritoneal neutrophils. PECs were cultured for 18 h with 1 µg/ml cycloheximide (CHX) at 37°C or 35°C. Proportion of apoptotic neutrophils was assessed by morphological observation of Giemsa-stained preparations. Each value is a mean ± SE (n = 10). * P < 0.01 compared with 37°C control and 35°C CHX by ANOVA/Fisher's PLSD.

Effect of incubating temperature on phagocytosis of peritoneal neutrophils. Finally, to examine whether changes in temperature regulate not only neutrophil apoptosis but also its phagocytic function, PECs (2 × 105/ml) were incubated with lumispheres (2 × 106/ml) in the presence or absence of 100 ng/ml rmTNF-alpha at 37°C or 35°C for 20 min. As shown in Table 2, in the absence of TNF-alpha , the percentage of lumisphere-phagocytosing cells was significantly higher at 35°C than at 37°C. We also counted the number of lumispheres per neutrophil, and the percentage of neutrophils phagocytosing more than four particles was significantly higher at 35°C than at 37°C (data not shown). Addition of TNF-alpha , although not significantly, tended to increase the phagocytosis by the same extents (~1.1 times controls) at 37°C and 35°C. Accordingly, the lower temperature was found to be beneficial in the promotion of phagocytosis of peritoneal neutrophils.

                              
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  		Table 2.   Effect of incubation temperature and rmTNF-alpha on peritoneal neutrophil phagocytosis (%)


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrated that intraperitoneal injection of 10% casein into mice induced a rapid decrease of rectal temperature, which reached a maximum at 1 h after injection, followed by gradual restoration by 15 h postinjection (Fig. 1B). During temperature restoration, neutrophil infiltration into the peritoneal cavity occurred and the population of neutrophils in total PECs reached a maximum after 5 h (Fig. 1A). Kitamura et al. (20) demonstrated the occurrence of a short-lasting fever after injecting 0.2% casein into rabbits that caused the rectal temperature to increase for 90 min after injection then peak at ~5 h (+0.8 ± 1.0 °C compared with a baseline) and finally decreased to the control level by 12-24 h. Neutrophil accumulation, in their experiments, was reported to reach a peak after 5 h, coinciding with our results. There are many reports indicating that LPS administration induced mono- or biphasic fever in various animals (22, 33). However, it has been recently reported that larger doses of LPS induced hypothermia and shock, whereas smaller doses caused fever in rats and mice (34). Therefore, we examined the effects of 2% and 5% casein in addition to 10% casein, but hyperthermia was not detected by the two smaller doses of casein (data not shown). In LPS-induced hypothermia, TNF-alpha has been considered to function as a cryogen, although it was originally thought to be a member of the endogenous pyrogens, because pretreatment of animals with neutralizing anti-TNF antibody before LPS injection resulted in enhancement of fever (26) or suppression of hypothermia (6). Moreover, intraperitoneal injection of TNF-alpha has been reported to show an antipyretic effect on febrile response to LPS (21). Attenuation of hypothermia was also observed in TNFR knockout mice in response to CLP, another model of sepsis (24). In our study, 10% casein induced a dramatic rise in intraperitoneal TNF-alpha level during the first hour, and a considerable amount of TNF-alpha was retained 5 h after the injection (Table 1). Although anti-TNF pretreatment 2 h before 10% casein injection did not block the casein-induced initial decline of body temperature, it significantly promoted the restoration of temperature to control values (Fig. 2A), indicating that endogenous TNF-alpha released by casein injection limited the increase in temperature during convalescence. It has been demonstrated that LPS-induced hypothermia occurred as a result of reduced thermogenesis (7), and Holt et al. (12) have reported that intracerebral injection of recombinant TNF-alpha decreased the activity of sympathetic efferent nerves to intercapular brown adipose tissue, a main effector of nonshivering thermogenesis. It should be clarified whether TNF-retained hypothermia in our inflammatory model occurred by the similar mechanism in a future study. The primary inducer of the hypothermia after casein injection is still uncertain, but other endogenous cryogens such as alpha -melanocyte-stimulating hormone, arginine-vasopressin, glucocorticoid, leukotrienes, or prostaglandin D2 might be involved in it (5, 6, 25, 27, 43).

Preinjection of anti-TNF partially decreased the 10% casein-induced neutrophil accumulation (Fig. 2B), implying that TNF-alpha is involved in the stimulation of neutrophils in the interstitial space during inflammation. Such involvement of TNF-alpha in neutrophil infiltration was also reported by others using immune complex-induced inflammation of the murine peritoneum (50). This effect of TNF-alpha is considered to be due to the stimulation of neutrophil-endothelial interaction, which is a pivotal event in neutrophil emigration from the intravascular to inflammatory sites (11, 19).

Neutrophils exudated to local tissue are destined to undergo apoptosis followed by recognition and phagocytosis by tissue macrophages (35). Therefore, suppression of apoptosis contributes to maintenance of neutrophil function, and many proinflammatory cytokines not only enhance neutrophil function but also suppress the apoptosis (3, 10, 28, 39). TNF-alpha , however, has been reported to promote neutrophil apoptosis (31, 39, 41, 42, 46) while enhancing the function (1, 28). Because changes in body temperature are considered to alter immune/inflammatory responses (22), we questioned whether they affect neutrophil apoptosis. When PECs isolated from peritoneal cavities 5 h after 10% casein injection were incubated at 35, 37, or 39°C, significant decrease of neutrophil apoptosis was observed at 35°C compared with 37°C (Fig. 3A) and at 37°C compared with 39°C (data not shown). Addition of 100 ng/ml rmTNF-alpha , which was the approximate concentration in PEFs (5 h after the casein injection), showed acceleration of neutrophil apoptosis when cultured at 37°C, but not at 35°C (Fig. 3A). Because the peritoneal neutrophils had already been exposed by various inflammatory mediators, some of which promote apoptosis, whereas others antagonize apoptosis, as stated above, we next did the same in vitro experiment by using nonactivated neutrophils isolated from peripheral blood of intact mice (Fig. 3B). Similar to the results of the peritoneal neutrophils, spontaneous and TNF-alpha -induced apoptosis of blood neutrophils were suppressed by the lower temperature, suggesting that apoptotic response of neutrophils to temperature is in an inflammatory activation- or neutrophil localization-independent manner. As we know, this is the first report that temperature (within a physiological range) affects neutrophil apoptosis, except for a report on heat shock-induced apoptosis (46).

We then asked how body temperature regulates neutrophil apoptosis. Neutrophils have been considered to synthesize apoptosis-inhibitory proteins, because addition of a protein synthesis inhibitor CHX to a culture medium of neutrophils has been reported to accelerate neutrophil apoptosis (31, 42). Thus we examined the involvement of protein synthesis in the lower temperature-induced suppression of neutrophil apoptosis by incubating PECs for 18 h with or without CHX. CHX apparently accelerated apoptosis of neutrophils at 37°C, but apoptosis was not significantly changed for the control in the absence of CHX compared with CHX at 35°C (Fig. 5), suggesting that suppression of apoptosis at 35°C was caused in a protein synthesis-independent manner. Tsuchida et al. (42) reported that during 3 h incubation at 37°C, CHX did not enhance apoptosis of rat peritoneal exudate neutrophils stimulated by proteose peptone injection. The discrepancy between their and our results on CHX action might be due to the difference of incubation time (3 vs. 18 h). Addition of CHX as well as TNF-alpha has been found to increase the concentration of ceramide and its catabolite, sphingosine, a potent endogenous protein kinase C inhibitor in neutrophils and other cells, and these second messengers are suggested to play critical roles in processes of apoptosis (32). Because the rate of sphingosine formation has been demonstrated to increase with a rise of temperature (from 30°C to 37°C) (36), the lower temperature suppression of apoptosis might involve the interruption of sphingosine formation.

Finally, we examined the effect of lower temperature on phagocytic function of the peritoneal neutrophils. A significant increase in lumisphere-ingesting neutrophils was obtained at 35°C, compared with 37°C (Table 2). TNF-alpha tended to slightly increase the percentage at both 37°C and 35°C. The effect of temperature on phagocytosis was examined earlier (15, 44, 45). Johansen et al. (15) reported that a moderate rise of temperature enhanced phagocytosis of some (but not all) kinds of bacterial organisms by human peripheral blood neutrophils, whereas Utoh and Harasaki (44) showed no change of phagocytosis of serum-opsonized latex particles by human and calf blood neutrophils below 42°C. The diverse results might be caused by obscurity in distinguishing phagocytosis from just attachment of the particles or organisms to cells. With the use of hydrophilic microsphere lumispheres, we recognized phagocytosis more easily and accurately by the difference in staining nature of the particles between intracellular and extracellular sites. As a possible mechanism by which neutrophil phagocytosis is upregulated by the lower temperature, less production of ceramide might be again related, because ceramide has been reported to inhibit neutrophil phagocytosis (38).

Taken together, we conclude that a rapid hypothermia occurs followed by neutrophil infiltration into peritoneum after injection of inflammatory stimuli in mice. Endogenously released TNF-alpha is suggested to regulate not only neutrophil infiltration, but also body temperature to maintain it slightly lower during neutrophil accumulation and function. Lowered body temperature would suppress neutrophil apoptosis without protein synthesis and upregulate neutrophil phagocytic function both by its own effect and by negating the undesirable effect of TNF-alpha on neutrophils. Further studies are essential to investigate the effect of body temperature on other neutrophil function, such as superoxide and cytokine production, and the mechanisms for how temperature regulates neutrophil apoptosis and function.

Perspectives

As to the temperature effect on neutrophil function other than phagocytosis, previous studies have shown that a moderate (up to 40°C) rise of temperature results in rapid neutrophil migration; the opposite results were obtained with a hypothermic situation (45, 48). Because neutrophil accumulation, in our study, occurred during restoration period of hypothermia, elevation of temperature may be involved in enhancement of neutrophil infiltration into a casein-injected site. However, when anti-TNF was applied prior to casein injection, the number of peritoneal neutrophils was decreased despite a slight increase of body temperature. Thus it is so far uncertain to what extent the changes of temperature actually influenced neutrophil infiltration in our inflammatory model.

Ensor et al. (8) have reported that high incubation temperature in a physiological range downregulates the expression of TNF-alpha in LPS-stimulated human macrophages derived from peripheral blood monocytes in vitro. Thus cytokine production of neutrophils might also be modulated by temperature. Moreover, on the basis of our data, it can be speculated that TNF-alpha may prevent a suspension of its own production by modulating a rise of body temperature in inflammation.

To investigate the temperature-induced intracellular events concerned in apoptosis or activation of neutrophils, concentrations and/or activity of ceramide, protein kinase C, or other second messengers should be assessed.


    ACKNOWLEDGEMENTS

We appreciate Dr. Azumi Hamasaki for kind instruction of neutrophil purification from blood and Drs. Tomoko Tajima and Shunji Kozaki for agreeable offers of their experimental equipment. We also thank Seiko Ishihara for technical help in the TNF-alpha bioassay.


    FOOTNOTES

This work was supported by the University-to-University Cooperative Research Program (08045058) by the Ministry of Education, Science, Sports, and Culture of Japan, Grant-in-Aid (09660324) for Scientific Research C by the Ministry of Education, Science, Sports, and Culture of Japan, and by the Naito Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: Yukiko Kannan, Dept. of Veterinary Physiology, College of Agriculture, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan (E-mail: kannan{at}vet.osakafu-u.ac.jp).

Received 26 January 1999; accepted in final form 11 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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