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Published ahead of print on May 8, 2003, doi:10.1164/rccm.200209-1077OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 158-164, (2003)
© 2003 American Thoracic Society

Original Article

Toll-like Receptor-mediated Tumor Necrosis Factor and Interleukin-10 Production Differ during Systemic Inflammation

Minou Adib-Conquy, Pierre Moine, Karim Asehnoune, Alain Edouard, Terje Espevik, Kensuke Miyake, Catherine Werts and Jean-Marc Cavaillon

UP Cytokines & Inflammation and Unité de Bactériologie Moléculaire et Médicale, Institut Pasteur; Département d'Anesthésie Réanimation, Hôpital Lariboisière, Paris; Département d'Anesthésie Réanimation, Hôpital du Kremlin Bicêtre, Le Kremlin Bicêtre, France; Institute of Cancer Research and Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway; and Institute of Medical Science, University of Tokyo, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Minou Adib-Conquy, UP Cytokines & Inflammation, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail: madib{at}pasteur.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Major trauma is associated with a decreased capacity of patients' leukocytes to produce proinflammatory cytokines on in vitro stimulation. We studied leukocytes from 48 patients with trauma and showed that this hyporeactivity was restricted to gram-negative bacteria, Escherichia coli endotoxin, and unmethylated bacterial DNA, whereas Leptospira interrogans endotoxin-induced tumor necrosis factor production was similar to that observed with healthy donors. As well, tumor necrosis factor and interleukin-6 production in response to gram-positive bacteria was not altered. The expression of toll-like receptor (TLR) 2 was not reduced on patients' monocytes as compared with healthy control subjects, whereas that of TLR4 was reduced. However, the hyporeactivity to gram-negative bacteria and E. coli endotoxin cannot be fully explained by the downregulation of TLR4. Indeed, unlike proinflammatory cytokines, after stimulation with these microbial products the release of antiinflammatory cytokines was increased as compared with healthy control subjects. The increased interleukin-10 production was analyzed in terms of intracellular signaling in peripheral blood mononuclear cells from patients with trauma: our results suggest the involvement of p38 mitogen-activated protein kinase, Sp-1 transcription factor, heterotrimeric Gi protein, and phosphatidylinositol-3'-kinase. In conclusion, the immunodysregulation described for patients with trauma is not a generalized phenomenon but depends on the stimulus and the signaling pathway.

Key Words: LPS • trauma • p38 mitogen-activated protein kinase • toll-like receptor –9

Despite significant advances in the fields of rescue and modern intensive care medicine and the development of new generation antibiotics, late sepsis remains the most frequent cause of complications and death in severely injured patients (1, 2). Sepsis (3, 4), hemorrhagic shock (5), and trauma (6) are associated with a marked depression of cell-mediated immune function, as assessed by a decreased capacity of patients' leukocytes to produce proinflammatory cytokines (tumor necrosis factor [TNF], interleukin [IL]-1ß, IL-6, IFN-{gamma}, and IL-8) in response to endotoxin ex vivo. In addition, anesthesia and surgery (7), blood transfusion (8), and various drugs (9) may have their own immunosuppressive effects. This immunodepression is also assessed by the downregulation of human leukocyte antigen–DR (HLA-DR) expression (10). These phenomena may lead to an increased susceptibility to sepsis and correlate with increased mortality in shocked animals subsequently challenged with a septic insult (5). However, the immune depression found in sepsis and trauma is not a generalized phenomenon. For instance, monocytes from such patients are hyporeactive to LPS but respond to Staphylococcus aureus (SAC) (11), muramyl-dipeptide (12), or phorbol 12-myristate 13-acetate stimulation (13), suggesting that some signaling pathways are defective, whereas others remain functional.

Toll-like receptors (TLR) have been shown to contribute to LPS-induced signaling (14, 15). TLR family members play a major role in sensing microbial molecular patterns. TLR2 is involved in the recognition of gram-positive and gram-negative bacteria, mycoplasma, lipoteichoic acid, and peptidoglycan (16–19). TLR4 is necessary for LPS signaling (14), except for LPS from Leptospira interrogans that signal through TLR2 (20). TLR9 is an intracellular receptor for unmethylated bacterial DNA (21).

The aim of this study was to identify the signaling pathways that are still functional in the monocytes of patients with major trauma. Thus, we analyzed the expression of TLR2 and TLR4 on monocytes from patients with trauma and simultaneously determined their reactivity to microbial products signaling through these receptors (L. interrogans and Escherichia coli LPS, respectively). We also analyzed the reactivity of the leukocytes of patients with trauma to an oligonucleotide carrying the CpG motif, which mimics unmethylated bacterial DNA and signals through TLR9. As infection also implies a contact with whole bacteria and not with purified microbial products, we analyzed the response of patients' leukocytes to several gram-negative and gram-positive bacteria. As a high ratio of IL-10 to TNF was associated with fatal outcome in patients with infection (22), we focused our study on the production of a proinflammatory (TNF) and an antiinflammatory cytokine (IL-10). It is well known that during systemic inflammation, TNF production by patients' leukocytes is defective in response to E. coli LPS (3, 4). We previously showed that the nuclear factor (NF)-{kappa}B pathway, with is necessary for TNF production, was altered in peripheral blood mononuclear cells (PBMC) from patients with sepsis and trauma (23, 24). In the present study, as an imbalance in favor of IL-10 production was noted for leukocytes of patients with trauma, we analyzed the activation of p38 mitogen-activated protein kinase (MAPK) and Sp-1 transcription factor because both are involved in the induction of IL-10 (25, 26). Furthermore, we examined the role of phosphatidylinositol-3'-kinase and of heterotrimeric Gi proteins in IL-10 production.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Selection
The study protocol was approved by the institutional review board for human experimentation. A total of 48 patients with major trauma as defined by an Injury Severity Score of 25 or more were included in this study (27). Exclusion criteria were patients who were under 17 years of age, were pregnant, had preexistence of any autoimmune or immune deficiency disease, or had used steroids or had undergone immune cell-ablative chemotherapy within the previous 30 days. Additional details are provided in an online supplement.

Flow Cytometry
Double staining was performed on PBMC, using an anti-CD14 IgG2b monoclonal antibody coupled to phycoerythrin and an anti-TLR4 (28) or an anti-TLR2 (19) or an anti–HLA-DR monoclonal antibody, all of IgG1 isotype and coupled with fluorescein isothiocyanate, as described (29). Mouse IgG2b-phycoerythrin and IgG1-fluorescein isothiocyanate were used as isotypic controls. The monocyte population was analyzed after gating on side and forward scatters and CD14-positive cells.

Whole Blood Cultures
Blood samples were diluted 1:5 in RPMI-1640 (Glutamax; Biowhittaker, Walkersville, MD) and stimulated with E. coli LPS (O111:B4) at 1 µg/ml (Sigma, St. Louis, MO), L. interrogans LPS (icterohaemorragial, strain Verdun, virulent isolate) at 10 µg/ml, heat-killed SAC at 100 µg/ml (Calbiochem, San Diego, CA), heat-killed E. coli O7 or Neisseria meningitidis group C or Streptococcus pyogenes A78 (105 bacteria/ml), TNF (10 ng/ml) or IL-1ß (20 ng/ml). Stimulation was also performed with an oligonucleotide carrying the CpG motif and its control non-CpG counterpart (6 µg/ml). In some experiments, whole blood was incubated with 10 µM SB203580 (a specific inhibitor of p38 MAPK), 10 µM PD98059 (a specific inhibitor of Erk MAPK), 100 ng/ml pertussis toxin (a specific inhibitor of heterotrimeric Gi proteins) or 10 µM LY294002 (a specific inhibitor of phosphatidylinositol-3'-kinase) (all from BioMol, Plymouth Meeting, PA) before SAC stimulation. Culture supernatants were harvested after 24 hours and frozen at -20°C until tested.

Cytokine Measurements in Culture Supernatants by ELISA
IL-6 and IL-10 concentrations were determined using ELISA kits (DuoSet; R&D Systems, Abingdon, UK). For TNF{alpha} and IL-1 receptor antagonist (IL-1ra), in-house ELISAs were performed (30, 31).

PBMC Isolation and Extract Preparations
PBMC were isolated from blood freshly collected in sodium citrate by centrifugation on Ficoll-Hypaque (MSL, Eurobio, Les Ulis, France) and cultured in the presence of E. coli LPS (0111:B4, 1 µg/ml) or SAC (100 µg/ml). After 45 minutes, the cells were washed with PBS and whole-cell extracts were prepared as described previously (24).

Western Blot
Whole-cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose sheets. Western blot analysis was performed using anti–phospho-p38 and anti-p38 antibodies (Cell Signaling, Beverly, MA). Densitometry analysis was performed using the NIH Image software.

Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay was performed on cellular extracts using a double-stranded oligonucleotide corresponding to the Sp-1 sequence (Promega, Madison, WI). Specificity of binding was assessed by competition with an excess of cold oligonucleotide. Gels were dried and subjected to autoradiography. The Sp-1 complexes were quantified using a PhosphorImager and the ImageQuant software (Amersham Biosciences, Saclay, France).

Statistical Analysis
Data are given as mean ± SEM, except for flow cytometry where the median is indicated. Statistical analyses were performed using the analysis of variance for repeated measurements, the nonparametric Mann–Whitney U test, or the Wilcoxon test. A p value less than 0.05 was considered significant (32).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics
Thirty-five male and 13 female patients with trauma and severe injuries (Injury Severity Score: 40 ± 12 [mean ± SD], range 25–66; Simplified Acute Physiology Score II: 37 ± 15, range 8–78) were enrolled. Their mean age was 35 ± 15 (range 17–80). On admission, 59% of the patients had hemorrhagic shock, 84% received red blood cells units, and catecholaminergic support was necessary for 84% of them. In the posttraumatic course, acute respiratory distress syndrome occurred in 18% of the patients and 59% developed infections. Twenty one percent of the patients died during their stay in the intensive care unit. The mean age of the nonsurvivors was 49 ± 22 (range 20–80), with a mean Injury Severity Score of 36 ± 10 (range 25–57) and a mean Simplified Acute Physiology Score II of 51 ± 12 (range 32–78). Their deaths were attributable to severe progressive brain injury (75%) and to multiple organ failure (25%). The patients enrolled in the different sets of experiments were homogenous, in terms of severity (Injury Severity Score and Simplified Acute Physiology Score II), age (Kruskal–Wallis test), transfusion, catecholamine usage and outcome (Fisher's exact test). A table, indicating the characteristics of the patients in each group of experiments is available in the online supplement (Table 1E). One blood sample was collected from each patient within 48 hours after arrival in the intensive care unit. Some patients developed secondary nosocomial infections, but all the infections occurred after the inclusion of the patients in this study. The patients were compared with 15 healthy control subjects (mean age 36 ± 10, range 25–49), 12 of whom were male.

Surface Expression of TLR2, TLR4, and HLA-DR on Monocytes from Patients with Major Trauma
The expression of TLR2 and TLR4 on the surface of monocytes from patients with major trauma and healthy control subjects was analyzed by flow cytometry. The expression of HLA-DR was also measured because this molecule is a good marker of immunodepression and is downregulated during systemic inflammation (10). Only the mean fluorescence values were compared because the shifts obtained for TLR4- and HLA-DR–positive populations were not enough to allow an accurate determination of the percentage of positive cells (Figure 1A) . As expected, the mean fluorescence intensity for HLA-DR was significantly lower for monocytes of patients with trauma as compared with healthy volunteers (Figure 1B). In addition, the mean fluorescence intensity obtained for TLR4 was also found to be profoundly decreased for patients with trauma versus control subjects, whereas TLR2 expression, though diminished, did not reach significant modification.



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  		Figure 1. The expression of HLA-DR, toll-like receptor (TLR) 2, and TLR4 was analyzed in CD14-positive peripheral blood mononuclear cells (PBMC) from healthy control subjects and patients with trauma. (A) Representative flow cytometry analysis for a healthy control subject and a patient with trauma are shown: gray lines represent the staining with the isotype control antibody, black lines show the specific staining. (B) Mean fluorescence intensities (MFI) are shown for six healthy control subjects and 11 patients with trauma (each of them is represented by a dot). The gray bars represent median values for each group. p Values versus healthy control subjects are indicated.

 
Production of TNF and IL-10 in Response to TLR2-, TLR4-, or TLR9-dependent Stimuli
The production of TNF and IL-10 was analyzed in whole blood assays. We compared the effect of E. coli LPS, which is TLR4-dependent, with that of LPS from L. interrogans, which is TLR2-dependent. The reactivity of the cells to an oligonucleotide carrying the CpG motif, which signals through TLR9, was also analyzed. As shown in Figure 2A , TNF was not produced by unstimulated cells or in the presence of the control oligonucleotide that does not carry the CpG motif (GpC). In contrast, a strong production of TNF was obtained for leukocytes of healthy control subjects stimulated with E. coli LPS. TNF was also produced in response to L. interrogans LPS and CpG oligonucleotide, although the latter two molecules were less potent inducers than E. coli LPS. In the case of patients with trauma, the TNF production in response to E. coli LPS and CpG oligonucleotide was significantly impaired as compared with healthy volunteers, whereas the response to L. interrogans LPS was not affected. We also analyzed the production of IL-10 (Figure 2B). Similar to TNF, we found no spontaneous or GpC-induced IL-10 production. For healthy control subjects, the IL-10 production in response to both types of LPS was very low and mostly below the limit of detection, whereas the CpG oligonucleotide could induce the production of this cytokine. In contrast, the leukocytes of patients with trauma produced IL-10 in response to all three stimuli. The concentration of IL-10 was comparable with that of healthy control subjects in response to CpG oligonucleotide but was higher in response to E. coli or L. interrogans LPS.



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  		Figure 2. Tumor necrosis factor (TNF) and IL-10 production analyzed by ELISA. Whole blood samples from healthy control subjects (white bars, n = 8–14 depending on the stimulation) and patients with trauma (black bars, n = 8–23 depending on the stimulation) were incubated with Escherichia coli or Leptospira interrogans LPS, CpG, or an oligonucleotide that does not contain CpG motifs (GpC), and heat-killed bacteria. p Values versus control values are indicated: *p value less than 0.05, **p value less than 0.01, and ***p value less than 0.005. Ec = E. coli; Nm = Neisseria meningitidis; SAC = Staphylococcus aureus; Strep = Streptococcus pyogenes.

 
Production of TNF and IL-10 in Response to Whole Bacteria
As shown in Figures 2C and 2D, stimulation with whole bacteria resulted in an important production of TNF in the case of healthy control subjects. For patients with trauma, the amounts of TNF measured after stimulation with gram-positive bacteria (SAC, S. pyogenes) was comparable with that obtained with healthy control subjects. Patients' leukocytes were also responsive to E. coli and N. meningitidis. However, TNF production was lower than that obtained with healthy control subjects, especially for N. meningitidis (a 68.5% decrease vs. a 35.7% decrease for E. coli). Gram-positive and gram-negative bacteria induced low level of IL-10 in healthy control subjects, whereas far higher levels of this cytokine were produced by the leukocytes of patients with trauma.

Dose–Response Curves
In the in vitro experiments described previously, our goal was not to mimic any in vivo encounter with bacteria or bacterial-derived products; in contrast, high amounts of activators were used to induce a strong cytokine response in healthy control subjects. However, we performed a dose–response analysis to confirm our results with lower concentrations of the same microbial products (see online supplement, Figures 1E and 2E).

Production of IL-6 and IL-1ra in Response to Various Stimuli
As shown in Figure 3 , IL-6 production, in response to E. coli LPS and CpG, was found to be defective in leukocytes from patients with trauma as compared with healthy control subjects. In contrast, the leukocytes from patients with trauma and healthy control subjects responded similarly to whole gram-positive bacteria. Indeed, IL-6 production was even higher for patients with trauma in response to SAC. Because IL-1 and LPS share a common signaling pathway (MyD88, IRAK, TRAF6) upstream of NF-{kappa}B, which is not the case for TNF, it was of interest to compare the activation of the leukocytes from patients with trauma by both cytokines. This was done by measuring IL-6 release, and it appeared that the production of this cytokine by patients' whole blood was impaired in response to both TNF and IL-1. In contrast to IL-6, and similar to IL-10, IL-1ra production in response to E. coli LPS, SAC, or S. pyogenes was increased for patients with trauma as compared with healthy control subjects. CpG was a poor inducer of IL-1ra, and no differences were seen between patients and healthy volunteers. The stimulation with TNF and IL-1 resulted in the production of comparable amounts of IL-1ra by leukocytes from patients with trauma and those from healthy control subjects.



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  		Figure 3. Interleukin-1 receptor antagonist (IL-1ra) and IL-6 production analyzed by ELISA. Whole blood samples from healthy control subjects (white bars, n = 8–14 depending on the stimulation) and patients with trauma (black bars, n = 9–23 depending on the stimulation) were incubated with LPS from Escherichia coli, heat-killed Staphylococcus aureus (SAC), or Streptococcus pyogenes (Strept), with an oligonucleotide carrying the CpG motif or a control oligonucleotide (GpC), with tumor necrosis factor (TNF) or IL-1. p Values versus control values are indicated: *p value less than 0.05, **p value less than 0.01, and ***p value less than 0.005.

 
Analysis of the Signaling Pathways Involved in IL-10 Production
We previously showed that the NF-{kappa}B pathway, which is necessary for TNF production, was altered in PBMC from patients with trauma after E. coli LPS stimulation but not in response to SAC (24). In the present study, as an imbalance in favor of IL-10 production was noted for patients with trauma, we analyzed the signaling pathways leading to the production of this cytokine. The activation of p38 MAPK, which is important for the induction of IL-10 (25, 26), was measured by analyzing its phosphorylation by Western blot analysis. As shown in Figure 4 , some phosphorylated p38 was detected in unstimulated PBMC. After stimulation with E. coli LPS or SAC, significantly more phospho-p38 was detected. However, the amounts of phospho-p38 found for patients with trauma were higher than those for healthy control subjects: the difference did not reach significance for unstimulated or LPS-challenged cells but was significant for SAC. In addition, we tested the effect of inhibitors of various signaling pathways on SAC-induced IL-10 production. As shown in Figure 5 , the incubation of whole blood samples with SB203850, a specific inhibitor of the p38 MAPK (33), resulted in reduced IL-10 production. Similar results were obtained with LY294002, a specific inhibitor of the phosphatidylinositol-3'-kinase pathway (34), and with pertussis toxin, which inhibits heterotrimeric Gi proteins (35). In contrast, preincubation with PD98059, an inhibitor of the Erk MAPK pathway, did not inhibit IL-10 production in response to SAC, whereas it inhibited TNF production in the same culture conditions (data not shown).



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  		Figure 4. Expression and phosphorylation of the p38 mitogen-activated protein kinase (MAPK) analyzed by Western blot. Whole-cell extracts were prepared from peripheral blood mononuclear cells (PBMC) of healthy control subjects and patients with trauma, after incubation with Escherichia coli LPS or heat-killed Staphylococcus aureus (SAC). (A) Representative results obtained for four healthy control subjects and four patients with trauma. (B) Densitometric analysis of the Western blots (mean ± SEM, n = 6). Phospho-p38 values were normalized against total p38. Unstimulated PBMC: white bars, LPS-stimulated PBMC: gray bars, and SAC-stimulated PBMC: black bars. *p Value less than 0.05 for LPS or heat-killed S. aureus (SAC) versus unstimulated cells (Wilcoxon test), and #p value less than 0.05 for patients with trauma versus healthy control patients (Mann–Whitney U test).

 


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  		Figure 5. Signaling pathways involved in interleukin (IL)-10 production. Whole blood samples from patients with trauma were incubated with Ly294002 (a specific inhibitor of phosphatidylinositol-3'-kinase), SB203580 (a specific inhibitor of p38 mitogen-activated protein kinase [MAPK]), PD98059 (a specific inhibitor of Erk MAPK) for 15 minutes or pertussis toxin (PTX) (a specific inhibitor of heterotrimeric Gi proteins) for 3 hours, before stimulation for 24 hours with heat-killed Staphylococcus aureus (SAC). The presence of IL-10 in culture supernatants was assessed by ELISA. *p Value less 0.05 for inhibitor-treated blood versus whole blood stimulated with SAC without inhibitors (Wilcoxon test, n = 6).

 
Analysis of the Activation of Sp-1 Transcription Factor in PBMC from Healthy Control Subjects and Patients with Trauma
Finally, we studied the activation of Sp-1, a transcription factor known to play a key role in the induction of IL-10 in human monocyte/macrophages (25, 26). Sp-1 activation was analyzed by electrophoretic mobility shift assay, and two Sp-1-binding complexes were detected (Figure 6A) . The specificity of these complexes was assessed by incubating the extracts with an excess of cold oligonucleotides. Whereas the addition of the cold Sp-1 probe abolished DNA binding, no effect was observed after the addition of cold cAMP response element–binding protein (Figure 6B). Sp-1-binding complexes were quantified. As shown in Figure 6C, in unstimulated PBMC from healthy control subjects, some basal DNA binding was found. LPS stimulation failed to increase the amount of Sp-1-binding complexes for healthy control subjects, in accordance with its weak capacity to induce IL-10. In contrast, SAC induced an increase of the upper but not of the lower complex. Extracts from unstimulated PBMC of patients with trauma contained more Sp-1-binding factors, and these complexes were increased by LPS or SAC stimulation.



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  		Figure 6. Activation of the Sp-1 transcription factor assessed by electrophoretic mobility shift assay (EMSA). Whole-cell extracts from peripheral blood mononuclear cells (PBMC) of healthy control subjects and patients with trauma were prepared after a 45-minute stimulation with Escherichia coli LPS or heat-killed Staphylococcus aureus (SAC). (A) Representative EMSA gels for two healthy control subjects and two patients with trauma. (B) The specificity of the detected complexes was assessed using an excess of cold Sp-1 or an oligonucleotide recognized by cAMP response element–binding protein (CREB). (C) Quantification of the upper and lower Sp-1 complexes for healthy control subjects (white bars) and patients with trauma (black bars). *p Value less than 0.05 for patients with trauma versus healthy control subjects (Mann–Whitney U test; healthy control subjects n = 4, patients with trauma n = 8).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have shown that monocytes from patients with major trauma express less TLR4 at their surface and have an impaired TNF production in response to E. coli LPS. The expression of TLR2 was not significantly diminished, and TNF was produced in amounts comparable with those obtained for healthy control subjects in response to L. interrogans LPS, which signals through TLR2 (20). Thus, the defect in TNF production in response to endotoxin, regularly reported during systemic inflammatory response syndrome, may be in part explained by the downregulation of TLR4 and is in accordance with previous endotoxin tolerization experiments showing a downregulation of this receptor (36, 37). Until now, TLR expression during systemic inflammatory response syndrome has been poorly studied: an increase in TLR2 and TLR4 expression was recently reported in monocytes of patients undergoing cardiopulmonary bypass (38). However, in the case of patients with trauma, the decreased TLR4 expression does not entirely explain all our results. Indeed, as will be discussed later, the release of antiinflammatory cytokines in response to E. coli LPS was not diminished, and some intracellular pathways shared with TNF and IL-1 were also altered.

Another important result of this study is that the defect in TNF and IL-6 production is not a generalized phenomenon and depends on the nature of the activating signal. The leukocyte hyporeactivity seemed to be restricted to some microbial products, such as E. coli LPS and CpG, whereas other pathogen-specific molecular patterns, such as L. interrogans LPS, induced unaltered levels of TNF production. In addition, leukocytes of patients with trauma were fully responsive to whole gram-positive heat-killed bacteria and produced large amounts of TNF. In contrast, the reactivity to gram-negative bacteria was decreased as compared with healthy control subjects. Bacteria can activate leukocytes via several pathways, after binding to TLR and/or other types of receptors and after internalization. Studies dealing with a mutant of N. meningitidis devoid of LPS have shown that this mutant signals through the TLR2 molecule (17). Thus, whole gram-negative bacteria can stimulate cells through different TLR pathways, the TLR4 receptor for LPS, and the TLR2 for other membrane components. In contrast, gram-positive bacteria mainly signal via TLR2 (16). When an infection occurs, the innate immune system is the first line to address whole bacteria. Thus, one can be surprised by the higher susceptibility of patients with systemic inflammatory response syndrome to nosocomial infections, as their leukocytes seem to be immunocompetent for responses to whole bacteria, especially in the case of gram-positive bacteria. The increased susceptibility of patients with trauma to infections might be linked to the imbalance between proinflammatory and antiinflammatory cytokines. Indeed, whereas the signaling events leading to the production of cytokines of inflammation, such as TNF and IL-6, were found to be altered in their leukocytes, those leading to the release of antiinflammatory cytokines, such as IL-10 and IL-1ra, were functional and even hyperactive. For all tested stimuli, TNF production by patients' leukocytes was always lower or at the same level to that obtained for healthy control subjects. In contrast, IL-10 production was much higher to that found for healthy control subjects, and thus may have induced some immunodepression or cellular deactivation despite TNF production. In addition, the downregulation of HLA-DR expression on their monocytes may also contribute to the increased susceptibility of these patients to infections due to a less potent antigen presentation that does not allow an efficient adaptive response.

We showed that p38 MAPK and Sp-1 transcription factor were both activated and found in higher levels in the PBMC of patients with trauma as compared with healthy volunteers and that the addition of SB203580, an inhibitor of p38, inhibited IL-10 production in response to SAC. Our data are in agreement with several studies showing the prominent role of Sp-1 in IL-10 induction (25, 39) and the regulation of its activation by p38 MAPK (26). p38 MAPK is mostly known for its proinflammatory properties. Indeed, this MAPK is necessary in association with NF-{kappa}B and Erk for TNF production, and its inhibition leads to a defective TNF release (40). However, our results suggest that p38 MAPK can also contribute to antiinflammatory signaling in the context of an altered NF-{kappa}B activation such as that found in sepsis and trauma (23, 24). On the contrary, in healthy control subjects where the NF-{kappa}B pathway is not defective, p38 was phosphorylated after LPS or SAC stimulation, but it induced a lower Sp-1 activation and failed to induce significant amounts of IL-10.

To characterize the signaling pathways upstream of p38 MAPK, we tested the effects of inhibitors of phosphatidylinositol-3'-kinase and heterotrimeric Gi proteins. Indeed, it has been shown that p38 can be activated through G protein–coupled receptors (41) and Gi heterotrimeric G proteins were coprecipitated with CD14 (42). Phosphatidylinositol-3'-kinase was also shown to be involved in p38 MAPK activation in different cell types (43, 44). Its expression is increased in endotoxin-tolerant macrophages (45), and it contributes to the elevated translation of soluble IL-1ra in leukocytes from patients with sepsis (46). Our results show that preincubation with pertussis toxin inhibited IL-10 production, suggesting the involvement of heterotrimeric Gi proteins in the induction of this cytokine. Similarly, phosphatidylinositol-3'-kinase seems to be involved in IL-10 induction because its inhibition by LY294002 resulted in a decreased IL-10 production.

In conclusion, this study shows that the leukocyte deactivation described for patients with trauma is not a generalized phenomenon but depends on the stimulus and the signaling pathway under study. As summarized in Figure 7 , TLR4- and TLR9-mediated TNF induction, but not TLR2-mediated, were defective. This is in accordance with our previous study showing an impaired NF-{kappa}B activation in PBMC from patients with trauma in response to E. coli LPS but not after SAC stimulation (24). In contrast, TLR2-, TLR4-, and TLR9-mediated IL-10 induction were fully functional in leukocytes from patients with trauma. Our results further suggest that p38 MAPK, heterotrimeric Gi proteins and phosphatidylinositol-3'-kinase are involved in IL-10 induction, probably by increasing the activation of Sp-1 transcription factor.



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  		Figure 7. Schematic illustration of the functional (black), increased (thick), and altered (gray) signaling pathways in leukocytes from patients with trauma. The dotted arrows refer to the literature showing the association of Gi proteins with CD14 (42). Concerning nuclear factor (NF)-{kappa}B, the figure refers to our previous study on the peripheral blood mononuclear cells (PBMC) from patients with trauma (24). BG - = gram-negative bacteria; BG + = gram-positive bacteria; CpG = unmethylated bacterial DNA; Pi3-K = phosphatidylinositol-3'-kinase; TLR = toll-like receptor.

 

   FOOTNOTES
 
Supported in part by a "Program Transversal de Recherche" (PTR), grant 94 from the Institut Pasteur. T.E. is financially supported by the Commission of the European Communities, RTD program: "QLK2-2000-336, HOSPATH."

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form September 20, 2002; accepted in final form May 4, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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