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Published ahead of print on August 15, 2002, doi:10.1164/rccm.200203-217OC
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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 1475-1482, (2002)
© 2002 American Thoracic Society

Original Article

Role of Interleukin-10 in the Intracellular Sequestration of Human Leukocyte Antigen-DR in Monocytes during Septic Shock

Thierry Fumeaux and Jérôme Pugin

Departments of Internal Medicine, and Genetic and Microbiology, Division of Medical Intensive Care, and Faculty of Medicine, University Hospital of Geneva, Geneva, Switzerland

Correspondence and requests for reprints should be addressed to Jérôme Pugin, M.D., Division of Medical Intensive Care, University Hospital of Geneva, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: pugin{at}cmu.unige.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes from many critically ill patients show a low level of major histocompatibility complex type II (MHC II) expression. This phenomenon is believed to play a role in these patients' increased susceptibility to secondary infections. In the present study, we show that the level of monocyte human leukocyte antigen (HLA)-DR expression inversely correlates with the degree of severity of the sepsis syndrome. The defect of the monocyte HLA-DR expression resides in an intracellular sequestration of the MHC II molecules, a posttranslational effect. No significant decrease in the rate of transcription of HLA-DR, or its major transcriptional inducer, Class II transactivator, was noted. The levels of HLA-DR protein produced by monocytes from patients with septic shock were comparable to those from healthy volunteers. Plasma from patients with septic shock induced significant HLA-DR endocytosis resulting in decreased surface HLA-DR expression of normal donor monocytes. This effect was partially blocked by anti–interleukin (IL)-10 monoclonal antibody, but not by antagonists to transforming growth factor-ß1, prostaglandins, or ß-adrenergic agonists. Altogether, these data suggest that HLA-DR molecules are re-endocytosed and retained intracellularly in monocytes from patients with septic shock, and that this phenomenon is partially mediated by IL-10. IL-10 may represent a future target for immunomodulating patients with the sepsis syndrome or critically ill patients at risk of developing infections.

Key Words: human leukocyte antigen D • Class II transactivator protein • immune paralysis • major histocompatibility complex type II genes • antigen presentation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on experimental models, sepsis was considered until recently to be the consequence of an overwhelming proinflammatory reaction (1). The failure of the therapies aimed at blocking proinflammatory mediators led to a reconsideration of this hypothesis (2). A growing body of data now strongly suggests that the inflammatory response is compartmentalized during sepsis (3). Whereas a net proinflammatory response has been measured at the site of an infection and in target organs, a net antiinflammatory reaction dominates in the systemic compartment (2, 3). The mediators implicated in this response are interleukin (IL)-10, transforming growth factor (TGF)-ß1, IL-1 receptor antagonist, soluble tumor necrosis factor and IL-1 receptors, catecholamines, and cortisol (3, 4). It has recently been proposed that this antiinflammatory response could be immunosuppressive (3). Supporting this theory is the finding that patients with sepsis have impaired delayed-type hypersensitivity, cutaneous anergy to common antigens (5, 6), and are vulnerable to reactivated cytomegalovirus infection (7).

Immune suppression is accompanied by phenotypic changes of circulating leukocytes, such as impaired cytokine production in response to lipopolysaccharides (8), a situation reminiscent of "endotoxin tolerance" (9). Circulating monocytes have a low surface major histocompatibility complex type II (MHC II) expression in this situation (10–12). Taken together, these changes have been referred to as "immune paralysis" (10). The magnitude of the immune paralysis of patients with sepsis correlates with an increased susceptibility to secondary infections (13) and possibly with late mortality. These changes are also observed in other critically ill patients and may represent a risk factor for nosocomial infections, and thus worsen the prognosis of such patients (14).

The molecular mechanisms responsible for an "immune paralysis" are poorly understood. In vitro studies suggest that TGF-ß1 and IL-10 may play a role (15), particularly in MHC II downregulation (16, 17).

MHC II antigens are mainly expressed at the surface of antigen-presenting cells, and their biosynthesis has been extensively characterized. The transcription of MHC II genes is under the control of Class II transactivator (CIITA) and factors of the Regulatory Factor X family (18). MHC II protein chains are assembled in the Golgi and transported with the invariant chain to the Class II compartment (MIIC) (19). In this compartment, MHC II molecules are loaded with the antigenic peptide, a process dependent on HLA-DM and cathepsin S (20), and are targeted to the cell surface (21, 22). Recycling of the MHC II molecules by endosomal re-endocytosis to the MIIC followed by re-expression of antigenic peptide-loaded MHC II molecules is a characteristic of this pathway.

The aims of our study were: (1) to define which step(s) of MHC II biosynthesis was (were) involved in the observed downregulation of monocyte expression of MHC II molecules; and (2) to identify plasma factors which could induce this process. We show that circulating monocytes from patients with septic shock have a low HLA-DR expression due to an intracellular sequestration of the MHC II molecules, a process that is mediated, at least in part, by bioactive plasma IL-10.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Monocyte HLA-DR Expression
Monocytes were studied from whole blood samples obtained from patients presenting with the sepsis syndrome (23) and in healthy volunteers. In a first cohort of patients with the sepsis syndrome, only HLA-DR expression was studied, whereas in a second cohort of patients with septic shock, more extensive investigations were performed on circulating monocytes at the time of admission, and at Weeks 1, 2, and 3 after admission, when possible (see subsequent text). The institutional ethics committee approved the study protocol. Surface cluster determinant (CD)14 and HLA-DR expression were determined by flow cytometry in circulating monocytes after erythrocyte lysis using fluorescent monoclonal antibodies (mAb). Results were expressed as the ratio of anti–HLA-DR mAb mean fluorescence index over that of isotype control mAb mean fluorescence index and expressed as a percent of mean ratio of volunteers.

Quantitative Polymerase Chain Reaction
Monocytes were isolated with a hypertonic Ficoll technique (24) followed by plastic adherence. The purity and the viability of the monocyte population were found to be over 95% (CD14 and propidium iodide staining, respectively). After lysis in Trizol, monocyte total RNA was extracted and complementary DNAs were generated by reverse transcription. MHC II-related genes and cathepsin S complimentary DNAs were quantified using real-time TaqMan polymerase chain reaction, with primers and fluorescent probes (see the online data supplement). Results were expressed as a percent of the mean value of results from volunteers.

Western Blot
Monocytes isolated by Ficoll, magnetic beads, and plastic adherence were lysed. Lysates were immunoprecipitated with anti–HLA-DR mAb. Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene (PVDF) membrane. Bands were revealed using an anti–HLA-DR mAb, an horseradish peroxidase (HRP)-coupled secondary antibody and enhanced chemoluminescence.

Immunofluorescence Microscopy
Monocytes isolated from peripheral blood mononuclear cells using magnetic beads were cytospinned, fixed in 4% paraformaldehyde, permeabilized with 0.1% saponin, and stained with a mouse anti-human HLA-DR mAb, directly conjugated with fluorescein isothiocyanate (standard immunofluorescence microscopy) or using a goat anti-mouse mAb conjugated to Alexa 488 as secondary antibody (confocal microscopy). To determine the possible plasma-induced re-endocytosis of HLA-DR molecules, leukocytes were, in some experiments, incubated with an anti–HLA-DR mAb, followed by treatment with heterologous plasma from patients with septic shock and from healthy controls. Cells were then fixed, permeabilized, and incubated with an Alexa 488-congugated secondary antibody.

Incubation of Normal Leukocytes with Plasma
Leukocytes from normal donors were incubated with 30% of complement-inactivated plasma from patients with septic shock or from volunteers, in the presence or absence of the following inhibitors: anti–huIL-10 mAb, anti–huTGF-ß1 mAb, acetyl-salicylic lysinate, and 10-5 M metoprolol. In other experiments, leukocytes were incubated with recombinant human IL-10, TGF-ß1, and epinephrine. Monocyte HLA-DR expression was measured by flow cytometry after 24-hour incubation time. Results were expressed as percent of HLA-DR expression of monocytes exposed to heterologous plasma from a volunteer.

Measurements of Plasma IL-10, Procalcitonin, and C-Reactive Protein
Plasma concentrations were quantified using commercially available immunoassays according to the manufacturers protocols.

Statistical Analysis
Results are reported as percent of the mean value of the population of volunteers, unless otherwise specified, and expressed as median plus 90% interquartile range, unless otherwise specified. For the sake of comparison between patients and volunteers, only the values measured during the first 48 hours were considered. Box plots, with representation of median, 75th and 90th percentiles, and outliers, were used for graphic representation of the data. Results between groups were compared with nonparametric paired t test, Mann-Whitney U test, Kruskal-Wallis test, or Spearman correlation, where appropriate. A p < 0.05 was considered significant.

A detailed description of METHODS can be found in the online data supplement.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients
Unless otherwise specified, values presented here are mean ± SD. In the first cohort, 15 volunteers (5 women, 10 men, 35 ± 7 years of age) were compared with 21 patients (sepsis, n = 7; severe sepsis, n = 6; septic shock, n = 8; 10 women, 11 men, 52 ± 25 years of age). In the second cohort, 11 volunteers (3 women, 8 men, 33 ± 2 years of age) and 31 patients with septic shock (8 women, 23 men, 66 ± 3 years of age, mean simplified acute physiology score (SAPS) II score of 47.2 ± 2.6) were included. In this group, septic shock was the consequence of pneumonia (n = 19), urinary tract infection (n = 3), severe skin infections (n = 2), and other infections (biliary tract, meningitis, mediastinitis, and septic arthritis, n = 4). The site of infection could not be determined in three cases. A microorganism was isolated in 26 patients (gram-negative bacteria, n = 17; gram-positive bacteria, n = 9). The most frequently isolated bacteria were Escherichia coli (n = 7), Streptococcus pneumoniae (n = 4), Legionella pneumophila (n = 3) and Pseudomonas aeruginosa (n = 3), Group A Streptococcus pyogenes (n = 2), other Streptococci (n = 2), and methicillin-resistant Staphylococcus aureus (n = 1). Other gram-negative bacteria (Klebsiella pneumoniae, Stenotrophomonas maltophilia, Serratia marcesens, and Moraxella catarrhalis) were cultured in four patients. Mean plasma concentrations of C-reactive protein and of procalcitonin were 287 ± 18 mg/L and 43.9 ± 18.8 ng/ml, respectively. The mean intensive care unit (ICU) length of stay was 15 ± 13 days. The ICU mortality rate was 32% (n = 10).

Surface Expression of HLA-DR in Monocytes
In the first cohort, the monocyte level of HLA-DR expression of patients with sepsis was significantly lower compared with volunteers and there was a trend towards even lower expression in patients presenting with severe sepsis and septic shock (Figure 1). In the second cohort, the level of monocyte HLA-DR expression measured during the first 48 hours was markedly decreased in patients with septic shock compared with volunteers (16%, range 9 to 38%, versus 90%, range 42 to 309%, p < 0.0001; Figure 2). No significant correlation was found between HLA-DR expression and any demographic or clinical variable (age, sex, type of infection, microorganism, SAPS II score, routine laboratory values, or outcome). The level of monocyte HLA-DR expression was measured once a week during the ICU stay, when possible. Although there was a trend towards an increase in the level of HLA-DR expression with time, this trend did not reach statistical significance (Figure 1B). In the first cohort of patients, the monocyte CD14 surface expression in patients with severe sepsis and septic shock was slightly decreased as compared with healthy control subjects and patients with sepsis (see Figure E1 in the online data supplement).



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  		Figure 1. (A) Surface expression of HLA-DR of circulating monocytes from volunteers (n = 15) and patients with the sepsis syndrome (sepsis, n = 7; severe sepsis, n = 6; septic shock, n = 8) measured by flow cytometry during the first 48 hours after ICU admission. (B) Monocytes expression of HLA-DR from volunteers (n = 11) and patients with septic shock (n = 31) at the time of admission (< 48 hr, n = 31), and during the ICU stay (Week 1, n = 22; Week 2, n = 16; Week 3, n = 10). Results are expressed as percent expression compared with the mean value of volunteers (100%).

 


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  		Figure 2. Monocytes levels of MHC II-related genes mRNA in volunteers and in patients with septic shock (less than 48 hours after admission). (A) HLA-DR{alpha} (DRA) mRNA; (B) CIITA mRNA; (C) HLA-DM mRNA; (D) Invariant chain (Ii) mRNA. Results are expressed as percent expression compared with the mean value of volunteers (100%).

 
Monocytes Levels of MHC II-related and Cathepsin S Messenger RNA
To test whether HLA-DR downregulation in patients with septic shock was a transcriptional effect, we measured messenger RNA (mRNA) levels of HLA-DR and of related molecules critical for HLA-DR expression by quantitative polymerase chain reaction. During the first 48 hours of the ICU stay, no significant difference in mRNA levels between patients with septic shock and volunteers was found for CIITA (71%, range 27 to 115%, versus 100%, range 24 to 168%, p = 0.09), DR-{alpha} chain (70%, range 28 to 128%, versus 129%, range 17 to 165%, p = 0.317), HLA-DM (57%, range 20 to 250%, versus 59%, range 33 to 154%, p = 0.93), and invariant chain (61%, range 33 to 172%, versus 93%, range 32 to 200%, p = 0.413) (Figure 2). Levels of mRNA for these genes had a tendency to increase with time during the ICU stay, but the small sample population and the fact that some patients were not tested at all time points limited our ability to perform statistical analysis (see Figure E2 in the online data supplement). No correlation was observed between monocytes MHC II-related genes mRNA levels and surface HLA-DR expression or with any demographic or clinical data. A highly significant correlation was found between DRA, CIITA, HLA-DM, and invariant chain mRNA levels in the patient population (r > 0.5; p < 0.0001, for all correlations). A higher median level of cathepsin S mRNA was measured in patients with septic shock compared with healthy volunteers (186%, range 60 to 961%, versus 79%, range 36 to 179%, p = 0.041) (see Figure E3 in the online data supplement).

Western Blot Quantification of Monocyte Total HLA-DR Protein Content
In an attempt to determine whether the HLA-DR downregulation was due to decreased protein production, we quantified total HLA-DR protein by Western blot of monocyte lysates. Monocytes from 12 patients with septic shock with low HLA-DR expression were compared with those from 6 volunteers with normal levels of expression. Although some visual differences were observed, when normalized for a nonrelevant band on the gel, no difference could be observed between band intensity ratios of monocytes from patients with sepsis and volunteers (0.77, range 0.67 to 0.87, versus 0.80, range 0.52 to 1.06, p = 0.80) (Figure 3). No significant correlation was found between HLA-DR protein content and surface HLA-DR expression.



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  		Figure 3. HLA-DR Western blots. (A) Representative immunoblots showing HLA-DR in monocyte lysates from patients with Septic shock and from Volunteers. The arrow indicates the positive control (HLA-DR in Raji cell lysates). (B) Quantification of HLA-DR band intensity from Volunteers (n = 8) and patients with Septic shock (n = 14). Results are expressed as ratios of relative band intensity (HLA-DR{alpha}/irrelevant band).

 
Cellular Localization of HLA-DR in Monocytes by Immunofluorescence Microscopy
Because both cellular HLA-DR mRNA and protein content were similar in patients with septic shock and in volunteers, the next step was to investigate the cellular localization of the HLA-DR protein. Monocytes from eight patients with septic shock with low surface HLA-DR expression and from four volunteers were permeabilized and immunostained on coverslips. All cells stained strongly with an anti-CD14 mAb. Monocytes from patients and from volunteers stained similarly in intensity with the anti–HLA-DR mAb, and there was no visible fluorescence of the cells incubated with the isotype control mAb for HLA-DR. However, the fluorescence distribution was characteristic of a membrane staining in monocytes from volunteers, whereas virtually all monocytes from patients with septic shock showed a punctuated fluorescence typical of intracellular compartment staining (Figure 4 and see Figure E4 in the online data supplement). Results were found to be very similar between standard and confocal immunofluorescence microscopy. In pulse–chase labeling experiments with anti–HLA-DR mAb, treatment of monocytes from healthy volunteers with septic plasma for 4 hours induced an intracellular redistribution of HLA-DR, which was not found with normal plasma or when cells were incubated with buffers only (Figure 5). Taken together, these data strongly suggest that the intracellular localization of HLA-DR in monocytes from patients with sepsis results from both re-endocytosis and endosomal sequestration mechanisms.



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  		Figure 4. HLA-DR localization in permeabilized monocytes by confocal immunoflurescence microscopy. (A) Representative images of monocytes from two healthy volunteers. (B) Representative images of monocytes from four patients with septic shock.

 


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  		Figure 5. HLA-DR localization in monocytes from healthy volunteers incubated with heterologous plasma for 4 hours (representative experiments). (A) Incubation with plasma from healthy volunteers. (B) Incubation with plasma from patients with septic shock.

 
Plasma IL-10 Concentrations
Because IL-10 is known for deactivating monocytes and causing sequestration of MHC II molecules, we investigated the role of this cytokine in this process. IL-10 levels were found to be elevated in the majority of patients with septic shock, whereas all volunteers had circulating IL-10 levels below the detection limit of the assay (median 11 pg/ml, 90% interquartile range 3 to 97 pg/ml, versus less than 3.9 pg/ml, p = 0.0065, see Figure E5 in the online data supplement). IL-10 plasma concentrations did not correlate with monocyte levels of HLA-DR expression, levels of MHC II-related genes mRNA, or any demographic or clinical data.

Incubation of Normal Leukocytes with Plasma from Volunteers and from Septic Patients, and Effect of Various Antagonists
To test whether IL-10 or other circulating molecules known for modulating MHC II expression were involved in the downregulation of HLA-DR expression in patients with septic shock, we incubated normal donor monocytes with plasma from patients with septic shock or from healthy volunteers. After incubation with "septic" plasma, the level of monocyte HLA-DR expression was decreased compared with incubation with "normal" plasma (p = 0.0027, Figure 6). The addition of a blocking anti-human IL-10 mAb to plasma from patients with septic shock significantly inhibited the plasma-induced HLA-DR downregulation (p = 0.014, Figure 6). The antibody had no effect on HLA-DR expression when monocytes were incubated with plasma from volunteers. Recombinant human IL-10 induced a decrease in monocyte HLA-DR expression in a dose-dependent manner (see Figure E6 in the online data supplement) and the mAb completely blocked the effects of recombinant human IL-10 in our system (data not shown). TGF-ß1, prostaglandins, and ß-adrenergic agonists were also candidate mediators for causing monocyte HLA-DR downregulation. In experiments not shown here, we confirmed in our system that the addition of these mediators induced a significant decrease in HLA-DR expression. The addition to "septic" plasma of a blocking anti–TGF-ß1 mAb, acetyl-salicylic lysinate to block prostaglandins, or metoprolol to block ß-adrenergic agonists, had no effect on the levels of HLA-DR expression.



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  		Figure 6. HLA-DR expression in monocytes from healthy volunteers, incubated 24 hours with heterologous human plasma, or with plasma from patients with septic shock, in the presence or absence of blocking anti–IL-10 mAb. Results are expressed as percent expression compared with the mean value of volunteers (100%).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Typical phenotypic changes of monocytes from critically ill patients include "endotoxin tolerance" and decreased surface expression of MHC II molecules (11). These changes are now commonly referred to as "immune paralysis" (10). Monocyte deactivation and decreased antigen-presenting capacity are believed to play an important role in the immune dysfunction of the critically ill patient (3) and in the development of secondary microbial infections (13).

In the present study, we show that the decreased monocyte expression of HLA-DR, such as that observed in patients with septic shock, is a posttranslational effect. The monocyte production of HLA-DR mRNA and protein chains seems only slightly affected. Re-endocytosis and cellular sequestration of HLA-DR molecules are the prominent mechanisms responsible for this phenotype. The observed HLA-DR downregulation can be reproduced in vitro by incubating normal monocytes with plasma from patients with sepsis, an effect that is partially blocked by anti–IL-10 antibodies.

Our data indicate that MHC II downregulation is a very proximal phenomenon in the development of sepsis. Most of our patients were sampled on the day of admission to the ICU, and already had profound monocyte MHC II downregulation. MHC II levels remained usually very low throughout the ICU stay. It remains unclear whether this phenotype is due to the systemic injury caused by the infectious process or if the MHC II downregulation precedes the bacterial infection, and therefore represents a "risk factor" for the development of sepsis. Sepsis is known to be associated with impaired cellular immunity, such as cutaneous anergy, and decreased delayed-type hypersensitivity (5, 6). Although very likely, a direct relationship between impaired MHC II expression and antigen-presenting capacity remains to be demonstrated in patients with sepsis. Recent reports show that such impaired responses of lipopolysaccharide-treated or "sepsis" peripheral blood mononuclear cells to recall antigens coincides with decreased HLA-DR and/or costimulatory molecules expression (25, 26).

In our first cohort of patients with the sepsis syndrome, the monocyte surface level of MHC II seemed to parallel the disease severity, the lowest levels being found in patients with septic shock. This is not a general phenomenon, because the expression of another monocyte surface receptor, CD14, was only slightly decreased in the same patients. Unlike other studies in critically ill patients (12, 14), we did not find a significant correlation between the monocyte surface level of MHC II and the outcome in patients with septic shock. This may be due to an insufficient number of patients studied or to the fact that all of our septic shock patients had very low MHC II monocyte levels to start with.

The analysis of the MHC II biosynthetic pathway allowed us to identify a likely mechanism responsible for the HLA-DR downregulation in monocytes from patients with septic shock. A small, although not statistically significant, decrease of monocyte HLA-DR {alpha}-chain mRNA levels was observed, and suggested that decreased transcription of the {alpha}-chain may play a limited role. This contrasts with other in vitro studies where the mechanism of MHC II downregulation seems to be directly related to HLA-DR transcription. Landmann and coworkers, for example, have shown that the treatment of monocyte-derived dendritic cells with endotoxin or proinflammatory cytokines was associated with a profound decrease in MHC II production, due to the shutdown of CIITA transcription (27). In monocytes from patients with septic shock, levels of CIITA mRNA were not found to be significantly different from those of volunteers. An important inter-individual variability of mRNA levels characterized both populations, a phenomenon that was reported for other transcripts. In monocytes from patients with septic shock, HLA-DR molecules seemed to be synthesized correctly but were found to reside predominantly in intracellular compartments, most likely in endosomes, as shown by immunofluorescence microscopy. Intracellular sequestration of MHC II molecules has been described in the promonocytic THP-1 cell line infected in vitro with bacteria (28, 29). This phenomenon could be secondary to a block of transport of Class II molecules to the cell surface, enhanced re-endocytosis of surface-expressed complexes, or both. We were able to demonstrate that re-endocytosis and cellular sequestration of Class II molecules could be induced in normal monocytes by incubation with septic plasma. Interestingly, IL-10 induces similar phenotypical changes in monocytes (30) and dendritic cells (31).

The transport of MHC II molecules from the endoplasmic reticulum to the cell surface is a complex process (19). After a short transit through the trans-Golgi network, HLA heterodimers associated with the invariant chain are transported to the Class II compartment, where the antigen loading occurs (20), before being expressed at the cell surface. Alternatively, immature unloaded HLA-DR molecules may be directly transported to the cell surface, together with the invariant chain (32). In a recycling pathway, loaded and unloaded surface HLA-DR molecules are re-endocytosed to the MHC II compartment, where antigenic peptides can be loaded or exchanged, before re-expression to the cell surface (33).

HLA-DM and cathepsin S are important factors implicated in MHC II cellular trafficking. HLA-DM catalyzes the exchange between invariant chain and the antigenic peptide (20) in a pH-dependant manner (34). Cathepsin S, expressed mostly in antigen-presenting cells, seems to play a critical role in the peptide loading and the surface targeting of loaded MHC II molecules (35). Supporting this, cathepsin S inhibition results in a profound defect of MHC II molecules expression in vitro (36) as well as in cathepsin S-deficient mice (37). We did not find significant differences in the monocyte levels of mRNA for invariant chain or HLA-DM in patients with septic shock compared with healthy volunteers. However, this does not rule out the possible involvement of these two molecules in the observed sequestration of MHC II molecules intracellularly, as no studies were done at the protein or functional levels. The mRNA levels for cathepsin S were elevated in monocytes from patients with septic shock. Again, without precise measurement of enzymatic activities, it is not possible to conclude on the role of cathepsins. A narrow acidic pH range of the Class II compartment was described as essential for antigenic peptide loading, and thus for the surface expression of MHC II molecules (34). Additional studies are needed to investigate whether the pH of the MHC Class II compartment is modified in monocytes from patients with septic shock.

Various circulating antiinflammatory mediators have been identified in critically ill patients. Among the most important are IL-10, TGF-ß1, cortisol, catecholamines, prostaglandin E2, and soluble receptors or receptor antagonists blocking the effects of proinflammatory cytokines such as tumor necrosis factor and IL-1 (3). All of these antiinflammatory mediators were at some point implicated in the phenomenon of the "immune paralysis" (15). IL-10 was a likely candidate for mediating the observed monocyte HLA-DR downregulation. This "monocyte-deactivating cytokine" was shown to be elevated in plasma from patients with septic shock (38) and has been implicated in the inhibition of proinflammatory cytokine synthesis, nitrous oxide production, expression of various surface molecules, and the phenomenon of endotoxin tolerance (39). In addition, IL-10 induces in vitro the downregulation of HLA-DR surface expression of "normal" monocytes and of immature dendritic cells obtained from peripheral blood mononuclear cells, with an accumulation of MHC II molecules in intracellular compartments (30, 31), a picture very similar if not identical to that observed in the present study.

It was recently recognized that several biological effects of IL-10 could be posttranscriptional (39). In the case of MHC Class II expression, one possible explanation is that IL-10 modifies the pH of the MHC Class II compartment, as recently proposed (40), and blocks the activity of the cathepsin enzymes required for efficient peptide loading of MHC II molecules and surface expression. Alternatively, IL-10 may increase the activity of cystatin C, a pH-dependent inhibitor of cathepsins (41), or enhance macropinocytosis (42–44).

The signaling molecules activated by the ligation of IL-10 to its receptor and responsible for the posttranslational effect of IL-10 remain to be identified (39). Interestingly, Döcke and coworkers found that interferon-{gamma} restored both monocyte MHC II expression and endotoxin-induced proinflammatory responses in patients with septic shock (16). The competition between interferon-{gamma} and IL-10 to differentially activate signal transducer and activator of transcription (STAT)-1 and STAT-3 may explain the various biological effects of IL-10 (45). Whether interferon-{gamma} overrides the IL-10–induced MHC II downregulation by simply increasing the rate of MHC II transcription or counteracts the "sequestration effects" of IL-10 remains to be determined. Nevertheless, a therapeutic strategy aimed at restoring monocyte function based on IL-10 blockade during sepsis could be proposed, and may compare favorably with interferon-{gamma} treatment, a treatment associated with several potentially harmful side effects. Such a strategy is supported by recent studies showing that IL-10 plasma levels correlated with mortality in various populations of critically ill patients (46–51) and that IL-10 induced an immune dysfunction in infectious animal models (52, 53).

Supporting the role of IL-10 in the downmodulation of monocytes during sepsis, Sfeir and colleagues showed that IL-10 in plasma from patients with septic shock also accounted for the observed decreased monocyte production of proinflammatory cytokines (38). Several hypotheses could be advanced for the lack of correlation between plasma IL-10 levels and the magnitude of HLA-DR downregulation in our patients with septic shock. Many investigators have found that plasma levels of a mediator poorly reflected its bioactivity, due to partial degradation, the presence of circulating natural inhibitors, or alternatively, co-activators. Finally, IL-10 can act in an autocrine manner (54), and IL-10 plasma levels may thus not reflect those found in the monocytic microenvironment.

The incomplete effect of anti-IL-10 antibody to block septic plasma-induced monocyte HLA-DR downregulation prompted us to look for additional soluble factors that may mediate this effect. TGF-ß1 carries both antiinflammatory and profibrotic activities (55) and was shown, in vitro, to inhibit interferon-{gamma}–induced HLA-DR surface expression of various cell types through a transcriptional effect (56, 57). TGF-ß1 has also been described to downmodulate the monocyte surface expression of HLA-DR in association with IL-10 (16). In the present study, although a purified form of TGF-ß1 was able to downregulate donor monocyte HLA-DR expression, its blockade in "septic" plasma had no significant effect on HLA-DR expression. Interestingly, TGF-ß1 did not seem to play a significant role in the decreased secretion of proinflammatory cytokines of monocytes during sepsis either (38). Since aspirin and ß-blockers did not influence "septic" plasma-induced monocyte HLA-DR expression, a participating effect of prostaglandins and ß-adrenergic agonists is dubious. Interestingly, these agonists also seem to inhibit MHC II expression at the transcriptional level (58).

In conclusion, we show here that the observed decrease in HLA-DR surface expression in monocytes from patients with septic shock is due to re-endocytosis and intracellular sequestration of the MHC Class II molecules. This forms the basis for the so-called immune paralysis such as that observed in various populations of critically ill patients. We found that this effect was at least partially mediated by circulating bioactive IL-10. Further studies are needed to investigate whether antagonizing IL-10 may prevent superinfection and favorably influence outcome in critically ill patients.


   Acknowledgments
 
The authors wish to thank the staff and nurses of the medical Intensive Care Unit (Geneva University Hospital); Irène Dunn, Marie-Luce Piallat, and Séverine Oudin, for their help and technical assistance; Salomé Landmann and Walter Reith for stimulating discussions and assistance with RT-polymerase chain reaction; Jean-Claude Chevrolet and Philippe Jolliet for their constant support and help in the preparation of the manuscript; and Michael S. Pepper (University of Geneva) for providing human TGF-ß1.


   FOOTNOTES
 
Supported by a grant from the Swiss National Foundation for Scientific Research #32–50764, and by the Stanley Thomas Johnson Foundation.

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

Received in original form March 18, 2002; accepted in final form July 30, 2002


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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