Cutting Edge: Fever-Associated Temperatures Enhance Neutrophil Responses to Lipopolysaccharide: A Potential Mechanism Involving Cell Metabolism 1

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Cutting Edge: Fever-Associated Temperatures Enhance Neutrophil Responses to Lipopolysaccharide: A Potential Mechanism Involving Cell Metabolism Allen J. Rosenspire, Andrei L. Kindzelskii and Howard R. Petty

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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J Immunol 2002; 169:5396-5400; ; doi: 10.4049/jimmunol.169.10.5396

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Cutting Edge: Fever-Associated Temperatures Enhance Neutrophil Responses to Lipopolysaccharide: A Potential Mechanism Involving Cell Metabolism1 Allen J. Rosenspire,2 Andrei L. Kindzelskii, and Howard R. Petty


ince antiquity it has been recognized that fever is associated with illness. Bacteria and their by-products trigger cytokine (e.g., TNF-␣) production by the host, leading to a rise in core body temperature (1, 2). Although the rise in temperature is the best known aspect of fever, it is perhaps the least understood. Several studies have suggested that a rise in core body temperature, independently of other variables, enhances host resistance to microbes. For example, perioperative hypothermia, a frequent complication of anesthesia and surgery, is associated with impaired mitogenic responses and an increased frequency of wound infections, which can be reversed when patient care includes forced-air warming to elevate core temperatures (3). It has

also been shown that increasing core temperature led to a reduced bacterial load and enhanced survival in a mouse bacterial peritonitis model. In the latter study, it was clear that the findings could not be the result of a direct temperature effect on bacterial proliferation, but rather reflected enhanced host defense (4). The first line of defense against invading microorganisms generally involves innate immunity, exemplified by neutrophil-mediated recognition and ingestion of bacterial and fungal pathogens. One key mechanism of microbial killing involves the production of reactive oxygen intermediates (ROIs)3 (5) and reactive nitrogen intermediates (RNI) (6). The production of ROI and RNI begin with the synthesis of superoxide and NO, respectively. Superoxide is produced by the NADPH oxidase according to the equation: 1/2 NADPH ⫹ O2 3 1/2 NADP⫹ 1/2 H⫹ O2. The NO synthase catalyzes the formation of NO according to the reactions: Larginine ⫹ NADPH ⫹ H⫹ ⫹ O2 3 NG-hydroxy-L-arginine ⫹ NADP⫹ ⫹H2O and NG-hydroxy-L-arginine ⫹ 1/2 NADPH ⫹ 1/2 H⫹ ⫹ O2 3 L-citrulline ⫹ 1/2 NADP⫹ ⫹ H2O ⫹ NO. Thus, ROI and RNI production are biochemically linked to NADPH. ROI, and RNI are individually cytotoxic, as they can damage pathogen membranes and DNA. However, their combined effect is more lethal than one would predict based upon an analysis of their individual properties due to the formation of additional reactive species (7). In this study, we have used new fluorescence techniques to analyze NAD(P)H, ROI, and NO production by individual neutrophils. We have found that both ROI and RNI production levels are temporally linked. Moreover, production is temperature dependent, and febrile temperatures serve to moderately increase background levels. Exposure of neutrophils to LPS predictably up-regulates ROI and RNI production. However, we demonstrate that febrile temperatures and LPS act together to greatly increase ROI/ RNI production, which suggests a physiological explanation for the evolution and utility of the thermal component of fever.

Department of Biological Sciences, Wayne State University, Detroit, MI 48202 Received for publication May 31, 2002. Accepted for publication September 23, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Materials and Methods Materials LPS (Escherichia coli serotype 026:B6) and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise.

1 This work has been supported by in part by the Fetzer Institute, the National Multiple Sclerosis Society and National Institutes of Health Grants AI51789 and ES11000. 2 Address correspondence and reprint requests to Dr. Allen J. Rosenspire, Department of Biological Sciences, Wayne State University, Detroit, MI 48202. E-mail address: [email protected]

Copyright © 2002 by The American Association of Immunologists, Inc.

3 Abbreviations used in this paper: ROI, reactive oxygen intermediate; RNI, reactive nitrogen intermediate; H2TMRos, dihydrotetramethylrosamine; TMRos, tetramethylrosamine; DAF-2DA, diaminofluorescein-2-diacetate.


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Although much progress has been made in elucidating the mechanisms underlying the physiological regulation of fever, there is little understanding of the biological utility of fever’s thermal component. Considering the evolutionary co-conservation of fever and innate immunity, we hypothesize that fever’s thermal component might in general augment innate immune function and, in particular, neutrophil activation. Accordingly, we have evaluated the effect of febrile temperatures on neutrophil function at the single-cell level. We find that reactive oxygen intermediates and NO release are greatly enhanced at febrile temperatures for unstimulated as well as LPS-stimulated adherent human neutrophils. Furthermore, our studies suggest that these changes in oxidant release are linked to upstream changes in NADPH dynamics. Inasmuch as reactive oxygen intermediates and NO production are important elements in innate immune responses to bacterial pathogens, we suggest that the febrile rise in core temperature is a broad-based systemic signaling mechanism to enhance innate immunity. The Journal of Immunology, 2002, 169: 5396 –5400.

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Cells Neutrophils were purified from the blood of healthy individuals by step gradient centrifugation over Histopaque 1077 and 1119 according to the manufacturer’s directions.

Microscopic assay for ROI release from adherent cells Pericellular release of ROI from single cells was monitored as described previously (8). Briefly, neutrophils suspended in HBSS (Life Technologies, Grand Island, NY), were allowed to adhere to a glass coverslip. HBSS containing 2% fluid-phase low melting gelatin and 100 ng/ml dihydrotetramethylrosamine (H2TMRos; Molecular Probes, Eugene, OR) at 45°C was placed on the coverslip. The gelatin on the coverslip was then allowed to quickly cool and gel. Next, the coverslip was mounted on a slide, which was in turn placed on a temperature-controlled microscope stage (Zeiss, New York, NY). At each temperature setting, ROI released by the cells diffused into and were trapped within the gelatin matrix, so that H2TMRos was oxidized to tetramethylrosamine (TMRos). After the slide and stage were equilibrated at the set temperature (⬃60 s), TMRos detection was begun by fluorescence microscopy.

Microscopic assay for NO release from adherent cells

Microscopy and fluorescence quantification Cells were individually illuminated and examined using an axiovert fluorescence microscope (Zeiss). Variable wavelength excitation illumination was provided by a xenon lamp coupled to a model 101 monochromator (Photon Technology International; Lawrenceville, NJ). NAD(P)H autofluorescence was excited using a wavelength of 365 nm and its emission was detected using a 405DF35 filter and a 405 long-pass dichroic mirror. TMRos fluorescence was excited at 540 nm and its emission was detected using a 590DF30 filter with a 560 long-pass dichroic mirror. DAF2DA fluorescence was studied using 485 nm for excitation and a 520EFLP filter with a 505 long-pass dichroic mirror for emission. The fluorescence intensity was measured and analyzed using a photomultiplier tube (Hamamastu, Bridgewater, NJ) housed in a model D104 fluorescence microscope detection system interfaced with a Pentium III computer running FeliX software (Photon Technology International).

Statistical analysis Statistical significance was assessed by one-way ANOVA analysis using Excel (Microsoft, Redmond, WA)

Results and Discussion We have recently developed sensitive fluorescence techniques that allow us to examine ROI and NO production in real time from single cells (10 –12). Polarized and adherent neutrophils produce large quantities of ROI and NO (8, 13, 14). Pericellular production of ROI and NO is not steady, but rather takes place in discrete uniformly timed bursts (8, 12), with an approximate 20-s period at 37°C. Using this method, we tested the hypothesis that temperature affects the rate of oxidant release. Fig. 1 shows representative examples of several neutrophils at different temperatures (n ⱖ 5 for each plot), where a qualitative analysis of the effect of temperature on NAD(P)H, NO, and ROI production is shown. Fluorescence intensity (in arbitrary units) is proportional to NAD(P)H, NO, or ROI as indicated, and is plotted as a function of time for each cell. At each temperature examined, NAD(P)H fluorescence exhibited an approximate sinusoidal pattern, while NO- and ROI-dependent fluorescence exhibited a “stepped” pattern, with each step interpretable as an additional production burst (15). As the temperature was increased, the frequency of the NAD(P)H oscillation as well as that of the NO and ROI bursts increased, so that the rate of NO and ROI production is enhanced. For the cells examined, NAD(P)H periods of 24.8, 22.3, 20.6, 19.7, and 17.1 s were found for temperatures of 29, 33, 37, 39, and 43°C, respectively. Control

experiments have demonstrated that the NO and ROI fluorescence assays themselves are not affected by temperature in the range of 22– 45°C (results not shown). The generation of ROI depends upon the production of superoxide following the enzymatic oxidation of NADPH by NADPH oxidase, whereas the generation of RNI follows the production of NO, which depends upon the enzymatic oxidation of L-arginine in the presence of oxygen and NADPH by NO synthase (16). Inasmuch as NADPH is central to the production of ROIs and RNIs, we hypothesized that the changes in oxidant production frequency were due to the dynamics of NADPH availability. At 37°C, polarized or adherent cells displayed an NAD(P)H oscillation period of ⬃20 s (12). Intracellular NAD(P)H levels were measured with epifluorescent microscopy by limiting the field of view to a single polarized cell and then directly monitoring NAD(P)H autofluorescence at 420 nm after excitation at 360 nm (12). We found that the timing between steps or bursts of oxidant release correlated with the NAD(P)H frequency, so that a cell exhibiting an NAD(P)H oscillation with a period of about 20 s will also exhibit production bursts of NO and ROI every 20 s. Thus, the temperature-dependent behavior of the intracellular NAD(P)H concentration may account for the increased production of oxidants at higher temperatures. LPS, a product of Gram-negative bacteria, stimulates neutrophil activation, including the production of NO and ROI. Since we had shown in Fig. 1 that NO and ROI production rates are temperature dependent and appear to be linked to the NAD(P)H oscillation frequency, we analyzed the combined effect of LPS and temperature on the levels of these metabolites in individual neutrophils. The results are shown in Fig. 2, where cells at the identical temperatures as in Fig. 1 were pretreated with LPS (50 ng/ml) for 30 min before the beginning of the experiment. As in Fig. 1, at each temperature, representative neutrophils were selected (n ⱖ 5), and NO, ROI, and NAD(P)H were measured as function of time. In this instance for the cells chosen, we measured NAD(P)H periods of 12.3, 11.7, 11.3, 10, and 9.3 s at temperatures of 29, 33, 37, 39, and 43°C, respectively. The figure confirms the connection between NAD(P)H frequency and NO and ROI output. It is apparent that at each

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Single-cell pericellular NO production was monitored in exactly the same manner as single-cell production of ROI, with the exception that the gelatin was mixed with 15 ␮M diaminofluorescein-2 diacetate (DAF-2DA; Daiichi Kagaku Yakuhin, Tokyo) in place of H2TMRos. DAF-2DA has been previously shown to become fluorescent upon exposure to NO, but not to ROI (9).

FIGURE 1. The production of NO and ROI is linked to NAD(P)H metabolism and is a function of temperature in adherent neutrophils. Human peripheral blood neutrophils were allowed to adhere to glass coverslips, mounted on a slide, and then placed on a temperature-controlled microscope stage on an epifluorescent microscope. At the indicated temperatures, NAD(P)H in individual neutrophils was monitored by autofluorescence and individual fluorescent photons were counted with a photomultiplier tube. For each temperature, photon flux vs time is plotted. In a similar manner, pericellular NO and ROI production at identical temperatures was measured in other neutrophils by epifluorescence of specific indicator probes.



temperature, the effect of LPS is to increase the NAD(P)H frequency. Concomitantly, the “pulse frequency” of NO and ROI production is elevated as well, so as to increase the total pericellular NO and ROI yields. Our findings suggest that the controlling parameter in NO and ROI production is the NADPH oscillation frequency. Accordingly, in Fig. 3, we have conducted a detailed analysis of the effect of temperature and LPS on neutrophil NAD(P)H levels. Neutrophils were maintained at various temperatures between 23 and 45°C, and internal NADPH concentrations were measured as a function

FIGURE 3. When combined, LPS and febrile temperatures act synergistically to increase the frequency of NAD(P)H oscillations in adherent neutrophils. The frequency of internal NAD(P)H oscillations in neutrophils, which were (f) or were not (⽧) exposed to LPS, was measured over a spectrum of temperatures from 23 to 45°C. Error bars associated with each symbol correspond to the SEM, and, on the scale of this figure, are often of comparable size to the symbols. At all temperatures, LPS increases the NAD(P)H frequency, but the effect is greatly exacerbated at temperatures above 37°C.

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FIGURE 2. NAD(P)H oscillation as well as NO and ROI production frequency increases in response to LPS and temperature. Peripheral blood neutrophils were incubated with LPS (50 ng/ml) and then allowed to adhere to glass coverslips. As in Fig. 1, NAD(P)H, NO, and ROI production in individual cells was monitored by epifluorescence microscopy at several temperatures. In each plot, photon flux vs time is recorded.

of time (see Fig. 1). For each temperature, six neutrophils in the presence or absence of LPS were analyzed. The average frequency of the NAD(P)H oscillation (with or without LPS) was then plotted as a function of temperature. Several things are of interest. First, in the absence of LPS, there is a general increase in NAD(P)H frequency with temperature. From 23 to 37°C, frequency increases slowly with temperature. However, between 37 and 41°C, there is an inflection, and the frequency increases much more rapidly, so that by 41°C it has increased by ⬃50% from its value at 37°C. After 41°C, the rate of increase in frequency with temperature levels out and approaches that found below 37°C. At all temperatures, LPS increased NAD(P)H frequency. But once again there is a distinct difference in the behavior of the system at temperatures ⬎37°C from that found at lower temperatures. In the physiologically relevant temperature range between 35 and 41°C, the temperature effect is highly significant, both in the presence and absence of LPS ( p ⬍ 0.00001 as judged by ANOVA analysis.) At or below 37°C, LPS essentially multiplies the NAD(P)H frequency by a factor of ⬃1.8, although there does seem to be a slight rise in this factor with temperature. However, at temperatures above 37°C, there is clearly a much more dramatic rise in frequency. One way of looking at this data is to consider that in the presence of LPS, the modest increase in frequency with temperature seen above 37°C is “magnified.” Put another way, we can see that at 37°C LPS serves to approximately double the NADPH frequency, but at febrile range temperatures above 37°C in the presence of LPS, the NAD(P)H frequency is increased approximately by a factor of 4 from what it was at 37°C in the absence of LPS. Our concern with the NAD(P)H frequency is of course based upon the connection that we have established between NAD(P)H frequency and the pericellular production rates of NO and ROI. From Figs. 1 and 2, we expect that the production of NO and ROI should both be proportional to the NAD(P)H frequency. If this is so, and considering Fig. 3, we would predict that LPS and febrile temperatures should act together to stimulate NO and ROI production. This is in fact exactly what we have found in Fig. 4, where we directly compared the rate of neutrophil ROI and NO production as a function of temperature in the presence or absence of LPS. Neutrophils were, or were not, treated with LPS for 30 min and then, as in Fig. 1, placed on a coverslip to adhere before warm fluid-phase gelatin containing H2TMRos was added. After cooling and solidification of the gelatin, the coverslip was mounted on a slide, which was then placed on a heated microscope stage. As described above, pericellular ROI production in single neutrophils was plotted as a function of time by recording TMRos fluorescence in the immediate vicinity of each cell. As in Figs. 1 and 2, a “step pattern” was obtained, showing that ROIs are produced in repetitive bursts, with the burst frequency increasing in LPS-treated cells. We also found that as expected, the burst frequency increased with increasing temperature, both in the presence and absence of LPS. To quantitatively compare ROI production between cells at different temperatures in a statistically meaningful manner, we determined the average rate of ROI production for groups of cells (n ⫽ 6 for each group). Each group was either treated or not treated with LPS, and each cell within the group was examined at the same temperature. First, the average rate of ROI production for each cell within a group was individually determined. This was done by measuring the change in TMRos fluorescence (measured as the change in counts per second detected by the photomultiplier) over three complete steps in the TMRos fluorescence vs time plot, and then dividing this number by the time equal to three periods (steps). Finally, the average rate of change of fluorescence for all

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cells within the group were averaged and plotted vs temperature (Fig. 4A). It is clearly seen that at all temperatures the rate of ROI production is enhanced in LPS-treated cells. Furthermore, in both LPS-treated as well as untreated cells, the rate of ROI production was increased at higher temperatures. As the NADPH measurements suggested (Fig. 3), the effect of temperature to increase ROI production in the physiologically relevant temperature range of 35– 41°C is statistically significant ( p ⬍ 0.00001) and more dramatic in LPS-treated cells. In Fig. 4B, we have performed an identical analysis for pericellular NO production, with the exception that the fluorescent indicator has been changed to DAF-2DA. As expected, the results for NO are consistent with those found for ROI. NO production is generally enhanced by a change in temperature, especially above 37°C. As in the case for ROI, in cells treated with LPS the enhancement is magnified, and the difference in production at elevated temperatures is statistically significant by ANOVA analysis with p ⬍ 0.00001. Several earlier investigators have looked at the influence of temperature on bacterial killing by neutrophils with mixed results. Roberts and Steigbigel (17) reported statistically enhanced killing of E. coli, Salmonella typhimurium, and Listeria monocytogenes at 40°C vs 37°C, but found that results with Staphylococcus aureus were too variable to draw a statistically significant conclusion. Sebag et al. (18) found enhanced killing of pneumococcus at 39°C

vs 37°C, but decreased killing of E. coli at 41oC vs 37°C. In the same study Sebag et al. (18) also reported that the nitroblue tetrazolium reduction assay for neutrophil superoxide production appeared to give similar results for all temperatures. Even earlier work by Craig and Suter (19) seemed to show that at temperatures below 36°C, neutrophil killing of S. aureus was enhanced by increases in temperature, but that there was no effect above 36°C. Although straightforward, there are several reasons why experiments in bulk or tissue culture systems designed to measure neutrophil killing or ROI production using classical methods are not directly comparable to the experiments reported here using newer fluorometric techniques. First, bacterial killing by neutrophils is a complex process that depends not only on ROI and NO production, but also on phagocytosis and opsonization. These latter processes are surely temperature dependent, with the possibility that temperature effects here may be bacteria specific. A second and perhaps more fundamental difference is that the fluorometric methods are essentially single-cell assays. Peripheral blood neutrophils are necessarily a heterogeneous cell population. Starting with this population, we have used the microscope to select for and then focus on and assay only adherent and polarized cells. It is precisely the adherent, polarized, and motile cell fraction which has extravasated from the blood to an infection site that is likely most relevant with respect to antimicrobial activity (13). In the absence of selection, the older techniques necessarily average results over hundreds of thousands, if not millions of different cells, many of which may or may not be responsive to temperature in the same way. Human neutrophils possess several endogenous oscillators including: intracellular calcium, cell shape, membrane potential, NADPH, ROIs, and NO (20). Due to their important role in inflammation, the present study has focused on ROI, NO, and NADPH, the source of reducing equivalents in the synthesis of ROIs and NO. Previously, we have shown that IFN-␥ increases the amplitude of these oscillations, whereas a variety of other neutrophil-activating stimuli such as immune complexes, LPS, TNF-␣, etc. increase the frequencies and enhance the downstream functional responses of these oscillations (12, 15, 17). In this study, we have shown that the frequencies are also strongly dependent on temperature as well as external cytokine-like stimuli. In contrast, we have found that oscillation amplitudes are relatively independent of temperature over normal physiological and febrile temperature ranges (results not shown). Recently, a robust and predictive computer model of NADPH and superoxide oscillations during neutrophil activation has been constructed (24). The present study has shown that a purely physical stimuli, temperature, affects the behavior of dynamic chemical processes in living neutrophils. Although it is well known that in many instances individual enzyme activities may be a function of temperature (21), these experiments demonstrate that dynamic metabolic oscillations, which are dependent on a network of many transporters and enzymes, remain stable at and are a function of higher temperatures. This stability, in the absence of chemical signals, allow febrile temperatures to enhance pericellular NO and ROI release. Importantly, our data suggest that during an inflammatory response to a Gram-negative bacterium, febrile temperatures serve to substantially enhance ROI and NO release. Previous studies have suggested that elevated temperatures enhance various aspects of innate and adaptive immunity, including neutrophil function (2, 22, 23). The mechanism responsible for these changes may be identical to that described herein, which relies not upon the properties of a single molecule, but rather the collective properties of many molecules. For example, the changes in energy metabolism may also contribute to enhanced cell speed during chemotaxis

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FIGURE 4. When combined, LPS and febrile temperatures act synergistically to increase pericellular neutrophil production of NO and ROI. A, The average rate of production of ROI as a function of temperature was measured in individual adherent neutrophils. Before measurement, cells were (f) or were not (Œ) exposed to LPS. The average rate of change of fluorescent photon flux (proportional to the rate of change of ROI) was determined for each cell and then averaged over all cells at a given temperature. The averages (measured in units of photon counts per second) are plotted vs temperature. Error bars associated with each symbol correspond to the SEM, and, on the scale of this figure, are of comparable size to the symbols. B, The average rate of pericellular production of NO as a function of temperature was measured in individual adherent neutrophils. As in A, cells were (f) or were not (Œ) exposed to LPS before the determination of the average rate of change of NO, and the average rate determined for individual cells was averaged over all cells at the same temperature. These final averages and the corresponding SEM (measured in units of photon counts per second) are plotted vs temperature.




(22, 23). Thus, fever may behave as an endocrine-like signal to enhance innate responses of the host.


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