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International Immunology Advance Access originally published online on November 10, 2006
International Immunology 2007 19(1):41-50; doi:10.1093/intimm/dxl119
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Effects of TREM-1 activation in human neutrophils: activation of signaling pathways, recruitment into lipid rafts and association with TLR4

Carl F. Fortin1, Olivier Lesur1,2 and Tamas Fulop, Jr1,2,3,4

1 Immunology Graduate Program, Clinical Research Center, Faculty of Medicine, University of Sherbrooke, Québec J1H 4C4, Canada
2 Department of Medicine, Pneumology Division, University of Sherbrooke, Québec J1H 4C4, Canada
3 Department of Medicine, Geriatrics Division, Faculty of Medicine, University of Sherbrooke, Québec J1H 4C4, Canada
4 Present address: Laboratory of Immunology, Research Center on Aging, University of Sherbrooke, 1036 rue Belvédère sud Sherbrooke, Québec J1H 4C4, Canada

Correspondence to: T. Fulop Jr; E-mail: tamas.fulop{at}usherbrooke.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Neutrophilic polymorphonuclears (PMNs) play an important role in the progression of sepsis-related inflammation and become highly activated by a wide array of ligands on the site. The triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently described receptor that has many effects on human PMN. The engagement of TREM-1 on PMN can induce phagocytosis, reactive oxygen species production and release of myeloperoxidase and IL-8. LPS has a priming effect on these functions. We show in this paper that Lyn, AKT, extracellular signal-regulated kinase 1/2 and Jak2 signaling pathways are elicited following TREM-1 engagement and activation by a monoclonal agonist antibody (anti-TREM-1) in human PMN, leading to the phosphorylation of STAT5 and RelA, a subunit of the nuclear factor-kappa B family. We also show that TREM-1 is recruited to ganglioside M1-lipid rafts in PMN upon stimulation with LPS or anti-TREM-1. Moreover, we observed that Toll-like receptor 4 and TREM-1 co-localize upon stimulation and TREM-1 engagement resulted in the phosphorylation of IL-1R-associated kinase 1, but not its stimulant-induced degradation. These data shed a new light on how various receptors implicated in the innate immune response could interact to insure an efficient inflammatory response upon pathogens-associated aggression.

Keywords: inflammation, lipid rafts, PMN, TLR4, TREM-1


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Neutrophilic polymorphonuclears (PMNs) play an important role in the progression of sepsis-related inflammation. These are the first cells recruited to the site of aggression and become highly activated by a wide array of ligands. Receptor-activated PMN release a wide array of chemokines and cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}), IL-1ß, IL-8, macrophage inflammatory protein (MIP)-1{alpha} and MIP-1ß (1, 2). These products of activated PMN not only recruit more cells to the site of inflammation but also contribute actively to the modulation of the adaptive immune response.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently described receptor on myeloid cells, and most of our knowledge for this receptor comes from experimentations done in monocytes (3, 4). TREM-1-induced effects in monocytes are mediated by tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), phospholipase C-{gamma} (PLC{gamma}), AKT/PKB and the release of intracellular calcium (3, 4). However, activation of TREM-1 on PMN also induces several functional changes. The engagement of TREM-1 by this yet unknown ligand can induce phagocytosis, reactive oxygen species (ROS) production and release of myeloperoxidase and IL-8 in PMN. TREM-1-induced degranulation was strongly potentiated by priming with LPS, like granulocyte macrophage colony-stimulating factor (GM-CSF) is able to prime PMN for fMLP activation (3, 5, 6). Septic shock develops when the appropriate initial host response to systemic infection becomes uncontrolled and the effects of TREM-1 on PMN is one of the possible pathways leading to such an unrestrained inflammatory response (7). This fact was underlined by the changes of TREM-1 expression on the surface of myeloid cells (PMNs and monocytes/macrophages) during sepsis, both at the site of infection and in peripheral circulation, and its usefulness was proved as a diagnostic tool and as a potential therapeutic target for intervention (8–12).

One of the mechanism by which innate immunity can achieve some specificity is with the recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs). To date, 10 human and 11 murine TLRs have been identified and these receptors signal principally by using the adaptor myeloid differentiation factor 88 (MyD88) and IL-1R-associated kinases (IRAKs). Recently, it has been shown that TLR2 and TLR4 are present in lipid rafts in untreated PMN and LPS could induce more recruitment of TLR4 (13). Stimulation of TLRs leads to the activation of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3 kinase (PI3K) (14). The effects of TLR2 and TLR4 have been assessed using highly purified human PMN. Activation of these TLR resulted in shedding of L-selectin (CD62L) and up-regulation of CD11b on the PMN surface, down-regulation of CXCR2 and reduction of chemotaxis towards IL-8, increased ROS production, degranulation and phagocytosis, activation of nuclear factor-kappa B (NF-{kappa}B) and cytokine generation, but modest effect on the delay of constitutive apoptosis (15). This outlines the important effects of TLR agonists on PMN pro-inflammatory functions. Thus, TLRs may also play a role in the pathophysiology of sepsis (16). However, the interrelation of TREM-1 and TLRs remains largely unknown (12).

Lipid rafts are relatively ordered membrane domains that float in the disordered glycerophospholipid bilayer and their central feature is that they allow the lateral segregation of proteins within the plasma membrane (17). Upon cross-linking of signaling receptors, lipid rafts become larger and more stable structure often attached to the cytoskeleton, a phenomenon called coalescence. Lipid rafts serve to spatially segregate signaling components in the plasma membrane and as such to regulate the initiation and prolongation of signaling (17, 18). In PMN, the importance of lipid rafts has been shown by the recruitment of various molecules into lipid rafts such as NADPH cytochrome b558, proteinase 3 (19), death receptors and DISC assembly (20), Src kinases, like Lyn upon GM-CSF stimulation (21) or Fc{gamma} ligation (22).

As many aspects of the effects of TREM-1 ligation in PMN have not yet been studied, in this paper our aim was to investigate its signal transduction pathways in relation to lipid rafts and TLR4. We showed in PMN that Lyn, AKT, ERK1/2, Jak2 and PLC{gamma} pathways are elicited following TREM-1 activation by a monoclonal agonist antibody (anti-TREM-1) leading to the phosphorylation of STAT5 and RelA (P65), a subunit of the NF-{kappa}B family. We also demonstrated that this molecule was recruited to lipid rafts and could bind to TLR4 in human PMN upon stimulation with LPS or an anti-TREM-1 antibody.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents and antibodies
Anti-human TREM-1 (clone 193015) and PE-conjugated anti-human TREM-1 (clone 193015-PE labeled) were from R&D Systems (Minneapolis, MN, USA) and are herein indicated as anti-TREM-1 and TREM-1–PE, respectively. Anti-phospho ERK1/2, anti-phospho AKT, anti-Lyn, anti-phospho STAT5 and anti-STAT5, anti-TLR4 and irrelevant mouse IgG1 isotype control were from Santa Cruz (Santa Cruz, CA, USA). Anti-phospho Lyn, anti-phospho IRAK1 and anti-phospho P65 were from Cell Signaling Technologies (Beverly, MA, USA). Anti-P65 and anti-phospho Jak2 were from Chemicon International (Temecula, CA, USA). PD98059 (MEK inhibitor), LY294002 (PI3K inhibitor) and AG490 (Jak2 inhibitor) were from Calbiochem (San Diego, CA, USA). DCFDA (carboxy-H2DCFDA) and cholera toxin coupled to AlexaFluor 488, herein indicated as CT-488, from vybrant lipid raft labeling kit were from Molecular Probes (Burlington, Ontario, Canada). Bradford assay reagent was from Bio-Rad (Hercules, CA, USA). Methyl-beta-cyclodextrin (CD) was from Cyclodextrin Technologies Development (High Springs, FL, USA). LPS from Escherichia coli O55:B5 and all other reagents, not otherwise stated, were from Sigma–Aldrich (St Louis, MO, USA).

PMN separation
Citrated blood was obtained by venipuncture from young donors aged 19–25 years (mean age 22 years) and the lipid profile was determined by routine biochemical analysis; all the subjects were in good health and normolipidemic. PMNs were isolated by Ficoll–Hypaque density sedimentation as already described (21). Activation of PMN by the separation method was assessed by the measure of ROS production by cytochrome oxidation (23). Cell viability was >95% as measured by Trypan blue exclusion.

Cell simulation and preparation of cell lysates
Purified PMNs were stimulated with 1 µg ml–1 of anti-TREM-1 (against the extracellular domain of TREM-1) for 2 min, or indicated times, and with 1 µg ml–1 of LPS for 5 min, or indicated times, in RPMI containing 10% fetal bovine serum before subsequent experiments or sample preparations as described (21). The anti-TREM-1 concentrations used were found to be optimal in our experimental settings and are in accordance to manufacturer's instructions (data not shown). The specificity of the anti-TREM-1 stimulation was controlled by a mouse isotype control antibody (data not shown). Where specified, PMNs were incubated with various inhibitors to assess the specificity of the immunodetection before stimulation with anti-TREM-1. The inhibitors were each diluted in medium as to give a final concentration of 10 µM for LY294002 (PI3K inhibitor), 20 µM for PD98059 (MEK inhibitor) and 100 µM for AG490 (Jak2 inhibitor). For cell lysates, neutrophils (5 x 106 cells) were sedimented rapidly, left on ice for 5 min and re-suspended in ice-cold lysis buffer [150 mM NaCl, 10 mM ethyleneglycol-bis (aminoethylether)-tetraacetic acid, 5 mM EDTA, 100 mM NaF, 2 mM NaVO4, 1 mM phenylmethylsulfonylfluoride, 10 mM NaPP, 1% Triton, anti-proteases cocktail in 50 mM HEPES, pH 7.4]. Lysates were cleared of cell debris by centrifugation at 10 000 x g for 5 min and protein concentration was determined by the Bradford assay.

Measurement of ROS production by DCFDA cleavage
PMNs (1 x 106) in 500 µl of complete RPMI were loaded with 20 µM DCFDA in PBS at 37°C for 15 min, and then cells were stimulated and fluorescence in the FL-1 channel was read with a FACScalibur from Becton Dickinson (Franklin Lakes, NJ, USA) as described (21). For the experiments with CD, PMNs were incubated for 1 h at 37°C with 10 mM CD, to deplete the cell membrane cholesterol and disrupt lipid rafts, loaded with DCFDA and stimulated with anti-TREM-1 or isotype control. Stimulations were done with anti-TREM-1 (1 µg ml–1), LPS (1 µg ml–1), GM-CSF (20 ng ml–1) or in combination. If required, PMNs were pre-incubated for 30 min at 37°C with inhibitors preceding DCFDA loading and cell stimulation. Data are shown as stimulation index: mean fluorescence intensity (MFI) of stimulated cells relative to MFI of loaded, but resting, cells.

Immunoblotting
The cell lysates were resolved by SDS-PAGE under denaturing conditions and blotted onto a polyvinylidene fluoride membrane. The strips were blocked with 5% milk, or 3% BSA for anti-phospho P65, for 1 h at room temperature. The strips were then probed with the appropriate primary antibodies and analyzed using the enhanced chemiluminescence detection system as already described (24).

Immunofluorescence staining
Freshly purified PMNs were left unstimulated or stimulated accordingly and fixed by 1% paraformaldehyde as described (25). The cells were then blocked with 10% serum for 20 min on ice and stained on ice, after washing, with a 1/50 dilution of the primary antibody (TREM-1–PE or indicated) on ice for 1 h in 1.5% serum and 2 µg of CT-488 where necessary. The cells were then washed and stained with the appropriate secondary antibody (1/75) in 2% serum for 45 min. After washing, cells were put on slides by cytospin and mounted with Prolong antifade kit, Molecular Probes. The images were obtained with a Nikon TE2000-S and analyzed using simplePCI software.

Recruitment to lipid rafts measured by PI–PLC
The detection of recruitment to lipid rafts by using bacterial PI-specific PLC was done essentially as described (19). PI–PLC cleaves the glycosylphosphatidylinositol (GPI) anchors, a structure used frequently as a mean of recruitment to lipid rafts in the cells, thus displacing the proteins that are recruited to lipid rafts by a GPI anchor-dependent mechanism. Freshly purified PMNs (5 x 106) were incubated for 1 h at 37°C with 1 U ml–1 of PI–PLC and thereafter left untreated or treated with 1 µg ml–1 of anti-TREM-1 for 2 min. The resulting cells are stained as for immunofluorescence with anti-TREM-1–PE or isotype antibodies and cell-surface expression of TREM-1 was analyzed directly by FACScalibur.

Statistical analysis
All the statistical calculations were done by GraphPad PRISM (San Diego, CA, USA). Data were analyzed by one-way analysis of variance using the Bonferroni correction. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Anti-TREM-1 elicits respiratory burst in human PMN
The newly described receptor TREM-1 is a known inducer of human PMN functions (3, 5). To further confirm these data on the respiratory burst, we measured DCFDA oxidation by anti-TREM-1-stimulated PMN. Anti-TREM-1 can significantly stimulate the respiratory burst already at 2 min following receptor engagement and the increase in respiratory burst is time dependent (Fig. 1A). Stimulation with phorbol myristate acetate (PMA) was used as a positive control and it was the most powerful activator of the respiratory burst in this setting (P < 0.01). Our results confirm the findings of Radsak et al. (5) who observed that anti-TREM-1 was able to induce over time a 4-fold respiratory burst increase in human PMN.


Figure 1
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  		Fig. 1 Anti-TREM-1 induces ROS production in peripheral PMN. PMNs (2 x 106 cells ml–1) from human donors were incubated with 1 µg ml–1 of anti-TREM-1 and PMA (4 ng ml–1, as a positive control) for the indicated times (A) and respiratory burst was measured by DCFDA cleavage as described in Methods. Alternatively, PMNs were incubated with GM-CSF (20 ng ml–1) and LPS (1 µg ml–1) in combination with anti-TREM-1 where indicated (B) and respiratory burst was measured. In (C), PMNs were incubated with 10 µM LY294002 (PI3K inhibitor), 20 µM PD98059 (MEK inhibitor) and 100 µM AG490 (Jak2 inhibitor) before loading with DCFDA and stimulation with anti-TREM-1. Stimulation with anti-TREM-1 for 10 min was included as mean of comparison to see the individual contribution of the inhibitors. The asterisk indicates significant differences (P <0.05) compared with the value of TREM-1 at 10 min. The results show the mean MFI ± SEM from five different donors and statistical differences are depicted as follows: *P < 0.05.

 
GM-CSF was shown to prime the fMLP-induced ROS production, but no data exist in connection to TREM-1. In contrast, it was shown that LPS synergizes with anti-TREM-1 to enhance the functional response of PMN (3, 5). We also observed this phenomenon as a small but significant (P < 0.05) increase in the respiratory burst was observed when using LPS in combination with anti-TREM-1 and the same was observed with GM-CSF in combination with anti-TREM-1 (Fig. 1B). To be sure that this was not caused by the LPS-induced increase in TREM-1 expression, we verified by FACScalibur that incubation of PMN with LPS prior to TREM-1 stimulation did not induce the expression of TREM-1 on the cell surface. We found no increase of TREM-1 expression with LPS up to 1 h of stimulation; however, the expression of TREM-1 doubled 2 h after the addition of LPS and stayed the same for up to 6 h after the LPS stimulation (data not shown).

Having observed that anti-TREM-1 could induce respiratory burst and have an additive effect with LPS and GM-CSF, we sought to study the possible contribution of individual signaling pathways important in PMN functions. Using LY294002 (PI3K inhibitor), PD98059 (MEK inhibitor) and AG490 (Jak2 inhibitor), we showed that all these kinases could be implicated in the respiratory burst mediated by anti-TREM-1 measured by DCFDA oxidation (Fig. 1C). DCFDA oxidation derives from several reactive intermediates but is an adequate probe to assess the overall index of oxidative stress (26). Given this, we cannot be certain that the inhibitors used act on the TREM-1-elicited pathways specifically or on the respiratory burst in general. Nevertheless, none of the individual inhibitors used could totally impede the respiratory burst, indicating that redundant pathways in human PMN elicit this function.

Signal transduction pathways elicited by anti-TREM-1 in human PMN
TREM-1 has no functional cytoplasmic tail to initiate signal transduction in cells (27), and it must therefore associate to the immune receptor tyrosine activation motif (ITAM)-containing molecule DAP12 which is expressed in PMNs, NK cells, monocytes and dendritic and mast cells (3, 4, 6, 28). However, the signal transduction pathways elicited by TREM-1 ligation has exclusively been studied in human monocytes and not in PMN (3). We show in this paper, for the first time in human PMN, that the engagement of TREM-1 by anti-TREM-1 results in a phosphorylation of diverse signaling molecules. Figure 2(A–D) shows that the phosphorylation of the Src kinase Lyn (A), Jak2 (B), AKT (C) and ERK1/2 (D) occurs following stimulation of human PMN with anti-TREM-1. AKT and ERK1/2 were phosphorylated after 2 min of stimulation with anti-TREM-1 and the maximum was attained after 10 min for ERK1/2 and 30 min for AKT. Lyn was already phosphorylated after 1 or 2 min of the stimulation and (Fig. 2B and data not shown) Jak2 was phosphorylated 10 min after receptor engagement (Fig. 2B). In a study done in monocytes, ERK1/2 was phosphorylated transiently at 2 min and PLC{gamma} was phosphorylated at 1 min (3). On the contrary, our result in PMN shows a sustained phosphorylation up to 30 min for ERK1/2 (Fig. 2D), and the phosphorylation of PLC{gamma} was only detected at 10 min whereas we did not detect the phosphorylation of p38 (data not shown). As the Lyn and Jak2 kinases are important for human PMN functions (21) and were phosphorylated by anti-TREM-1, we wondered if TREM-1 engagement resulted in the phosphorylation of some downstream transcription factors. Figure 2(E and F) shows that the maximum phosphorylation of STAT5 (E), STAT3 (data not shown) and RelA (F) occurs 10 min after anti-TREM-1 stimulation.


Figure 2
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  		Fig. 2 Anti-TREM-1 induces the phosphorylation of several signaling molecules in human PMN. Purified PMNs (5 x 106 cells ml–1) were stimulated with anti-TREM-1 (1 µg ml–1) or isotype control for indicated times. Inhibitors were used before stimulation to assess the specificity of the immunodetection. The resulting cell lysates were resolved by SDS-PAGE and blotted onto a polyvinylidene fluoride membrane and analyzed using the enhanced chemiluminescence detection system. The membranes were probed with anti-phospho Lyn (A), Jak2 (B), AKT (C), ERK1/2 (D), STAT5 (E) or RelA (F) and antibodies against the non-phosphorylated form were used to assess the equal loading. The blots represent a typical image from five different individuals. Results of densitometric analyses of the phosphorylation from these five different donors are shown as the mean arbitrary unit ± SEM and statistical differences are depicted as follows: **P < 0.01, ***P < 0.001.

 
TREM-1 is recruited to lipid rafts in human PMN upon stimulation
The importance of lipid rafts in PMN and T cells is shown by the recruitment of essential signaling molecules for their functions. Examples are given by the recruitment of Lck and LAT following TCR engagement (29) or in human PMN Src kinases, like Lyn, upon GM-CSF stimulation (21) or under Fc{gamma} ligation (22). Given the wide effects of the engagement of TREM-1 on human PMN, we therefore studied its potential location to the lipid rafts in these cells. Using cholera toxin coupled to fluorescence (CT-488) as a maker that stain ganglioside M1 (GM1) in lipid rafts, we found that TREM-1 is not recruited in the GM1-lipid rafts in resting PMN (Fig. 3A, upper third panel). However, 2 min of stimulation with anti-TREM-1 was able to induce a strong recruitment of this molecule to the GM1-lipid rafts (Fig. 3A, lower third panel). This is the first demonstration that TREM-1 is recruited to lipid rafts in human PMN upon its engagement. The use of bacterial PI–PLC to cleave the GPI anchors, a structure used frequently as a mean of recruitment to lipid rafts in the cells, revealed that TREM-1 was recruited to GM1-lipid rafts by a GPI anchor-dependent mechanism following stimulation (Fig. 3B and C) and confirmed the results of fluorescence microscopy staining. Given the synergistic effects of the stimulation of PMN with TLR ligands and anti-TREM-1, we tested if LPS could cause the recruitment of TREM-1 in the GM1-lipid rafts of human PMN. In resting PMN no recruitment was observed, whereas TREM-1 was recruited to GM1-lipid rafts after up to 1 h of LPS stimulation (data not shown). These results show that TREM-1 is recruited to the GM1-lipid rafts of the human PMN following its activation which results in the well-known effects on PMN functions (3, 5, 30). To test whether this recruitment into the lipid rafts was important to PMN functions, we treated the cells with CD to deplete cholesterol from the membrane. Disrupting the lipid rafts with CD totally abrogate the TEM-1-induced respiratory burst in human PMN (Fig. 3D), indicating the importance of lipid rafts (including GM1-lipid rafts) for TREM-1 activation and functions.


Figure 3
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  		Fig. 3 Anti-TREM-1 induces the redistribution of TREM-1 into the lipid rafts of human PMN. In (A), Purified PMNs were stimulated with 1 µg ml–1 of anti-TREM-1 for 2 min before immunofluorescence staining with TREM-1 coupled to PE and cholera toxin coupled to AlexaFluor 488 to stain GM1 in lipid rafts. These experiments were done with four different donors and at least 10 individual cells were examined for each experiment. The detection of the recruitment into lipid rafts by using bacterial PI–PLC was carried out as described in Methods. Freshly purified PMNs were incubated for 1 h at 37°C with 1 U ml–1 of PI–PLC and thereafter left untreated (B) or treated with 1 µg ml–1 of anti-TREM-1 for 2 min (C). The resulting cells were stained as for immunofluorescence with anti-TREM-1-PE or isotype antibodies and analyzed directly by FACSCalibur. This experiment was done with four different donors and representative results are shown. In (D), PMNs (2 x 106 cells ml–1) were treated with 10 mM CD 1 h 37°C before the measurement of TREM-1-induced respiratory burst by DCFDA oxidation as described earlier for Fig. 1. Statistical differences are depicted as follows: ***P < 0.001 relative to the 10 min stimulation with anti-TREM-1.

 
TREM-1 and TLR4 co-localize upon stimulation of human PMN
It is well known that TREM-1 and TLRs synergize in the production of pro-inflammatory cytokines in monocytes (12, 30, 31) and in PMNs (3, 5). These data indicate a link between the two receptors in cell activation and since a complex of proteins recognizes LPS, we investigated if TREM-1 and TLR4 could be part of the same complex whose ligands could possibly include those of TLR4 and TREM-1. Our data indicate that in resting PMN, TLR4–FITC and TREM-1–PE are not close enough to create an energy transfer. However, upon stimulation with anti-TREM-1 or LPS, a yellow color appears in the overlay (Fig. 4A). Immunoprecipitation studies done with anti-TREM-1 revealed that TLR4 was recruited to TREM-1 immunoprecipitates upon PMN stimulation with 5 min LPS or 2 min anti-TREM-1, but the latter triggers more recruitment (data not shown). Our results indicate that stimulation of human PMN either with LPS or anti-TREM-1 caused TLR4 and TREM-1 to co-localize.


Figure 4
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  		Fig. 4 TREM-1 and TLR4 associate together in the lipid rafts upon PMN stimulation and activate IRAK1 in human PMN. Purified PMNs were stimulated with anti-TREM-1 (1 µg ml–1) for 2 min or with LPS (1 µg ml–1) for 5 min before the immunofluorescence staining as described in Methods. In (A), PMNs were stained with anti-TREM-1 coupled to PE and TLR4 was detected using FITC. In (B), TLR4 was detected using PE and cholera toxin coupled to AlexaFluor 488 to show the recruitment of TLR4 to the GM1-lipid rafts. These experiments were done with four different donors and at least 20 individual cells were examined for each experiment. Purified PMNs were stimulated with anti-TREM-1 (1 µg ml–1) (C) or with LPS (1 µg ml–1) (D). The resulting cell lysates were resolved by SDS-PAGE and blotted onto a polyvinylidene fluoride membrane. The membranes were probed with anti-phospho-IRAK1 and anti-IRAK1 antibodies and analyzed using the enhanced chemiluminescence detection system. Results of densitometric analyses from different donors are shown as arbitrary unit (AU) directly above the lanes, as mean ± SEM. This experiment was done with four different donors and a representative result is shown.

 
Having shown earlier that TREM-1 was recruited to GM1-lipid rafts upon stimulation with anti-TREM-1 or LPS (Fig. 3), we sought to determine if TLR4 was also recruited to lipid rafts. The results show that TLR4 was recruited by LPS stimulation in the GM1-lipid rafts of human PMN (Fig. 4B, lower third panel). Interestingly, using ultracentrifugation as a mean to isolate lipid rafts fractions and western blotting as a detection technique, our group recently described that TLR4 was already present in the lipid rafts of resting human PMN where it was further recruited by LPS stimulation (13). This discrepancy is explained by the differences in the methodology. Immunoblotting reveals the content of all subtypes of lipid rafts, whereas staining for GM1 only reveals the content of that specific subtype of lipid rafts.

We then tested if the stimulation of human PMN by anti-TREM-1 could activate some of the kinases associated with the LPS-signaling pathway. We choose to study IRAK1, as it is the kinase directly activated by phosphorylation via IRAK4, downstream of the adaptor MyD88 (32, 33). The results show that anti-TREM-1 was able to induce the phosphorylation of IRAK1 after 2 min in PMN like LPS did in our experimental settings (Fig. 4C and D, upper panels, second lanes). Interestingly, in contrast to LPS, anti-TREM-1 was not able to induce the degradation of IRAK1 (Fig. 4C and D, lower panels, fourth lanes) as observed after LPS stimulation (34) or IL-1 signaling (33). These results indicate that TREM-1 and TLR4 associate in the GM1-lipid rafts in PMN upon stimulation with LPS or anti-TREM-1 and that TREM-1 is able to drive TLR4 to the lipid rafts when stimulation occurs.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this paper, we sought to investigate the putative effects of TREM-1 activation in PMNs in relation with lipid rafts and TLR4.

It was shown that a longer period of incubation of PMN with agonist antibody against TREM-1 offered no protection from apoptosis, and even suppressed the rescue from apoptosis given by TLR ligands like LPS, Pam3Cys and R-8484. In the same paper, the authors showed that the TREM-1-induced acceleration of apoptosis was not caused by soluble factors (5 and our unpublished data). A possible explanation is that engagement of TREM-1 can activate the Caspase machinery or some negative regulatory phosphatases like SHP-1. This indicates that engagement of TREM-1 on human PMN has opposing effects on their functions. The earlier responses include phagocytosis, respiratory burst, degranulation and enhanced TLR-mediated activation while causing increased apoptosis at long term (3, 5, 6). It is possible that activation of TREM-1 on PMNs and the concurrent ROS production could be related to the acceleration of apoptosis in a manner similar to the ROS-mediated DISC assembly in lipid rafts and activation of Caspase 8 in human spontaneous PMN apoptosis (20). We confirmed the effects of anti-TREM-1 on the respiratory burst in human PMN (Fig. 1A). Interestingly, the similarity between TREM-1 and GM-CSF effects on the respiratory burst observed in this paper probably reflects their common pro-inflammatory nature. These data point out the important and pleiotropic roles of TREM-1 on human PMN functions.

TREM-1 has been shown to associate to DAP12, an ITAM-containing adaptor protein, for the initiation of the signaling cascades in monocytes (3). However, the signal transduction pathways elicited by TREM-1 ligation were not studied in PMNs. We show in this paper data concerning the signal transduction elicited by TREM-1 in human PMN. The data presented throughout Fig. 2 show that engagement of TREM-1 by an agonist elicits similar pathways than that induced by GM-CSFR or TLR stimulations, as well as those induced via DAP12 in monocytes. The pathways studied may contribute to some extent to the phosphorylation of subunits of the NADPH oxidase in the same manner than p38, ERK, protein kinase C (PKC) and PKA were shown to phosphorylate p47phox after fMLP or PMA stimulation of human PMN (35, 36). Likewise, the p110{gamma} subunit of the PI3K was essential for TNF-induced oxidants generation in mice endothelial cells (37), and some data indicate a role for MAPK in the production of ROS following serum-opsonized zymosan stimulation of bovine PMN (38). We also demonstrate here that the Jak2 kinase contributes to respiratory burst activation in human PMN. It is of interest that TLRs and GM-CSFR also activate the MAPKs, PI3K and NF-{kappa}B in human PMN (14, 24, 39). Although the transduction-specific signals and the link to downstream effectors through ITAM are incompletely understood, it seems clear that both the complexity and the strength of the signals activated allow cells to appropriately respond to various ligands by effector functions, anergy, growth, survival or apoptosis (40).

TREM-1 is recruited to GM1-lipid rafts upon stimulation by its agonist or after LPS stimulation (Fig. 3A and D), and this recruitment is dependent on GPI, which anchors proteins in lipid rafts (Fig. 3B and C). Recent data support a model where lipid rafts and their associated GPI-anchored proteins are critical for C1q-triggered superoxide production in PMN (41). It is of note that some of the NADPH oxidase components are already resident of lipid rafts and its cytosolic components are recruited as it has been shown under Fc{gamma}R activation (19, 42). Kinetic analysis of NADPH oxidase activation revealed that lipid rafts determine the onset, but not the maximal rate of enzyme activity. Thus, in that case, lipid rafts served to physically juxtapose the NADPH oxidase effector, PKC and Fc{gamma}R (42) in lipid rafts. We suggest that in human PMN, this recruitment of TREM-1 to lipid rafts could be coupled to the NADPH oxidase assembly in lipid rafts resulting in superoxide anion production, as evidenced by the decrease of the TREM-1-induced respiratory burst when lipid rafts are disrupted by cholesterol extraction with CD (Fig. 3D).

As TLR4 and TREM-1 have been both implicated in sepsis, we investigated whether an interaction exists between these two receptors. We present evidences here that TREM-1 and TLR4 associate together in the GM1-lipid rafts in PMN upon stimulation either with LPS or with anti-TREM-1 (Figs. 3 and 4). It is likely that TREM-1 is able to drive TLR4 to the lipid rafts when stimulation occurs, explaining in part the additive effects on the functions of PMN observed when both receptors are activated as in the case of the respiratory burst (Fig. 1B). Moreover, we found that TREM-1 engagement could induce the phosphorylation of IRAK1, a component of the LPS-signaling cascade. It is thus possible that the TIR domain-containing adaptors of the TLR pathways (MyD88, TIRAP/Mal, TRIF and TRAM) or the scaffolding proteins could mediate this binding. The scaffolding protein Pellino2, for example, can be phosphorylated by IRAK1/4 (43) and links engagements of IL-1 and TLRs to MAPK activation (44) and TRIF has an unsuspected pro-apoptotic activity initiated by Fas/Apo-1 (45). Furthermore, Park et al. (46) showed recently that the high-mobility group box 1 protein could interact with both TLR2 and TLR4 in RAW264.7 macrophage, resulting in a cellular activation similar to that of LPS. The lipid rafts-recruited IRAK1 could be directly activated by TREM-1 or via DAP12, and, also possible, the yet unknown TREM-1 ligand could bind to TLR4 or to the structures responsible for LPS recognition. It is also possible that the TLR4 ligands interact directly with TREM-1 (presented in Fig. 5). All these molecules could contribute to the creation of an 'innateosome' in the membrane of PMN and monocytes, based on lipid rafts, which could recognize and respond efficiently to invading micro-organisms.


Figure 5
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  		Fig. 5 Signaling of TREM-1 and putative interactions between TREM-1 and TLR4 in human PMN. TREM-1 activation on PMN can induce the phosphorylation of IRAK1, a component of the LPS-signaling cascade. Activation of PMN results in the recruitment and association (not depicted) of TREM-1 and TLR4 in lipid rafts. It is thus possible that the TIR domain-containing adaptors of the TLR pathway can mediate the IRAK1 activation, or the lipid rafts-recruited IRAK1 can be directly activated by TREM-1 or DAP12. Moreover, the natural ligands of TREM-1 could also bind to TLR4 or the structures responsible for LPS recognition. This activation of IRAK1 leads via the MAPK pathway to the phosphorylation of NF-{kappa}B. Dotted arrows represent putative interactions.

 
These data presented here shed a new light on how various receptors implicated in the innate immune response could interact to insure an efficient inflammatory response upon pathogens-associated aggression.


   Acknowledgements
 
This work was supported by a grant-in-aid from the Canadian Institute of Health Research (No. 63149) and the Clinical Research Center. The authors would like to thank Jean-François Tessier for critical reading of the manuscript and helpful discussion.


   Abbreviations
 
CD, cyclodextrin
ERK, extracellular signal-regulated kinase
GM-CSF, granulocyte macrophage colony-stimulating factor
GM1, ganglioside M1
GPI, glycosylphosphatidylinositol
IRAK, IL-1R-associated kinase
ITAM, immune receptor tyrosine activation motif
MAPK, mitogen-activated protein kinase
MFI, mean fluorescence intensity
MIP, macrophage inflammatory protein
MyD88, myeloid differentiation factor 88
NF-{kappa}B, nuclear factor-kappa B
PI, phosphatidylinositol
PI3K, phosphatidylinositol 3 kinase
PLC, phospholipase C
PMA, phorbol myristate acetate
PMN, neutrophilic polymorphonuclear
ROS, reactive oxygen species
TLR, Toll-like receptor
TNF, tumor necrosis factor
TREM, triggering receptor expressed on myeloid cell

   Notes
 
Transmitting editor: A. Falus

Received 28 July 2006, accepted 13 October 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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