INTRODUCTION
Lipopolysaccharide (LPS)-induced cellular activation can be modulated in vivo , and hyper-response and hyporesponse can be induced in a susceptible host (1, 2 3 4 5, 6
Accordingly, cellular response to LPS and other microbial components is modulated during clinical sepsis, yet the mechanisms are only partially understood. Downregulation of inflammatory response was described in ex vivo experiments with whole blood (7 8 9 10 11 12 13
Monocytes and neutrophils play a central role in the pathophysiology of sepsis and are involved in the protective response as well as in the sequelae of the syndrome (1, 6, 14 15, 16
The induction of LPS signaling is complex and includes interaction with proteins in serum, which may either enhance (LPS binding protein) or decrease LPS activity (bactericidal permeability/increasing protein), and binding to the cell via the glycosyl-phosphatidil-inositol-anchored receptor CD14, which complex to TLR4/MD-2, triggering the LPS signal (15 17 18 19
LPS and other bacterial products may modulate the TLRs expression on the cell surface, which in turn will modulate the cellular adaptation to the insult. Thus, the TLR4 expression extent in mice is associated with the extent of inflammatory response (20 in vitro (21 22
Thus, we evaluated the expression of CD11b, CD11c, CD14, TLR2, and TLR4 on the cell surface from monocytes in whole blood from patients in the different stages of sepsis. To assess these receptors modulation impact on the cellular response to LPS, we measured the production of inflammatory and anti-inflammatory cytokines in PBMC supernatants. We further assessed the cellular response to TNF-α and IL-1β looking for cross-tolerance or a state of cellular refractoriness that would not depend on the expression of TLR2 and TLR4 on the cell surface but rather on intracellular signaling common pathways.
MATERIALS AND METHODS
Patients and Healthy Volunteers
The study was approved by the University and Hospital Ethics Committee of Hospital Sao Paulo and Hospital Santa Marcelina. Written informed consent was provided by volunteers. Forty-one patients with clinical diagnosis of sepsis, severe sepsis, and septic shock were enrolled in the study, according to the criteria of the American College of Chest Physicians/Society of Critical Care Medicine consensus conference (23 24 ) were used as control.
LPS and Recombinant Cytokines
LPS from Salmonella abortus equi , prepared by the phenol-water method and further purified with phenol-chloroform-petroleum ether, as previously described (25
Blood Samples
Sixty milliliters of blood was drawn from both healthy volunteers and patients with sepsis into heparin-treated vacuum tubes, 5 mL into EDTA-treated tubes, and 10 mL into dry tubes (Becton Dickinson, Plymouth, UK). Immunophenotyping was performed in EDTA-treated blood; LPS- and cytokines-induced IL-6, TNF-α, and IL-10 were performed in PBMCs from heparin-treated blood; and detection of sCD14 was done in serum.
Cell Separation and Stimulation
PBMCs were obtained by the ficoll density gradient method (ficoll-paque plus, Amersham Bioscience GE Healthcare, Uppsala, Sweden) and suspended in RPMI 1640 medium (Sigma, St Louis, Miss) supplemented with 10% fetal calf serum, 10 IU/mL penicillin, 10 μg/mL streptomycin (Gibco, Gaisthersburg, Md), and 200 mM L-glutamine (Sigma). The cells viability and count were made with trypan blue dye using a hemocytometer chamber. Standard cell concentration was 4 × 106 cell/mL.
Five hundred of cell suspension was cultured in 24-well plates (Nunclon, Nalge Nunc Int, Denmark). After preliminary dose-response experiments (data not shown), cells were stimulated with 100 ng/2×106 cell of LPS, 10 pg/2×106 cell of IL-1β, or 10 pg/2×106 cell of TNF-α. Cells without stimulus were used to measure the unspecific stimulation. Supernatants were collected after 24 h of incubation at 37°C with 5% CO2 , and cells were discarded after centrifugation at 250× g for 5 min. Cell-free supernatants were aliquoted and stored at −80°C until used for cytokine determination.
Measurement of Cytokines
TNF-α, IL-6, and IL-10 were measured by capture enzyme-linked immunosorbent assays (ELISA). Antibody pairs and reagents were obtained from BD Bioscience Pharmingen for TNF-α and IL-10 assay and obtained from BD OptEIA sets for IL-6 assays. Samples were tested in duplicates, and a standard curve with human recombinant cytokine was built in each plate. Tests were performed according to the manufacturer's instructions. Sensitivity was 10 pg/mL for all cytokines measured. The results obtained with nonstimulated supernatants were deducted from LPS, IL-1β, and TNF-α stimuli.
Measurement of sCD14
Measurement of sCD14 was performed using commercially available ELISA kits (R&D Systems, Minneapolis, Minn). Sensitivity was 250 pg/mL.
Flow Cytometry
Monoclonal antibodies used were the following: CD66b-fluorescein isothiocyanate, clone G1OF5; CD14-peridinin-chlorophyll-protein, clone MφP9; CD11b-allophycocyanin (APC), clone D12; CD11c-APC, clone S-HCL-3; and isotype control mIgG2b-APC, clone 27-35 obtained from BD Bioscience Pharmingen, and TLR2-PE, clone TL2.1; TLR4-PE, clone HTA125; and isotype control mIgG2a-PE, clone MOPC-173 obtained from e Bioscience (San Diego, Calif).
The expression of cell surface receptors on monocytes was performed in whole blood. One-hundred microliters of whole blood from patients and controls were stained with 4 μL of CD14-peridinin-chlorophyll-protein and 5 μL of CD660-fluorescein isothiocyanate. The tubes were also stained with the isotypes 10 μL of mIgG2a-PE and 2 μL of mIgG2b-APC (tube 1); 10 μL of TLR2-PE and 2 μL of CD11c-APC (tube 2), or 20 μL of TLR4-PE and 2 μL of CD11b-APC (tube 3). Samples were incubated with fluorochrome-conjugated monoclonal antibodies for surface staining for 15 min in the dark at room temperature. Red blood cells were ruptured with 2 mL lysis solution (FACS lysing solution, BD Bioscience) for 10 min in the dark at room temperature. Two milliliters of phosphate-buffered saline were added to each tube, followed by centrifugation at 2500× g for 5 min at 4°C. Supernatants were discarded, and cells were resuspended in 0.3 mL phosphate-buffered saline 1% sodium azide.
Events Acquisition and Analyses were performed by CellQuest software (BD Bioscience) in a FACSCalibur 4-color flow cytometer (BD Bioscience). For each condition, 5000 events were counted in forward and side scatter parameters combined with CD14-positive cells, and all events were acquired and stored. Monocyte analyses were performed using forward and side scatter parameters combined with CD14-positive- and CD66b-negative-stained cells. The surface receptor expression was measured as the geometric mean fluorescence intensity, and results were expressed as the difference between the fluorescence obtained with specific antibodies and isotype controls, except for CD14.
Statistical Analysis
The results were analyzed using SPSS software (version 13.0, SPSS, Chicago, Ill). Group comparisons were performed by using the Kruskal-Wallis test. All values, including outliers, were analyzed. The variables that showed differences among the 4 groups were compared group with group by the Mann-Whitney test. The correlation between variables was analyzed by the Pearson correlation test. A P value less than 0.05 was set as statistically significant.
RESULTS
There was no difference in the expression of TLR2 and TLR4 on monocytes among the 4 groups (P = 0.615 and P = 0.873, respectively) (Fig. 1,A and B ). The same was true for CD11c (P = 0.598) (Fig. 1C ). A trend toward differential expression of CD11b was found (P = 0.092), with higher values found in patients with sepsis as compared with healthy volunteers (P = 0.032) (Fig. 1 ). A striking difference was found in the CD14 expression on monocytes among the groups, the highest values being found in healthy volunteers in relation to patients with sepsis (P = 0.001), severe sepsis (P < 0.001), and septic shock (P < 0.001) (Fig. 1E ). This finding contrasted with the sera levels of CD14, the highest values of which were found in the groups of patients with sepsis (P < 0.001), severe sepsis (P = 0.001), and septic shock (P = 0.002) compared with healthy volunteers (Fig. 1F ).
Fig. 1: Surface markers expression on monocytes and sera levels of sCD14 in healthy volunteers, patients with sepsis, severe sepsis, and septic shock . TLR2 (A), TLR4 (B), CD11c (C), CD11b (D), and CD14 (E) are expressed as the geometric mean fluorescence intensity. Sera levels of sCD14 (F) were measured by ELISA and are expressed as ng/mL. Values, represented as box plots, are median, quartiles 25% to 75% (box), and minimum and maximum values (bars). In the cases that the values are higher than 1.5 (Q3−Q1), they are represented as outliers (?). * P < 0.05 compared with healthy volunteers Kruskal-Wallis followed by Mann-Whitney test).
LPS-induced TNF-α differed among the 4 groups (P = 0.001). Patients with sepsis presented higher PBMC supernatant levels (median, 1123.1 pg/mL; range, 107.8-31569.7 pg/mL) as compared with healthy volunteers (median, 341.1 pg/mL; range, 43.9-3328.6 pg/mL) (P = 0.121), whereas those levels were strikingly reduced in patients with severe sepsis (median, 80.9pg/mL; range, 0-1506.7 pg/mL; P = 0.043 compared with healthy volunteers and P = 0.01 compared with patients with sepsis) and septic shock (median, 54.6 pg/mL; range, 0-3436.6 pg/mL; P = 0.002 compared with healthy volunteers and P = 0.001 compared with patients with sepsis) (Fig. 2A ).
Fig. 2: Cytokines levels in PBMC supernatants from healthy volunteers, patients with sepsis, severe sepsis, and septic shock, following LPS, IL-1β, and TNF-α stimulation . TNF-α (A), IL-6 (B), and IL-10 (C) were measured by ELISA and were expressed in pg/mL (differences between stimulated, LPS, IL-1β, and TNF-α, and unstimulated cells, medium alone). Values, represented in the figures as box plots, are median, quartiles 25% to 75% (box), and minimum and maximum values (bars). In the cases that the values are higher than 1.5 (Q3−Q1), they are represented as outliers (?). * P < 0.05 compared with healthy volunteers and # P < 0.05 compared with the sepsis group (Kruskal-Wallis followed by Mann-Whitney test).
The same trend was found with IL-6, with the highest concentrations being detected in patients with sepsis (Fig. 2B ). There was no significant difference among the groups regarding the levels of LPS-induced IL-10 (P = 0.076), and similar values were found in healthy volunteers (median, 384.8 pg/mL; range, 9.1-7786.0 pg/mL) and patients with sepsis (median, 876.1 pg/mL; range, 0-3.700.2 pg/mL) (P = 0.484) (Fig. 2C ). The same pattern of cytokine production was found when IL-1β and TNF-α were used as stimuli, with levels similar with LPS being obtained in healthy volunteers and patients with severe sepsis and septic shock (Fig. 2 ). Accordingly, we found a strong correlation between the levels of cytokines induced by the different stimuli. Correlation between LPS- and IL-1β-induced cytokine was 0.874 for TNF-α production. The same pattern was found with IL-6 and IL-10 induced by LPS, IL-1β, and TNF-α. However, the difference of IL-6 production between sepsis and healthy volunteers observed with LPS (P = 0.041) decreased with IL-1β (P = 0.087) and was not significant with TNF-α (P = 0.737).
There was a correlation between LPS-induced cytokines and the expression of receptors in patients with sepsis, severe sepsis, and septic shock. Interestingly, no correlation was found with TLR2 (Table 1 ). Surprisingly, we also found a correlation between IL-1β- and TNF-α-induced cytokines and the same receptors as in LPS-induced cytokines (Table 1 ).
Table 1: Correlation Among Monocytes Surface Receptors and Cytokine Production by PBMC After LPS, IL-1β, and TNF-α Challenge in vitro
DISCUSSION
Our results show that cellular surface receptors were differently regulated on monocytes across the continuum of sepsis disease. Thus, we observed no difference in the expression of TLR2 and TLR4 among healthy volunteers and patients with sepsis, severe sepsis, and septic shock, an upregulation of CD11b and a downregulation of CD14. We used whole blood for immunophenotyping aiming to describe the modulation going on in vivo in granulocytes (Martins PS et al., manuscript in preparation) and in monocytes. In patients with sepsis, the distinction between monocytes and granulocytes based only on forward and side scatter parameters may be difficult. In this study, we used CD66b and CD14, combined with forward scatter and side scatter, as a strategy to gate on monocytes (CD14+) and granulocytes (CD66b+, CD14−, or weakly positive). Both cell populations could be clearly defined with this approach. The advantages of the use of these antibodies to get well-defined monocytes population was described recently (26
The expression of TLR2 and TLR4 on monocytes during sepsis has driven increasing interest because they are considered the pathogen-associated pattern receptor for gram-positive (among other pathogens) and gram-negative (namely, LPS) products, respectively (16 21
However, studies with human beings have generated conflicting results. In an elegant study, Adib-Conquy et al. (27 28-30 28, 29, 31 30, 31 in vitro effects on the TLR4 expression on monocytes from healthy volunteers, with TNF-α inducing decreased expression and IL-6 enhancing the expression of TLR4, both at the mRNA level and on cell surface (22
Expression of CD14 proved to be lower in our patients than in healthy volunteers, but no difference was found among the different sepsis groups. Decreased CD14 on the cell membrane is likely to be caused by shedding of the receptor because the levels of soluble CD14 in plasma were higher in patients than in the controls, and they mirror the mCD14 results. Decreased expression of CD14 on monocytes and increased sCD14 (32 29, 30
Finally, we found a trend toward different expression of CD11b on the cell surface of monocytes, with the patients in the early stages of sepsis presenting a higher expression than the healthy volunteers. Enhanced CD11b expression on monocytes has been previously described in patients with severe sepsis or shock septic (13
Mononuclear functional activity was evaluated by the production of inflammatory, TNF-α and IL-6, and anti-inflammatory (IL-10) cytokines. Interestingly, we found an upregulation of inflammatory cytokines production in the earliest stage of sepsis, followed by dramatic downregulation in the later stages, that is, patients with severe sepsis and septic shock. Although this concept is accepted, it should be emphasized that it was supported mainly by experimental data, with scarce demonstration in clinical patients (33 34
We found a positive correlation between LPS-induced cytokines and some surface receptors on monocytes. These positive correlations were found for IL-6 with TLR4, CD14, and CD11c but not with TLR2. Although these results point out to the specificity of the finding, we found the same positive correlations also when IL-1β and TNF-α were used as stimuli. This may suggest a positive association with IL-1β and TNF-α receptors, which were not measured, or probably that the regulation is not limited to cell surface receptors. Clearly, the upregulation of LPS-induced inflammatory cytokines seen in patients with sepsis and the downregulation seen in patients with severe sepsis and septic shock, that is the dynamic process seen across the continuum of sepsis, cannot be directly associated with the pattern of receptors modulation found on the monocytes cell surface. As mentioned, no difference among healthy volunteers and the groups of patients with sepsis was found regarding TLR2 and TLR4 expression. The striking mCD14 decrease and enhanced sera levels of sCD14 would explain the decreased cytokine production in the later stages of sepsis, but no difference in their expression (mCD14) or levels (sCD14) was found between severe sepsis/septic shock and the sepsis group, which are markedly increased in LPS response. On the other hand, the increased CD11b expression on monocytes from patients with sepsis compared with healthy volunteers could be associated with enhanced LPS response, but the preserved expression of CD11b in patients with severe sepsis and septic shock would not explain the striking downregulation of cytokines production seen in these patients.
There is conclusive experimental data supporting the role of the receptors, which we evaluated, in LPS response. Accordingly, the levels of TLR4 expression have been shown to determine the LPS response and susceptibility in mice (20 21 18 10, 35 36 in vitro experiment demonstrated that tolerance is related to a decreased TLR4-MyD88 complex formation with impairment of IL-1 receptor-associated kinase 1 but not to the expression of TLR4 on the cell surface. The finding that monocytes from patients with severe sepsis and septic shock are also hyporesponsive to TNF-α and IL-1β point to an intracellular pathway modulation common to LPS, IL-1β, and TNF-α. One possible mechanism is the modulation of NF-κB, as Ziegler-Heitbrock et al. (37 38
In conclusion, our findings confirm the downregulation of inflammatory cytokines seen in severe sepsis and septic shock and add evidence of an upregulation in earlier stages of sepsis, which may be related to pathogen recognition because it is more pronounced for LPS than for IL-1β and TNF-α. In contrast, the downregulation observed in patients with severe sepsis and septic shock appears to be related to intracellular pathways common to LPS, IL-1β, and TNF-α.
ACKNOWLEDGEMENTS
Murillo Assunção and Camila Valentin for the help in the inclusion of patients; Maria da Luz Fernandes and Leandro S. W. Martos for excellent laboratory work, and Clovis de A. Peres and Esper G. Kallas for helpful discussions.
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