Abstract
Inflammation is necessary for survival, but it is also an important cause of human morbidity and mortality, as exemplified by sepsis. During inflammation, cells of the innate immune system are recruited and activated in response to infection, trauma or injury. These cells are activated through receptors, such as Toll-like receptors (TLRs), which recognize microbial ligands such as lipopolysaccharide (LPS). Triggering receptor expressed on myeloid cells (TREM)-1 amplifies the inflammatory response initiated by TLRs, and its expression on the surface of monocytes increases in the presence of TLR ligands. Here we have shown that in monocytes TREM-1 mRNA levels, measured by reverse transcription–polymerase chain reaction (RT–PCR), remained unchanged and TREM-1 protein levels, measured by flow cytometry, increased, indicating that LPS increases TREM-1 expression by a post-transcriptional mechanism. We also showed that TREM-1/Fc fusion protein decreased the ability of the sera of some patients with sepsis to activate monocytes, indicating that the TREM-1 ligand, whose identity is unknown, may be present in the sera of some of these patients. We describe a mechanism for the regulation of TREM-1 expression on monocytes and the possible presence of its ligand in serum; these findings help to explain the contribution of TREM-1 during systemic inflammation.
Keywords: DAP12, inflammation, sepsis, TREM-1
Introduction
During inflammation, cells of the innate immune system are recruited and activated in response to infection, trauma or injury. The initial response is local, and the anti-inflammatory response restores homeostasis [1]. The inflammatory response is initiated when innate receptors, such as Toll-like receptors (TLRs), recognize microorganism-associated molecules, such as lipopolysaccharide (LPS) [2–4]. The normal course of an inflammatory response leads to elimination of the causal agent, tissue repair and clearance of the cells involved in the response [5]. However, sometimes the inflammatory response escapes control and proinflammatory mediators are released systematically, causing a generalized inflammatory response in sites remote from the initial injury site. This is known as the systemic inflammatory response syndrome (SIRS) [6]. During SIRS, the anti-inflammatory response can also become systemic, leading to the compensatory anti-inflammatory response syndrome (CARS) [7–9]. CARS is characterized by a low level of human leucocyte antigen (HLA)-DR expression on blood monocytes [10], probably in response to the effects of interleukin (IL)-10 [11,12], and a low level of production of tumour necrosis factor (TNF)-α when these monocytes are challenged with LPS ex vivo [13,14]. Sepsis is defined as a systemic inflammatory response to infection [15].
Triggering receptor expressed on myeloid cells (TREM)-1 is expressed on monocytes and amplifies the inflammatory response initiated by TLRs, signalling through the adaptor molecule DAP12 (DNAX protein of 12 kilodaltons) [16–18]. Our previous studies showed that TREM-1 expression in monocytes is higher and HLA-DR expression is lower in patients with sepsis than in healthy volunteers [19]. We also found that monocytes of patients with sepsis express more of the alternatively spliced form of TREM-1 mRNA (svTREM-1) [19], which is thought to produce a soluble form of TREM-1 [20]. The purpose of this study was to determine whether TLR ligands are capable of increasing the level of TREM-1 mRNA in monocytes. We found that LPS increases the expression of TREM-1 on the surface of monocytes and that this effect depends on the dose of LPS used. Our findings show that LPS increases TREM-1 expression by a post-transcriptional mechanism, because the mRNA levels remained unchanged while the protein levels increased. We also found evidence that the TREM-1 ligand, whose chemical structure remains to be identified, might be present in the sera of some septic patients, because TREM-1/Fc fusion protein decreased the ability of their sera to activate monocytes. These findings confirm that TREM-1 is an important mediator of the systemic inflammatory response and may help to identify the mechanisms responsible for its proinflammatory role.
Materials and methods
Antibodies
Fluorescein isothiocyanate (FITC)-labelled anti-CD14 monoclonal antibody (MoAb; mouse IgG2a, κ, clone M5E2) was purchased from Pharmingen (San Diego, CA, USA). Phycoerythrin (PE)-labelled anti-TREM-1 was from R&D Systems (Minneapolis, MN, USA). PE-labelled mouse IgG1 and FITC-labelled mouse IgG2a (Pharmingen) were used as isotype-matched controls.
Culture of monocytes from buffy coats
Buffy coats were obtained from healthy volunteer donors according to institutional guidelines. Peripheral blood mononuclear cells were obtained by density centrifugation using Lymphoprep™ (Axis-Shield, Kimbolton, UK). A total of 30 × 106 cells were incubated in 10 ml of RPMI-1640 medium supplemented with 5% heat-inactivated fetal calf serum, 2 mM l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (all media and supplements were from Gibco-BRL, Grand Island, NY, USA) and were incubated at 37°C for 1 h. Non-adherent cells were removed by extensive washing, and the remaining adherent cells were recovered by scraping. The mean purity of the enriched CD14+ cells was greater than 85%. Cells were cultured subsequently in 12-well plates (1 × 106 cells/well) and stimulated with 100 ng/ml of Escherichia coli O111 : B4 LPS (Sigma, St Louis, MO, USA) for the indicated times. The supernatants were frozen at −70°C, and the cells were processed for flow cytometric analysis or RNA extraction.
Flow cytometric analysis
A suspension of 1 × 106 cells/ml was prepared in phosphate-buffered saline containing 0·1% sodium azide, 10 mg/ml bovine serum albumin and 200 µg/ml mouse IgG (Sigma), and incubated on ice for 20 min. Subsequent staining with either labelled MoAbs or appropriate isotypic controls was performed on ice for 20 min. The stained cells were washed, resuspended and analysed for two-colour immunofluorescence by flow cytometry (EPICS ALTRA Cytometer, Beckman Coulter, Tokyo, Japan). The gate that contained monocytes was verified by the presence of CD14, and a minimum of 104 cells were analysed for each sample. Results were processed using expo for altra analysis software V2·0 (Beckman Coulter).
Polymerase chain reaction (PCR) analysis
Total RNA of the cultured monocytes was obtained with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. cDNA was synthesized in first-strand buffer with 50 ng/µl oligo-dT primers, 0·5 mM deoxyribonucleosides triphosphate (dNTPs), 10 mM dithiothreitol (all reagents were from Promega, Madison, WI, USA) and 200 U SuperScript II reverse transcriptase (Invitrogen), according to the manufacturer’s protocol.
For TREM-1 and DAP12 amplification, each reaction consisted of Mg-free thermophilic buffer with 0·2 mM dNTPs, 0·5 mM MgCl2, 1 µM primers (5′-GGA CGG AGA GAT GCC CAA GAC C-3′ and 5′-ACC AGC CAG GAG AAT GAC AAT G-3′ for TREM-1 and 5′-ATC CCA CCG GCC CTT ACA CT-3′ and 5′-GGG GAG CGG TCT GGT CTC T-3′ for DAP12 [21]), 1·25 U Taq polymerase (all reagents were from Promega) and 1 µg cDNA. The amplification reaction included 32 cycles of 94°C for 15 s, 60°C for 15 s and 72°C for 20 s, followed by extension at 72°C for 5 min. To verify the RNA integrity, the β-actin gene was simultaneously amplified under previously described reaction conditions. The PCR products were visualized by gel electrophoresis in 2% agarose after ethidium bromide staining.
TREM-1 was also amplified by real-time PCR, with the LightCycler Fast Start DNA Master HybProbe System (Roche Molecular Biochemicals, Basel, Switzerland). Each reaction comprised LightCycler master mix with 2 mM MgCl2, 0·5 µM primers (5′-GCC TCA CAT GCT GTT CGA T-3′ and 5′-GGT ACA AAT GAC CTC AGC GT-3′), 0·2 µM fluorescein-labelled probe (5′-CAG CCA GGA GAA TGA CAA TGT TGA-Fl-3′), 0·2 µM red640-labelled probe (5′-Red640-CAC CGG AAC CCT GAT GAT ATC TGT-3′), 0·2 µM red705-labelled probe (5′-Red705-CAC CGG AAC CCC TTG GTC AC-3′) and 500 ng cDNA. The amplification reaction included one cycle of 95°C for 10 min, 45 cycles of 95°C for 10 s, 55°C for 15 s (single acquisition), 72°C for 14 s and a final cycle of 40°C for 30 s. To verify the RNA integrity, the hypoxanthine phosphoribosyltransferase (HPRT) gene was simultaneously amplified, using TIB MOLBIOL (Berlin, Germany) primers and probes, according to the manufacturer’s protocol. The fluorescence emitted by the samples during amplification was measured using LightCycler equipment (Roche Molecular Biochemicals).
Production of TNF-α and IL-10 from monocytes
The amount of TNF-α and IL-10 in each supernatant was measured using enzyme-linked immunoassay (ELISA) kits (Pharmingen) according to the manufacturer’s protocols. The minimum detectable concentration was 4·7 pg/ml for TNF-α and 7·8 pg/ml for IL-10.
Patient selection criteria
This study was approved by the Hospital Ethics and Research Committee. The patients or their legal representatives received detailed information about this protocol and, if they chose to participate, signed an informed consent form.
The inclusion criteria admitted male or female patients between 18 and 80 years of age who presented with sepsis according to the guidelines established by the American College of Chest Physicians and the Society of Critical Care Medicine [22,23]. The exclusion criteria were recent surgery, pregnancy, treatment with immune suppressors or human immunodeficiency virus (HIV) or hepatitis C virus (HCV) infection or the presence of a chronic disease. A group of 40 healthy volunteers was included in this study for comparison purposes; these individuals also signed an informed consent form.
Detection of TREM-1 ligand in serum
Five millilitres of blood was obtained by venipuncture or from the central catheter. The serum was separated by centrifugation and stored at −70°C.
Monocytes were cultured in 12-well plates (Corning, Corning, NY, USA) as described previously, and 125 µl of serum or 125 µl of serum incubated previously for 30 min at room temperature with TREM-1/Fc fusion protein (R&D Systems) was added. Cells were incubated for 3 h at 37°C, and 100 ng/ml of LPS was added. Cells were incubated for 18 h at 37°C, and the supernatants were collected and stored at −70°C until TNF-α was measured.
Statistical analysis
Data were analysed with the Mann–Whitney U-test, Student’s t- and χ2 tests; P less than 0·05 was considered significant.
Results
LPS increases TREM-1 expression at the protein level but not at the mRNA level
We examined the effect of different doses of LPS on TREM-1 expression on the surface of monocytes (Fig. 1). The addition of 100 ng/ml of LPS significantly increased the percentage of cells co-expressing CD14 and TREM-1 (CD14+/TREM-1+) (P < 0·05 compared with non-treated cells) but had no effect on the mean fluorescence intensity (MFI) of TREM-1. The addition of 10 ng/ml of LPS significantly increased the percentage of CD14+/TREM-1+ cells and the MFI of TREM-1 (both P < 0·05 compared with non-treated cells). The amount of CD14+/TREM-1+ cells induced by the low LPS dose was significantly higher (P < 0·05) than that induced by the high LPS dose.
Fig 1.
Effect of lipopolysaccharide (LPS) on surface triggering receptor expressed on myeloid cells (TREM)-1 expression by peripheral blood monocytes. Monocytes were stimulated with 10 or 100 ng/ml LPS, and TREM-1 expression was analysed by flow cytometry. The results are shown as individual data (cells from different donors) and mean. Significant (P < 0·05) differences between groups are shown (*Mann– Whitney U-test). MFI: mean fluorescence intensity.
We determined the amount of TNF-α and IL-10 produced by monocytes stimulated with 10 ng/ml of LPS for different periods of time. TNF-α was detected in the culture supernatants after 2 h of stimulation; the concentration peaked at 4 h (2100 ± 170 pg/ml) and then decreased. IL-10 presented a similar pattern, but the maximum concentration was reached after 8 h of stimulation (300 ± 40 pg/ml).
To investigate whether the increase in the protein level of TREM-1 resulted from an increase in its mRNA level, we examined the effect of different doses of LPS on the level of TREM-1 mRNA. The addition of LPS did not increase the amount of TREM-1 mRNA that could be detected by agarose gel electrophoresis or by real-time PCR (Fig. 2).
Fig 2.
Effect of lipopolysaccharide (LPS) on triggering receptor expressed on myeloid cells (TREM)-1 mRNA level I human peripheral blood monocytes. (a) Monocytes were stimulated with 10 ng/ml LPS for the indicated times, total mRNA was extracted with TRIZOL reagent, cDNA was synthesized and TREM-1 and DNAX protein of 12 kilodaltons (DAP12) were amplified with Taq polymerase, using the indicated primers. For TREM-1, two sequences [476 base pairs (bp) and 286 bp)] were detected by gel electrophoresis. β-actin was amplified simultaneously to verify RNA integrity and to ensure that equivalent amounts of template were used. (b, d) Monocytes were stimulated with 10 ng/ml LPS for 0, 5, 10 and 40 min, and 2 and 24 h, total RNA was extracted with TRIZOL reagent, cDNA was synthesized and the target genes were amplified by real-time polymerase chain reaction (PCR). The graphs represent the specific fluorescence (F2 or F3) divided by the background fluorescence (F1), plotted against the cycle number of the reaction. (c) Monocytes were treated as in (b), and the expression indices (E.I.) of membrane form of TREM-1 (mTREM-1) and the alternatively spliced form of TREM-1 mRNA (svTREM-1) relative to hypoxanthine phosphoribosyltransferase (HPRT) were calculated. The graphs represent the change in mTREM-1 and svTREM-1 E.I. relative to their E.I. in untreated monocytes. These results are representative of five independent experiments with cells from different donors.
The primers used in the reaction with Taq polymerase enzyme bound to the second and fourth exons of TREM-1 gene and therefore allowed the amplification of two sequences (Fig. 2a). The first one is 476 base pairs (bp) long and corresponds to the mRNA that codes the membrane form of TREM-1 (mTREM-1). The second one is 286 bp long and corresponds to the alternatively spliced form of TREM-1 mRNA (svTREM-1). This sequence does not include the third exon of the gene and is thought to produce a soluble form of TREM-1 [21]. The identity of the PCR products was verified by sequencing. The addition of 10 ng/ml of LPS did not increase the amount of mTREM-1 or the amount of svTREM-1 after 5, 10, 20 or 30 min of stimulation (Fig. 2a), nor did these conditions modify the amount of DAP12 mRNA (Fig. 2a). Similar results were obtained after 40 min and after 2, 12, 24, 48 and 72 h of stimulation with 10 ng/ml of LPS, and with 100 ng/ml of LPS, 10 µg/ml of peptidoglycan, 50 µg/ml poli(I:C) and 50 U/ml of granulocyte–macrophage colony-stimulating factor (GM-CSF) followed by 10 ng/ml of LPS (data not shown).
The previous results were confirmed by real-time PCR. Using this technique, we measured the expression of mTREM-1 and svTREM-1 against the expression of the housekeeping gene HPRT. The levels of HPRT mRNA were not altered by the activation of monocytes with LPS (Fig. 2b). The red640-labelled probe binds to the last nucleotides of the third exon and the first nucleotides of the fourth exon, and is therefore specific for mTREM-1. The red705-labelled probe binds to the last nucleotides of the second exon and the first nucleotides of the fourth exon, and is specific for svTREM-1. The expression indices of mTREM-1 and svTREM-1 relative to HPRT were not significantly modified by the assay conditions used in this study (Fig. 2c). The cDNA samples derived from monocytes activated with LPS for different periods of time had the same threshold cycle in the real-time PCR reaction (22 for mTREM-1 and 25 for svTREM-1) (Fig. 2d), indicating that they contained the same amount of mTREM-1 and svTREM-1.
TREM-1 ligand is present in the sera of some septic patients
There is evidence that the soluble form of TREM-1 is present in the sera of septic patients [24,25]. Our previous results [19] showed a higher level of svTREM-1 in monocytes from septic patients; this mRNA is thought to produce the soluble form of TREM-1. We tried to detect the presence of the TREM-1 ligand, whose chemical structure is unknown, in the sera of septic patients.
We analysed 19 septic patients and 40 healthy volunteers, and measured the level of TREM-1 expression on their monocytes (Table 1). The level of TREM-1 expression was significantly higher in septic patients than in healthy controls, and the level of TREM-1 did not differ between men and women in either group. The infection source was diverse in these septic patients: 92% of the patients who died and 83% of the patients who survived presented with sepsis of abdominal origin, whereas the rest had sepsis of thoracic origin. Positive haemocultures were obtained from 58% of these patients (62% of those who died and 50% of those who survived). Twenty-five per cent of the microorganisms recovered from the patients who died were Gram-negative (E. coli, Pseudomonas), 38% were Gram-positive (Staphylococcus epidermidis, S. aureus, Enterococcus faecium) and 38% were fungi (Aspergillus, Candida). All the microorganisms recovered from the surviving patients were Gram-negative (Pseudomonas, Klebsiella). Age and sex were distributed homogeneously in both groups of patients (Table 2).
Table 1.
Triggering receptor expressed on myeloid cells (TREM)-1 expression mean fluorescein intensity (MFI) on monocytes from healthy volunteers and septic patients.
| n | Mean | SD | Mean difference | P | CI 95% | |
|---|---|---|---|---|---|---|
| TREM-1 (MFI) | ||||||
| * | 40 | 33·6 | 12·6 | 20·4 | 0·026‡ | 3·6–37·4 |
| † | 19 | 54·0 | 34·3 | |||
| TREM-1 (MFI)* | ||||||
| F | 10 | 32·9 | 11·8 | − 0·88 | 0·852§ | − 10·3–8·6 |
| M | 30 | 33·8 | 13·1 | |||
| TREM-1 (MFI)† | ||||||
| F | 8 | 57·2 | 34·0 | 5·6 | 0·738§ | − 28·9–40·0 |
| M | 11 | 51·7 | 35·9 | |||
Healthy volunteers (n = 40)
septic patients (n = 19)
Mann–Whitney U-test
Student’s t-test. F, female; M, male.
Table 2.
Clinical characteristics of the septic patients included in this study (n = 19) and amount of tumour necrosis factor (TNF)-α produced by monocytes in response to their sera.
| TNF-α (pg/ml) | ||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Change | ||||||||||||||
| Without TREM-1/Fc | With TREM-1/Fc | pg/ml | ||||||||||||
| Outcome | A | SD | P* | S | % | P† | Mean | SD | Mean | SD | Mean | SD | % | P† |
| Death (n = 13) | 63·2 | 16·0 | 0·067 | F | 46·2 | 0·599 | 3013·7 | 2290·7 | 2846·8 | 2146·1 | 116·8 | 585·1 | 6·9 | 0·392 |
| M | 53·8 | |||||||||||||
| Survival (n = 6) | 46·5 | 20·2 | F | 33·4 | 4110·3 | 2752·8 | 3709·7 | 2387·1 | 400·6 | 756·2 | 7·8 | |||
| M | 66·6 | |||||||||||||
A, age in years; S, sex (male or female)
Student’s t-test
χ test.
Human peripheral blood monocytes were activated with serum and lipopolysaccharide (LPS) or with serum previously incubated with triggering receptor expressed on myeloid cells (TREM)-1 /Fc fusion protein and LPS for 18 h. The amount of TNF-α in each supernatant was measured by enzyme-linked immunosorbent assay and is reported in pg/ml.
The addition of sera from septic patients and LPS induced the production of TNF-α by monocytes, and the sera from patients who survived induced the production of higher amounts of this proinflammatory cytokine. The addition of TREM-1/Fc fusion protein (R&D Systems) did not significantly change the ability of the septic patients’ sera to activate monocytes, as assessed by TNF-α production. However, in four particular cases the addition of this fusion protein decreased the serum’s ability to activate monocytes by 21·5%, 23·5%, 25·8% and 60·8%, indicating that the TREM-1 ligand may have been neutralized by the fusion protein in these sera.
Discussion
Inflammation is a physiological process that is necessary for survival, but it is also an important cause of human morbidity and mortality [26]. Two dramatic examples of the pathological potential of inflammation are SIRS and sepsis [15,27]. A systemic inflammatory response can be caused by infection, trauma, surgery, burn injury or pancreatitis [1]. Recent intensive study has focused on the molecules involved in the development of the inflammatory response. TREM-1 was first described in 2000 [17] in a report that the expression of TREM-1 on the surface of human monocytes increased in response to Salmonella minnesota LPS, and heat-inactivated P. aeruginosa and S. aureus, but not in response to bacille Calmette–Guérin (BCG) or mycolic acids [18]. Our results are consistent with these previous data because we found that E. coli O111:B4 LPS increased the expression of TREM-1 on the surface of human monocytes in a dose-dependent manner. In our study, a high dose of LPS (100 ng/ml) increased the percentage of CD14+/TREM-1+ cells but not TREM-1 MFI, indicating an increase in the number of cells expressing this receptor but no change in the density of the receptor on each cell. In contrast, a low dose of LPS (10 ng/ml) increased both the number of cells expressing TREM-1 and the density of this receptor on each cell. The percentage of CD14+/TREM-1+ cells induced by a low-dose LPS was higher than the percentage induced by a high-dose LPS, probably because the high dose induced the production of a greater amount of IL-10, a cytokine that decreases TREM-1 expression [11]. TREM-1 signalling amplifies the inflammatory response, but this signalling has to be regulated negatively to restore homeostasis. It has been reported that a soluble form of TREM-1 is released by monocytes in response to LPS, and that this decreases inflammation by capturing and neutralizing TREM-1 ligand in the extracellular milieu [20]. TREM-1 down-regulation by high LPS doses could be another mechanism that regulates the activity of this proinflammatory receptor.
Our cultured monocytes produced typical amounts of TNF-α and IL-10 in response to LPS, but no alterations in TREM-1 mRNA level were detected in response to LPS. We measured the effect of LPS on TREM-1 mRNA expression by two different procedures: PCR with Taq enzyme, in which the products are visualized in agarose gels, and real-time PCR, in which the products are detected by the specific fluorescence emitted by the probes when they bind to their target sequence. In both cases, a housekeeping gene was simultaneously amplified to verify RNA integrity and to ensure that equal amounts of template were used in each case. These findings indicate that the levels of mTREM-1 and svTREM-1 are not altered by activation with LPS, peptidoglycan, poli(I:C), or GM-CSF and LPS. However, the amounts of LPS used in our experiments increased the expression of TREM-1 on the cell surface, suggesting that the expression of TREM-1 in this system is regulated at the post-transcriptional level. Post-transcriptional regulation is an important mechanism that controls the expression of many proinflammatory mediators, whose mRNAs are short-lived in resting cells but become stabilized and efficiently translated upon stimulation with TLR agonists, such as LPS [28]. TNF-α, IL-1β, IL-6 and IL-8 mRNAs contain adenosine and uridine-rich elements (AREs) within their 3′ untranslated regions. The presence of these AREs promotes degradation of the mRNAs unless stabilizing signals are provided through p38, a mitogen-activated protein kinase (MAPK) that is activated after LPS recognition [29]. IL-10, a cytokine induced by LPS, has also been described to destabilize the mRNA of several proinflammatory mediators [30]. It is possible that some of these mechanisms are responsible for the phenomenon that we report here.
The primers used in this study bind to the second and fourth exons of the TREM-1 gene [21] and amplify two sequences of 476 bp and 286 bp, respectively, in non-treated CD14+ monocytes. Begum etal. [31] found that LPS and BCG increase the amount of TREM-1 mRNA in monocytes. Their primers bound to the first and fourth exons of the TREM-1 gene, but they found the alternatively spliced form of TREM-1 only on BCG-treated monocytes and not in non-treated or LPS-treated monocytes. Bleharski et al. [16] reported that TREM-1 mRNA increases in response to LPS; their primers bound to the second and third exons of the gene and therefore detected only mTREM-1. These different results may be explained by the cell culture conditions used in each study and by differences in purity and cell density, which may alter the regulatory mechanisms acting upon monocytes and the signalling pathways that are activated in these cells [32]. During the course of an inflammatory response, the patients’ monocytes are exposed to a great diversity of microenvironments [33], which can alter TREM-1 expression at both the protein and mRNA levels. Our results showed that, even if the amount of RNA does not increase, monocytes can up-regulate TREM-1 expression by a post-transcriptional mechanism.
We also found that the levels of DAP12 mRNA were not altered by activation with LPS, in agreement with the report of Aoki et al. [34]. DAP12 is an immunoreceptor tyrosine-based activation motif (ITAM)-containing immunoreceptor expressed constitutively on various cell types, including natural killer (NK) cells, T and B lymphocytes, dendritic cells, monocytes, granulocytes, basophils, mast cells, osteoclasts and microglia, and it associates with many cell type-specific receptors that are involved in inflammation, anti-viral and anti-tumoral responses and bone remodelling [35]. DAP12 levels tend to remain constant while the levels of its associated receptors change, as is the case reported here with TREM-1.
We compared the expression levels of TREM-1 in monocytes of septic patients with those of healthy volunteers. Consistent with our previous observations [19], we found that TREM-1 levels were higher in septic patients. The larger number of cases analysed in our present study allowed us to determine that the level of TREM-1 is not associated with sex in septic patients or in healthy volunteers. We tried to evince the presence of the TREM-1 ligand in the sera of septic patients by adding TREM-1/Fc fusion protein, because this protein neutralizes the effect of such ligand on monocytes. Although the ability of sera from the septic patients, considered as a group, to activate monocytes in the presence of TREM-1/Fc fusion protein did not change significantly, the sera from four patients showed a decreased ability to activate monocytes. These data suggest that the TREM-1 ligand might have been neutralized by the fusion protein in these sera.
Patients diagnosed with sepsis generally constitute a heterogeneous group, and our patients were no exception. The diagnosis, causative microorganisms and serum concentrations of cytokines can differ between patients and between samples from the same patient obtained at different times during the process of sepsis. Other studies with larger groups of septic patients and with serum samples taken at different times are required to confirm our observations. The TREM-1 ligand could be a microbial product or an endogenous molecule induced by the inflammatory process and released into the bloodstream. In septic patients, microbial products such as LPS can reach serum concentrations of 10 ng/ml [36]. TREM-2, a related protein expressed mainly on dendritic cells, binds to E. coli and S. aureus, and this binding can be inhibited by anionic molecules such as dextran sulphate [37]. It is possible that the TREM-1 ligand is present in the sera of all septic patients but is bound in most by the soluble form of TREM-1 [20,24,25] and therefore cannot be detected by our experimental design. These results reflect the heterogeneity of the patients grouped under the diagnosis of sepsis.
Acknowledgments
The present study was supported financially by Coordinación de Investigación en Salud (CIS), IMSS (grant no. 2005/1/I/069). I. Wong-Baeza and N. González-Roldán received scholarships from Consejo Nacional de Ciencia y Tecnología (CONACyT) and CIS, and N. Esquivel-Callejas received financial support from CIS.
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