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Originally published online as doi:10.1189/jlb.0405236 on October 21, 2005

Published online before print October 21, 2005
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(Journal of Leukocyte Biology. 2005;78:1301-1305.)
© 2005 by Society for Leukocyte Biology

Endotoxin tolerance induces selective alterations in neutrophil function

Lisa C. Parker*,1, Elizabeth C. Jones*, Lynne R. Prince*, Steven K. Dower{dagger}, Moira K. B. Whyte* and Ian Sabroe*

Academic Units of
* Respiratory Medicine and
{dagger} Cell Biology, Section of Functional Genomics, Division of Genomic Medicine, University of Sheffield, United Kingdom

1 Correspondence: Academic Unit of Respiratory Medicine, Division of Genomic Medicine, University of Sheffield, M Floor, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK. E-mail: l.c.parker{at}sheffield.ac.uk


   ABSTRACT
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Endotoxin tolerance has the potential to limit phagocyte responses to Toll-like receptor (TLR) agonists, but the role of tolerance in regulating neutrophil responses is unknown. We investigated neutrophil responses to prolonged lipopolysaccharide (LPS) exposure and observed induction of tolerance in intracellular signaling pathways and respiratory burst. These effects were not prevented by granulocyte macrophage-colony stimulating factor (GM-CSF) pretreatment, and tolerized neutrophils retained the ability to respond to GM-CSF and other survival factors with a delay in apoptosis. In addition, LPS-exposed neutrophils showed continued generation of CXC chemokine ligand 8, which was not reduced in tolerized cells. Induction of tolerance was associated with a loss of TLR4 surface expression. Tolerance, therefore, induces a selective reprogramming of neutrophil function, but cells retain a predominantly proinflammatory phenotype.

Key Words: inflammation • LPS • TLR

Excessive or inappropriate neutrophil activation can result in vascular and tissue damage and thus, contribute to the pathology of a variety of inflammatory diseases including vasculitides [1 ] and septic shock. Circulating leukocytes from septic patients have a reduced capacity to produce cytokines compared with healthy controls, potentially representing a protective mechanism to limit vascular damage [2 ]. This resembles the in vitro phenomenon known as endotoxin tolerance, whereby activation of cells by lipopolysaccharide (LPS), acting on the receptor Toll-like receptor 4 (TLR4), renders them refractory to further challenges with the same agonist [3 ]. In monocytes, tolerance results in decreased transcription factor activation [4 , 5 ], decreased release of some but not all proinflammatory cytokines [6 7 8 9 ], and down-regulation of TLR4 signaling components [10 , 11 ]. Neutrophils from patients with sepsis also respond to ex vivo challenge with LPS with altered cytokine production [12 13 14 ]. As the neutrophil also plays a major role in TLR-driven inflammation, we investigated the functional consequences of prolonged activation of the neutrophil with LPS with respect to TLR signaling and cell survival.

Neutrophils were prepared from healthy volunteers (after informed consent) by density centrifugation and purified by negative magnetic selection [15 ]. Neutrophils (5x106/ml) were cultured in media (RPMI/10% fetal calf serum) alone or media containing 100 ng/ml purified Escherichia coli LPS [16 ] (a gift from Professor Stephanie Vogel, University of Maryland, College Park) for the times specified (primary stimulation); then, cells were washed twice and resuspended in media again, with or without 100 ng/ml LPS (secondary stimulation). In some experiments, neutrophils were treated with 50 U/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Roche Molecular Biochemicals, UK) for 1 h prior to the first stimulation with LPS. Western blot analysis was performed as described [17 ], using antibodies specific to inhibitor of {kappa}B-{alpha} (I{kappa}B-{alpha}) or the phosphorylated forms of p38, extracellular signal-regulated kinase (ERK)1/2, and Jun N-terminal kinase (JNK), followed by a horseradish peroxidase-coupled secondary antibody. Anti-total p38 or actin binding was used to control for loading. Representative blots are shown, except for the effects of GM-CSF pretreatment on p38 phosphorylation when films were analyzed densitometrically using National Institutes of Health Image (Version 1.62f) and presented as the ratio of phosphorylated versus nonphosphorylated forms. CXC chemokine ligand 8 (CXCL8) was measured by enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs from the National Institute for Biological Standards and Controls (Potters Bar, UK) as described [15 ]. Generation of H2O2 and reactive oxygen species (ROS) was detected using measurements of dichlorodihydrofluorescein diacetate (DCF; Sigma-Aldrich, UK) fluorescence as described [15 ]. Briefly, after primary stimulation with buffer or LPS for 5 h, neutrophils were washed and incubated with 5 µM DCF for 15 min at 37°C and then treated with buffer or LPS for 30 min before being washed in phosphate-buffered saline and analyzed immediately by flow cytometry. Apoptosis was quantified by morphology on Diff-Quik (Dade Behring, Marburg, Germany)-stained cytospins, counting >300 cells per slide on duplicate cytospins as described [18 ]. Neutrophil TLR4 surface expression was measured by flow cytometry using biotinylated-anti-TLR4 monoclonal antibody (clone HTA125) or an isotype control antibody from eBioscience (San Diego, CA) as described [19 ]. Data are presented as mean ± SEM of ≥3 independent experiments, analyzed using Prism 4.0b (GraphPad, San Diego, CA).

Although the majority of studies into endotoxin tolerance uses an initial exposure time of 20–24 h, tolerance of cytokine production has been described in murine macrophages in response to as little as 1 h prior exposure to LPS [20 ], and a 5-h LPS pretreatment was sufficient to inhibit nuclear factor (NF)-{kappa}B, mitogen-activated protein kinase (MAPK) activation, and I{kappa}B-{alpha} degradation in Chinese hamster ovary/CD14 cells [21 ]. We also determined that pretreatment of primary human monocytes with LPS for 5 h was sufficient to induce tolerance of MAPK activation and tumor necrosis factor {alpha} (TNF-{alpha}) release (data not shown). We observed that stimulation of neutrophils with LPS for 1 h was sufficient to render cells tolerant to subsequent LPS exposure, as determined by p38 phosphorylation, and tolerance was also detected following 5-, 10-, and 20-h primary stimulations (Fig. 1A ). Of note, phosphorylation of p38 increases in untreated cells as the neutrophil ages (Fig. 1A) , and I{kappa}B-{alpha} levels decrease gradually (data not shown). Thus, for all subsequent work, we chose to induce tolerance using a 5-h primary stimulation. We determined that exposure of neutrophils to LPS for 30 min or media for 5 h and then LPS for 30 min resulted in broad activation of the MAPKs, and cells treated with LPS for 5 h showed no response to a second LPS stimulus in these assays (Fig. 1B) . Activation of NF-{kappa}B was measured using degradation of I{kappa}B-{alpha} as a marker. LPS exposure for 30 min led to degradation of I{kappa}B-{alpha} (Fig. 1C) . I{kappa}B-{alpha} levels were restored to those observed in buffer-treated cells by 5 h; however, subsequent stimulation with LPS caused complete degradation of I{kappa}B-{alpha} only in neutrophils that had not previously been exposed to LPS (Fig. 1C) .



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  		Figure 1. Repeated exposure to LPS induces tolerance in the human neutrophil, which cannot be prevented by GM-CSF. (A) Neutrophils were cultured alone (–) or with 100 ng/ml purified LPS (+) for the specified times (primary stimulation), after which cells were washed twice and again cultured alone (–) or with 100 ng/ml LPS (+) for 0.5 h (secondary stimulation). Cell lysates were analyzed by Western blot 0.5 h after the secondary stimulation using an antibody specific to the phosphorylated form of p38 (P-p38), and actin was used as a loading control. Data shown are representative of three experiments, each from a separate donor. In all subsequent experiments, 5 h was chosen for the primary stimulation, after which the neutrophils were washed twice and received a second stimulation for 0.5 h unless otherwise stated. (B) Cell lysates were analyzed by Western blot 0.5 h after the primary or secondary stimulation, using antibodies specific to the phosphorylated forms of p38, ERK (p42/44), and JNK (p46/54) and total p38. Data shown are representative of three experiments, each from a separate donor. (C) Cell lysates were analyzed by Western blot using an antibody specific to I{kappa}B-{alpha} 0.5 h after the primary or secondary stimulation and also just prior to the second stimulation to check that I{kappa}B-{alpha} levels had been restored. Actin was used as a loading control. Data shown are representative of three experiments, each from a separate donor. (D) Neutrophils were pretreated with buffer (–) or GM-CSF (50 U/ml; +) for 1 h, at which point cells were stimulated as described above. Cell lysates were analyzed by Western blot for total and phosphorylated p38 and films densitometrically analyzed; data are presented as mean ± SEM of three experiments, each from a separate donor, showing the ratio of phosphorylated versus nonphosphorylated forms. ROS generation was measured by oxidation-mediated increases in DCF fluorescence using flow cytometry. Data are mean ± SEM of five experiments, each from a separate donor. Significant differences are indicated as follows: between the buffer and GM-CSF-pretreated groups, {phi}, P < 0.05 –/+ versus –/+; within the GM-CSF-pretreated group, ***, P < 0.001 –/– versus –/+; #, P < 0.05 –/+ versus +/+ or +/–, all analyzed by ANOVA and Tukey’s post-test.

 
GM-CSF is a potent, neutrophil-priming cytokine and broadly up-regulates neutrophil responses to TLR agonists [22 ]. Pretreatment of neutrophils with GM-CSF for 1 h failed to prevent the tolerizing effects of LPS with respect to p38 phosphorylation (Fig. 1D) . TLR4 activation also caused a modest increase in ROS generation, which was inhibited, although not significantly, by a tolerizing dose of LPS (Fig. 1D) . In contrast, GM-CSF-pretreated neutrophils showed significantly increased ROS production in response to LPS, which was significantly inhibited by prior exposure to LPS (Fig. 1D) . Therefore, tolerance can limit neutrophil proinflammatory responses even in the presence of the classical priming cytokine, GM-CSF. These mechanisms may serve to limit neutrophil responses in vivo, potentially preventing excessive cell activation, which could culminate in tissue damage.

The mechanisms triggering endotoxin tolerance remain to be fully elucidated in the monocyte/macrophage. It was hypothesized initially that down-regulation of TLR4 expression was the cause, as it has been shown to correlate with development of the tolerant phenotype in monocytic cells [20 , 23 ]. We found that LPS caused a rapid (within 30 min) decrease in TLR4 expression on the surface of neutrophils, which had not received prior stimulation with LPS, and this reduction was potentiated in cells that had received prior exposure to LPS (Fig. 2A ). It is interesting that we previously found that treatment with commercial LPS had little effect on neutrophil TLR4 expression [19 ]; however, here, we used a more potent, TLR4-selective, repurified LPS, which may account for the different results. It should be noted that decreased TLR4 expression alone is no longer thought to be sufficient to explain tolerance in monocytes [3 , 21 , 24 ], but as TLR4 is expressed at much lower levels on the neutrophil compared with the monocyte [19 ], regulation of TLR4 expression may have a greater potential to modify neutrophil responses to LPS. However, although neutrophils from patients with sepsis show impaired cytokine generation [12 13 14 ], recent evidence suggests that TLR2 and -4 expression can be increased on neutrophils from patients with sepsis [25 ]. These data suggest that induction of tolerance can proceed even when TLR expression is enhanced (potentially by proinflammatory cytokines [22 , 25 , 26 ]), which is in keeping with our data here showing that GM-CSF fails to prevent induction of tolerance in vitro. Control of neutrophil tolerance and function in vivo is thus likely to be a complex sum of actions of LPS and proinflammatory cytokines on TLR expression and signaling.



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  		Figure 2. Neutrophil tolerance is potentially mediated by down-regulation of TLR4 expression; however, tolerized neutrophils retain critical, proinflammatory functions. (A) Neutrophils were stimulated as described in Figure 1 . Cell surface expression of TLR4 was determined by flow cytometry 0.5 h after the secondary stimulation. An illustrative histogram, demonstrating the down-regulation of TLR4 surface expression in response to LPS, is shown above the graph of the mean ± SEM of four experiments, each from a separate donor. Data are expressed as specific mean fluorescence {after subtraction of nonspecific binding of isotype-matched [immunoglobulin G2a (IgG2a) control]}. *, P < 0.05 –/– versus +/+, analyzed by ANOVA and Tukey’s post-test. (B) Cell supernatants were removed at time 0 h, just prior to (time 5 h) and 20 h after (time 25 h) the secondary stimulation and assayed by ELISA. Data shown are mean ± SEM of three experiments, each from a separate donor, carried out in duplicate, and are expressed as pg/ml release. *, P < 0.05 –/– versus –/+ or +/+, analyzed by ANOVA and Tukey’s post-test. (C) Neutrophils (N{phi}) were cultured with (+) or without (–) LPS for 5 h, washed twice, and cultured for a further 20 h alone (open bars) or with 5% peripheral blood mononuclear cells (PBMCs; solid bars) again with (+) or without (–) LPS. Neutrophil cell death was then quantified by determination of the percentage showing apoptotic morphology by light microscopy. Data are mean ± SEM of three experiments, each from a separate donor; statistical differences between neutrophils alone (buffer) and LPS-treated cells at 5 h are indicated by *, P < 0.05, analyzed by paired t-test, and between neutrophils alone and neutrophils + PBMCs at 25 h, ***, P < 0.001, analyzed by ANOVA with Tukey’s post-test. (D) Neutrophils were cultured alone (open bars) or with LPS (solid bars) for 5 h, washed twice, and cultured for a further 20 h alone (buffer) or with GM-CSF (50 U/ml), TNF-{alpha} (10 ng/ml), or interferon-{gamma} (IFN-{gamma}; 10 ng/ml). Neutrophil cell death was quantified as in C. Data are mean ± SEM of four experiments, each from a separate donor; statistical differences between buffer and cytokine-treated groups are indicated by *, P < 0.05; **, P < 0.01; ***, P < 0.001, analyzed by ANOVA with Tukey’s post-test. No differences between buffer and LPS-treated (tolerized) neutrophils were detected.

 
Neutrophilic inflammation has the potential to be self-perpetuating through the production of CXCL8 by the neutrophils themselves [27 ]. Our results show that release of CXCL8 from neutrophils was not tolerized in response to prior exposure to LPS (Fig. 2B) , which confirms previous reports that LPS-induced CXCL8 release is not tolerized by LPS pretreatment in monocytic cells [28 ] or in neutrophils and monocytes from septic patients [25 ]. It is striking that neutrophils transiently activated with LPS for 5 h, followed by culture in media for 20 h, showed similar levels of CXCL8 generation to cells cultured throughout with LPS. Thus, a single activating stimulus can switch the neutrophil into a proinflammatory, cytokine-producing cell, whose cytokine production may persist after the inflammatory response is withdrawn. To remove these activated cells from resolving inflammatory sites, neutrophils undergo apoptosis and phagocytosis by macrophages. Apoptosis is inhibited by proinflammatory mediators such as GM-CSF, presumably extending the time in which these cells can execute their antimicrobial functions. We have shown that LPS, previously thought to be a major survival factor, is relatively ineffective at delaying apoptosis in highly purified neutrophils, and the presence of PBMCs markedly prolongs neutrophil survival in response to LPS [15 , 18 ]. As tolerance reduces respiratory burst but not CXCL8 generation, we investigated the extent to which tolerance would impair neutrophil survival responses. Figure 2C shows that consistent with our previous data [15 , 19 ], LPS enhanced early but not late neutrophil survival. Addition of PBMCs to washed neutrophils at the 5-h time-point (Fig. 2C ; solid bars) did not alter survival in the absence of any LPS stimulation (group –/–). In contrast, neutrophil survival was increased substantially when PBMCs were added in the presence of LPS, regardless of whether the neutrophils had undergone a tolerogenic stimulus (groups –/+, +/+). Similarly, tolerized and nontolerized neutrophils showed identical survival responses to GM-CSF, TNF-{alpha}, and IFN-{gamma} (Fig. 2D) .

These data reveal that tolerance induces important changes in neutrophil phenotype. Down-regulation of TLR4 signaling may limit neutrophil inflammation and potentially reduce the risks of inappropriate or excessive responses, which could damage tissue. However, a tolerized neutrophil can continue to recruit further neutrophils through production of CXCL8 and shows similar survival responses to PBMC-derived survival factors and specific survival cytokines, retaining the potential to act as an important driver of the inflammatory response. These data reinforce the potential for neutrophils to act in capacities beyond those of simple effector cells in a range of diseases from vasculitis to sepsis.


   ACKNOWLEDGEMENTS
 
This work was supported by an Arthritis Research Campaign (UK) project grant (SO666) and by the Medical Research Council (UK) through a Senior Clinical Fellowship (G116/170) to I. S.

Received April 29, 2005; revised August 12, 2005; accepted September 8, 2005.


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