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Cutting Edge |
Department of Biological Sciences, Wayne State University, Detroit, MI 48202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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 
1/2 NADP+ 1/2
H+ O2. The NO synthase
catalyzes the formation of NO according to the reactions:
L-arginine + NADPH + H+ +
O2
NG-hydroxy-L-arginine
+ NADP+ +H2O and
NG-hydroxy-L-arginine
+ 1/2 NADPH + 1/2 H+ + O2
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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LPS (Escherichia coli serotype 026:B6) and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise.
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
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 µM 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).
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. DAF-2DA 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)
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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shows representative examples of
several neutrophils at different temperatures (n 
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).
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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 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 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.
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, 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
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.
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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.
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A). 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 (22, 23). Thus, fever may behave as an endocrine-like signal to enhance innate responses of the host.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Allen J. Rosenspire, Department of Biological Sciences, Wayne State University, Detroit, MI 48202. E-mail address: arosensp{at}sun.science.wayne.edu
3 Abbreviations used in this paper: ROI, reactive oxygen intermediate; RNI, reactive nitrogen intermediate; H2TMRos, dihydrotetramethylrosamine; TMRos, tetramethylrosamine; DAF-2DA, diaminofluorescein-2-diacetate.
Received for publication May 31, 2002. Accepted for publication September 23, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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in IL-6- and IL-2-mediated cell activation. J. Immunol. 163:4367. and sinusoidal electric fields signal by modulating NAD(P)H oscillations in polarized neutrophils. Biophys. J. 79:3001.This article has been cited by other articles:
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) or were not (
) 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.
) 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 (