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The effect of fever-like temperatures on neutrophil signaling

Medical Faculty of the Charité, Department of Nephrology and Hypertension, Franz Volhard Clinic at the Max Delbrück Center for Molecular Medicine, HELIOS-Klinikum-Berlin, Germany

¹ Correspondence: Wiltbergstr. 50, 13125 Berlin, Germany. E-mail: kettritz{at}fvk.charite-buch.de

SPECIFIC AIMS

The purpose of fever is unknown. We determined the effect of fever-like temperature spikes on intracellular signaling and cellular functions of human neutrophils challenged with tumor necrosis factor- (TNF-) and investigated how heat exposure differentially affects neutrophils in suspension compared with neutrophils that interact with the extracellular matrix protein fibronectin.

PRINCIPAL FINDINGS

1. Temperature and TNF--induced pathways in suspended neutrophils
We exposed neutrophils in suspension to 42°C for 60 min or kept the cells at 37°C control for the same period. Then cells were allowed to adapt to 37°C for 30 min before 2 ng/mL TNF- or buffer control was added. Samples were harvested at different times and subjected to Western blot to assess activation of p38 MAPK, ERK, PI3-K/Akt, and NF-B. Short-term heat exposure inhibited TNF--induced activation of all four pathways (Fig. 1 A).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of short-term heat exposure on activation of ERK, p38 MAPK, PI3-kinase/Akt, and IB degradation and on apoptosis in suspension neutrophils. A) After exposure of cells to 37°C or 42°C for 60 min, neutrophils were stimulated with 2 ng/mL TNF- in suspension. Cells were harvested and subjected to Western blot experiments using phospho-specific antibodies to ERK, p38 MAPK, Akt, and an antibody to IB. These studies indicate that short-term heat exposure inhibits activation of all 4 signaling pathways by TNF-. B) Effects of p38 MAPK and NF-B inhibitors on TNF--delayed apoptosis by flow cytometry. Neutrophils were preincubated with buffer control, 10 µM SB202190 (SB), 10 µM NBD, or 10 µM of a mutant control (CTR) at 37°C, respectively. After incubation with TNF- for 20 h, samples were harvested and apoptosis was measured using propidium iodide staining and flow cytometry. The data demonstrate that TNF- did not delay apoptosis in the presence of the p38 MAPK and NF-B inhibitors (n=5, *P<0.05).

2. The effect of fever-like exposure on TNF--mediated neutrophil functions
P38 MAPK, ERK, PI3-kinase, and NF-B induce anti-apoptotic signals in neutrophils. We thus investigated the effect of pharmacological inhibitors on these pathways on TNF--delayed apoptosis. To inhibit NF-B, we used an 11 amino acid sequence NEMO binding domain (NBD) that selectively blocks IKK (NEMO) IKKß interaction. An HIV-TAT sequence served as a highly effective transduction shuttle. We observed that preincubation with the p38 MAPK blocker SB202190 or NBD resulted in significant inhibition of the ability of TNF- to delay apoptosis (Fig. 1B ). Pharmacological inhibition of ERK or PI3-K/Akt had no effect in parallel experiments (data not shown).

Because we had observed that heat exposure abrogates p38 MAPK and NF-B signaling and that both pathways transduce TNF--activated anti-apoptotic signals, we next studied the effect of short-term heat exposure on apoptosis in TNF--treated neutrophils. Short-term heat exposure abrogates TNF--delayed apoptosis at 20 h in a dose-dependent manner beginning already at 39°C. In contrast, when we studied the effect of short-term heat exposure on adhesion-dependent TNF--induced neutrophil functions—namely, respiratory burst and adhesion—we found no significant inhibition by prior heat exposure.

3. Temperature and TNF--induced pathways in adherent neutrophils on fibronectin
After exposing cells to 37°C or 42°C for 60 min in suspension, samples were stimulated with 2 ng/mL TNF- or buffer control on fibronectin at 37°C. Our results indicate that, in contrast to suspension conditions, exposure to 42°C did not affect TNF--induced activation of p38 MAPK, ERK, PI3-K/Akt, and NF-B when neutrophils were incubated on fibronectin. Figure 2 B depicts a typical EMSA to assess NF-B activation.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of short-term heat exposure on TNF--delayed apoptosis and on TNF--induced NF-B gene transcription in neutrophils in suspension and on fibronectin. After exposure of cells to 42°C (gray columns) or 37°C (black columns) for 60 min, samples were incubated with buffer control or TNF- (TNF) at 37°C in either suspension or adherent conditions, respectively. A) Apoptosis was estimated after 20 h by propidium iodide staining and flow cytometry. Delayed apoptosis by TNF- was prevented by prior 42°C preincubation when the assay was performed under suspension conditions but not when cells were cultured on fibronectin (n=5 parallel experiments, *P<0.01). B) After 60 min, nuclear extracts were prepared and analyzed by EMSA using an H2K binding site probe for NF-B (upper panel, n=3) or C) IB mRNA was assessed after 60 min by quantitative RT-PCR (lower panel, n=3). Total RNAs were isolated according to a Qiagen protocol including DNase treatment. Quantification was checked for each sample using probes for GAPDH mRNA. TNF- increased NF-B binding activity and IB mRNA, which was prevented by prior preincubation at 42°C when TNF- stimulation was performed under suspension conditions but not when cells were cultured on fibronectin.

4. Effect of fever-like exposure on TNF--induced neutrophil functions in suspended vs. neutrophils on fibronectin
We selected apoptosis after 20 h to directly compare functional consequences of short-term heat exposure in suspended neutrophils vs. neutrophils interacting with fibronectin. After short exposure to 37°C or 42°C, cells were incubated with buffer control or TNF-. TNF- delayed neutrophil apoptosis in suspension and under adhesion conditions (Fig. 2A ). Short-term heat exposure abrogated the TNF- effect in suspension cells but no effect was seen on fibronectin.

NF-B activation has implications beyond apoptosis by controlling a variety of genes that participate in the inflammatory response. Quantitative RT-PCR for IB mRNA indicated that TNF- increased IB mRNA in suspension neutrophils and in cells on fibronectin. Prior preincubation at 42°C prevented this effect completely when TNF- stimulation was performed under suspension conditions; no such temperature-mediated inhibition was observed when cells were cultured on fibronectin (Fig. 2C ).

5. Role of ß2-integrin mediated costimulation for TNF--induced NF-B activation after heat
We demonstrated previously that ß2-integrins provide costimulatory signals for NF-B activation in neutrophils. However, in the previous study TNF- induced NF-B independent of ß2-integrins. We considered the possibility that ß2-integrins may gain costimulatory functions after neutrophils are exposed to fever-like temperatures. We used activating anti-CD18 antibodies to study NF-B activation. By IB Western blot, we observed that a CD18-activating antibody provided costimulation, allowing TNF- to activate NF-B in suspended neutrophils after heat exposure. Thus, NF-B activation by TNF-, which was not ß2-integrin dependent at 37°C, became ß2-integrin dependent after short-term heat exposure (Fig. 3 ).

View larger version (30K): [in this window] [in a new window]   Figure 3. Proposed scheme of the differential effect of fever-like temperatures on TNF--mediated signaling and function of human neutrophils. Short-term heat exposure blocks activation of p38 MAPK and NF-B in response to TNF- and thereby prevents delayed apoptosis and initiation of gene transcription. This heat exposure-mediated inhibition does not occur when ß2-integrin costimulation is provided.

CONCLUSIONS AND SIGNIFICANCE

1. Fever-like temperatures down-regulate TNF- signaling in suspended neutrophils but not in neutrophils interacting with fibronectin
Our study indicates that exposure to fever-like temperatures affects intracellular signal transduction pathways in human neutrophils. With p38 MAPK, ERK, PI3-K/Akt, and NF-B, we selected signaling pathways that are strongly activated by TNF-. When we assayed suspended neutrophils, we noted that short-term heat exposure had profound inhibitory effects on all four pathways.

In contrast to the finding obtained with suspended cells, we found that heat exposure did not block TNF--induced activation of p38 MAPK, ERK, and NF-B in neutrophils interacting with fibronectin. Our results show that extracellular matrix-dependent signals prevented heat exposure-mediated blockade of p38 MAPK, ERK, and NF-B activation in neutrophils responding to TNF-. These data had not been previously observed to our knowledge.

2. Fever-like temperatures abrogate TNF--delayed apoptosis and gene transcription in suspended neutrophils, but not in neutrophils interacting with fibronectin
Neutrophil activation and apoptosis are important for an appropriate inflammatory response. Our data indicate that heat exposure does not significantly affect TNF--induced adhesion to fibronectin and respiratory burst activity in fibronectin-adherent neutrophils, which would suggest that after a fever spike TNF--stimulation might still activate neutrophil adhesion and generate reactive oxygen species. Both responses are important for an optimal neutrophilic inflammatory response. However, our data indicate that heat exposure affects apoptosis and that this effect depends on whether neutrophils were maintained in suspension or interacted with extracellular matrix. NF-B activation during inflammation has obvious implications beyond the neutrophil. NF-B controls generation of multiple inflammatory molecules that can be activated by TNF-. Our data demonstrate that, in contrast to adherent cells, heat exposure blocks TNF--stimulated NF-B gene transcription in suspended neutrophils. This temperature effect may promote termination of the NF-B-dependent gene program in neutrophils circulating in the bloodstream. In contrast, short-term heat exposure did not block TNF--induced NF-B activation in neutrophils on fibronectin. This effect may allow neutrophils that have emigrated from the circulation to generate and deliver inflammatory molecules.

3. ß2-Integrins provide costimulatory signals allowing TNF- to activate NF-B even after prior heat exposure
We found that a CD18-activating antibody provided costimulation, allowing TNF- to activate NF-B in suspension neutrophils even after heat exposure. Thus, NF-B activation by TNF-, which was not ß2-integrin dependent at 37°C, became ß2-integrin dependent after short-term heat exposure. ß2-Integrin costimulation may at least provide a partial underlying mechanism of how heat exposure differentially affects neutrophils in suspension vs. neutrophils that interact with matrix proteins, such as fibronectin (Fig. 3) .

We suggest that the heat temperature effect described in this study may become important in maintaining the inactivated state while neutrophils are circulating without inhibiting those that have migrated from the bloodstream into inflamed tissue during the inflammatory response. The data support the idea that fever is a physiologically parsimonious rather than a pathological event.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2983fje;

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