help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on March 17, 2004, doi:10.1164/rccm.200309-1329OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200309-1329OCv1
169/10/1144    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Le Tulzo, Y.
Right arrow Articles by Drénou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Le Tulzo, Y.
Right arrow Articles by Drénou, B.
American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 1144-1151, (2004)
© 2004 American Thoracic Society

Original Article

Monocyte Human Leukocyte Antigen–DR Transcriptional Downregulation by Cortisol during Septic Shock

Yves Le Tulzo, Celine Pangault, Laurence Amiot, Valérie Guilloux, Olivier Tribut, Cédric Arvieux, Christophe Camus, Renée Fauchet, Rémi Thomas and Bernard Drénou

Service de Réanimation Médicale et des Maladies Infectieuses, Laboratoire d'Hématologie et de Biologie des Cellules Sanguines UPRES-EA 22-33, Laboratoire de Pharmacologie Clinique et Expérimentale, Centre Hospitalier Universitaire de Rennes, Rennes, France

Correspondence and requests for reprints should be addressed to Yves Le Tulzo, M.D., Ph.D., Service de Réanimation Médicale et des Maladies Infectieuses, Hôpital Pontchaillou, Rue Henri Le Guilloux, CHU Rennes, 35033 Rennes, France. E-mail: yves.le-tulzo{at}univ-rennes1.fr


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocyte deactivation has been identified as a major factor of immunosuppression in sepsis and is associated with a loss of surface human leukocyte antigen–DR (HLA-DR) expression on circulating monocytes. Using flow cytometry, quantitative reverse transcription-polymerase chain reaction, we investigated this phenomenon in septic patients. We confirmed the early loss of monocyte HLA-DR expression in all infected patients and demonstrated that this persistent lowered expression at Day 6 correlated with severity scores, secondary infection, and death. This phenomenon occurred at a transcriptional level via a decrease in the class II transactivator A (CIITA) transcription. Furthermore, these abnormalities correlated with the high cortisol levels observed in sepsis and not with those of other putative factors such as catecholamines or interleukin-10. Finally, in vitro studies evidenced that glucocorticoids decrease HLA-DR expression at a transcriptional level via a decrease in CIITA mRNA levels, mainly by down modulating its isoforms I and III. We conclude that in human sepsis, the loss of HLA-DR expression on circulating monocytes is associated with a poor outcome. We suggest that the high endogenous cortisol level observed in septic shock may be a possible new factor involved in the loss of HLA-DR expression on monocytes via its effect on HLA-DR and CIITA transcription.

Key Words: major histocompatibility complex type II • class II transactivator A • immune paralysis • infection

Septic shock (SS) is an acute systemic disease that is caused by an inadequate response of the organism to microbial invasion and represents the main cause of mortality in adult intensive care units. Despite advance in supportive therapy, a recent report showed only a slight improvement of the outcome (1). Recent hypotheses to explain this persistent poor prognosis take account of the alterations observed in the immune response of SS patients. A systemic hyperinflammatory immune response is described at the very early stage of SS and is rapidly associated with an immune hyporeactivity state termed compensatory antiinflammatory response syndrome (2). This immunosuppressive response may explain the persistence of infection, late nosocomial infections, and death. The defect of the immune response observed in septic patients includes abnormalities of lymphocytes (3, 4) and functional monocyte alterations such as low cytokine production under stimulation (5) and reduced human leukocyte antigen–DR (HLA-DR) expression (6). Mechanisms leading to the loss of HLA-DR molecules on monocytes in SS are only partially determined. Proinflammatory cytokine production by monocyte can be modulated by endogenous mediators such as cortisol (7), catecholamines (8), and interleukin (IL)-10 (9, 10); however, the exact role of these mediators in the loss of HLA-DR expression is not fully elucidated even if several soluble factors such as catecholamines, IL-10, or lipopolysaccharide have been suspected (6, 11, 12). In vitro studies evidenced that constitutive and IFN-{gamma}–inducible expressions of HLA-DR are predominantly regulated at a transcriptional level (13–15), mainly by the non–DNA-binding class II transactivator A (CIITA). CIITA is recruited onto the class II promoter and interacts with the enhanceosome constituted by DNA-bound factors such as regulatory factor X, nuclear factor-Y, and cAMP response element-binding protein (CREB), which bind to the cis-acting elements X1, X2, and Y boxes identified in the enhancer of class II genes (14). Then CIITA activates transcription through the activation of other coactivators such as CREB-binding protein (CBP) or CBP-associated factor. Thus, CIITA is the major regulator of class II gene expression and is the target of factors that regulate positively or negatively these genes.

This study includes two parts: In the first, we demonstrated that the loss of HLA-DR expression on monocyte is an early event in sepsis and that the persistence of this alteration is associated with severity score, nosocomial infection, and death. In a second, we investigated the mechanisms involved in this downregulation. We evidenced that the loss of HLA-DR expression on monocyte observed in septic patients involves transcriptional regulation via a decrease in its transactivator CIITA. Furthermore, we evidenced an in vivo relationship between HLA-DR expression, HLA-DR mRNA, CIITA mRNA, and the high level of endogenous cortisol observed in septic patients. Finally, to confirm the role of the glucocorticoid hormone in the loss of HLA-DR molecules by the monocytes in this setting, we reproduced in vitro the down-modulating effect of glucocorticoids on the transcription of HLA-DR via CIITA. Preliminary results have been previously reported in the form of an abstract (16).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
A detailed description of METHODS is provided in the online supplement.

Patient Selection
This study represents an extension of a previous one in which we reported the significance of lymphocyte apoptosis in septic patients (4). In a first cohort of patients, monocyte HLA-DR expression and transcription were investigated in SS, sepsis without shock, and control subjects. Patients under 18 years with malignancies who received chemotherapy, immunosuppressive agents, steroids, or ß-adrenergic blockers were not included. We used standard definitions for sepsis and SS (2). Recent infection was considered when a blood culture or normally sterile body fluid obtained within 3 days before or 3 days after the day of inclusion grew pathogenic microorganisms or when sepsis was accompanied by an infectious focus requiring antibiotics. The Simplified Acute Physiology Score II at Day 1 (17) and the logistic organ dysfunction system at Day 6 (18) were used to assess severity. Nosocomial infections were collected according to Centers for Disease Control definitions (19). In addition, as we suspected time variations of HLA-DR expression, we compared HLA-DR expression in septic patients at Day 1 of the study with that observed 5 to 7 days after (Day 6). In a second cohort of septic patients selected according to the same criteria than in the first one, HLA-DR and CIITA expressions were evaluated on isolated monocytes (discussed later here) and compared with control subjects. This study was approved by the Institutional Review Board of the Rennes University Hospital.

Flow Cytometry Protocol
Venous samples were immediately processed and incubated with PE-Cy5 anti-CD14 and PE anti–HLA-DR monoclonal antibodies using saturating concentrations and analyzed using flow cytometry. HLA-DR cell surface density was expressed in linear mean fluorescence index. Nonviable cells were excluded using propidium iodide. The same protocol was used for all the patients. Multivariate data of 50,000 events were collected in list mode, stored, and analyzed.

Purification of Patient Monocytes
Monocytes from patients of the second cohort and from their controls were isolated by Ficoll density gradient centrifugation and magnetic microbeads (Miltenyi). The purity of positive fraction (monocytes) evaluated using anti-CD14 monoclonal antibodies was found to be over 90%.

Soluble Factors Measurement
Plasmas from heparinized blood samples were obtained between 7 and 8 A.M. and were evaluated for cortisol (chemiluminescence), epinephrine, norepinephrine (HPLC), and IL-10 (cytometric bead array).

Real-time Quantitative Polymerase Chain Reaction
Total RNA was isolated from total blood (first cohort of patients) or from isolated monocytes (second cohort and in vitro studies) using RNAble reagent. cDNA was prepared by reverse transcription. Polymerase chain reaction (PCR) amplifications of Abelson gene, CIITA (total, pI, pIII, pIV), or HLA-DR transcripts were performed using ready-to-use PCR Mastermix, sense and antisense primer, specific minor groove binding probe, or Sybergreen reagent and cDNA. Each PCR reaction was run concomitantly as duplicate under identical conditions. Sybergreen-amplified product specificity was confirmed by dissociation curve analysis. The transcript amount for each gene was normalized to the Abelson housekeeping gene expression and compared with control subjects.

In Vitro Stimulation of Normal Monocytes
Monocytes isolated from healthy blood donors by Ficoll and adherence were incubated with medium containing norepinephrine, IL-10, and dexamethasone with or without the glucocorticoid receptor antagonist RU 486 and harvested for flow cytometry analysis and RNA isolation as previously described.

Statistical Analysis
Data are given as means ± SEM. Differences were analyzed using analysis of variance and Fisher's test or nonparametric tests when appropriate. Relationships between continuous variables were analyzed using regression analysis.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics
In the first cohort of patients, forty-eight patients who fulfilled inclusion criteria entered the study over a 14-month period: 23 SS (average age, 64.05 years; range, 33–86 years; median, 68 years) and 25 sepsis patients without shock (average age, 55.16 years; range, 21–82 years; median, 59 years). They were compared with 25 control patients (2 chronic obstructive pulmonary diseases, 4 preoperative patients, 2 suicide attempts, and 17 healthy volunteers) (average age, 55.76 years; range, 22–80 years; median, 66 years). Among the 48 septic patients included in the study, 10 died after the first evaluation, and 8 were discharged before Day 6. Consequently, only 30 septic patients were evaluated at Day 6; among them, 24 survived and 6 died between Day 6 and Day 57. Other characteristics of septic patients and sepsis origins are shown in Tables 1 and 2 . The second cohort consist of nine consecutive septic patients (average age, 46 years; range, 20–71 years; median, 45 years) (Simplified Acute Physiology Score II, 49 ± 8). In this group, infections consist of endocarditis (1), mediastinitis (1), peritonitis (1), pneumoniae (3), and meningitis (3). Three were related to a Gram-positive agent, three to a Gram-negative agent, and one to miscellaneous agents, and two were not documented because of early prehospital antibiotic treatment.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Septic patient characteristics

 

View this table:
[in this window]
[in a new window]
  		TABLE 2. Sources of infection and microorganisms isolated in septic patients

 
Clinical Significance of the Loss of HLA-DR Expression on Monocytes in Sepsis and Relationship with Outcome
Using data from the first cohort, we demonstrate that in septic patients, the loss of the class II molecule HLA-DR expression on monocytes is linked to the severity of the disease and to its unfavorable evolution. At Day 1, monocyte HLA-DR expression evaluated by flow cytometry was reduced in all infected patients (Figure 1A) . Thirty septic patients were analyzed at Day 6, and the differences observed in the ability to restore HLA-DR were related to severity of the disease and outcome. Compared with Day 1, HLA-DR expression remained low at Day 6 in SS patients (7.5 ± 2 and 8 ± 1.5 mean fluorescence index, respectively, p = NS, n = 12), whereas in nonsevere sepsis, this expression significantly increased (10 ± 3 to 17 ± 4 mean fluorescence index, 68%, respectively, p < 0.01, Wilcoxon's test, n = 18). Furthermore, patients who acquired secondarily nosocomial infections (Figure 1B) or those who died (Figure 1C) exhibited a persistent loss of HLA-DR expression, whereas patients free from nosocomial infection or those who survived restored HLA-DR expression at Day 6. Confirming this link to severity, HLA-DR expression on monocytes at Day 6 correlated negatively with the number (Figure 2A) and the intensity of organ failures calculated at Day 6 (Figure 2B). These results were confirmed by those of the second cohort in which the levels of monocyte HLA-DR expression was reduced when compared with those of control subjects (29 ± 4 vs. 94 ± 3 mean fluorescence index, respectively, –69%, p < 0.001).



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Evolution of human leukocyte antigen–DR (HLA-DR) expression on circulating monocytes in septic patients according to severity. Monocyte HLA-DR expression evaluated by flow cytometry at Day 1 (A) was reduced both in nonsevere sepsis (S) and septic shock (SS) groups when compared with the control group (C) (–66% and –78%, respectively, p < 0.0001, analysis of variance, Fisher's test). Six patients acquired late nosocomial infections (secondary infection) (columns 1 and 2) in the unit and were evaluated at Day 6 (B): they exhibited a persistent loss of HLA-DR expression when compared with Day 1 (8 ± 3 vs. 6 ± 3 mean fluorescence index [MFI], respectively, p = NS). In contrast, patients free from nosocomial infection (no infection) (columns 3 and 4) partially restored HLA-DR expression (50%, p < 0.05, Wilcoxon's test). Monocyte HLA-DR expression increased between Day 1 and Day 6 from 10 ± 3 to 15 ± 3 MFI (50%, p < 0.05, Wilcoxon's test) in only patients who survived (n = 24) (columns 3 and 4) but not in patients who ultimately died (n = 6) (columns 1 and 2) (C).

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. HLA-DR expression on monocytes is related to severity scores. In septic patients, a significant relationship was observed between HLA-DR expression on monocytes at Day 6 and the number of organ failures (r = 0.573, p < 0.001) (A). A similar relationship was observed between HLA-DR expression on monocytes at Day 6 and the severity of the organ failures (Logistic Organ Dysfunction score) (r = 0.542, p = 0.002) (B).

 
The Loss of Monocyte HLA-DR Expression Involved a Transcriptional Regulatory Mechanism
Eighteen infected patients of the first cohort were evaluated for HLA-DR and CIITA transcription. In all of these patients, we observed a decrease in the level of HLA-DR transcription (Figure 3A) . Furthermore, the positive in vivo relationship observed between the levels of mRNA and those of the cell surface protein for HLA-DR (Figure 3B) confirmed that the loss of HLA-DR expression was under transcriptional regulation; we confirmed this hypothesis by evidencing a decreased transcription of the main class II transactivator CIITA in nonsevere sepsis and SS patients compared with control subjects (Figure 3C). Moreover, CIITA mRNA levels correlated with cell surface expression and mRNA HLA-DR levels (Figures 3D and 3E). To confirm these results, we evaluated HLA-DR and CIITA transcription on purified monocytes of the second cohort of nine patients after isolation by Ficoll and positive selection by microbeads (Figure 3F). We observed a comparable decrease in HLA-DR transcription (–82%, p < 0.001) and in CIITA transcription (–57%, p < 0.05) when compared with the set of control patients. Furthermore, this decrease was observed in all the CIITA isoforms I, III, and IV transcription (–97%, p < 0.001; –87%, p < 0.001; and –80%, p < 0.01, respectively).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. Analysis of HLA-DR regulation by quantitative polymerase chain reaction in septic patients. A significant decrease (up to –50%) in mRNA levels for HLA-DR (A) was observed in a first cohort of patients with nonsevere sepsis (S, gray circles) (n = 11) and in those with septic shock (SS, black circles) (n = 7) when compared with control patients (open circles, C) (n = 12). *p < 0.01, Fisher's test. A positive relationship (r = 0.764, p < 0.0001) was observed between HLA-DR mRNA levels and HLA-DR cell surface expression on monocytes (B). Total class II transactivator A (CIITA) mRNA levels were significantly reduced in patients with nonsevere sepsis (n = 11) (–43%, p < 0.01) and in patients with SS (n = 7) (–70%, p < 0.0001, Fisher's test) when compared with levels observed in control subjects (C). Positive relationships were observed between CIITA mRNA levels and HLA-DR cell surface expression (D) (r = 0.576, p < 0.001) and between CIITA and HLA-DR mRNA levels (E) (r = 0.599, p < 0.001). These results are confirmed in a second cohort of nine septic patients (F): HLA-DR, total CIITA, and types I, III, and IV CIITA isoforms mRNA levels measured after monocyte purification were significantly reduced when compared with control subjects (–82%, p < 0.001; –57%, p < 0.05; –97%, p < 0.001; –87%, p < 0.001; and –80%, p < 0.01, respectively).

 
Putative Circulating Factors Involved in the Loss of HLA-DR Expression on Monocytes
We measured cortisol, catecholamines, and IL-10 plasma levels, all factors that have been implicated in the antiinflammatory response observed in SS, and we determined their relationship with HLA-DR expression on circulating monocytes. Cumulative levels of epinephrine and norepinephrine significantly increased in SS patients (n = 21) when compared with nonsevere sepsis (n = 25) (5,258 ± 1,328 vs. 974 ± 192 pg/ml, p < 0.0001) or with control subjects (254 ± 17 pg/ml, p < 0.0001). IL-10 significantly increased in SS patients (n = 21) compared with nonsevere sepsis (n = 25) (414 ± 230 vs. 40 ± 24 pg/ml, respectively, p = 0.0001) and remained below the detection limit of the assay in control group, but we observed no relationship between these two factors and HLA-DR expression on circulating monocytes. Cortisol levels significantly increased in SS patients (n = 20) (248 ± 23 ng/ml, p < 0.01) and in nonsevere sepsis (n = 24) (211 ± 23 ng/ml, p < 0.05) compared with control subjects (147 ± 10 ng/ml) (Figure 4A) , and interestingly, cortisol levels correlated negatively with HLA-DR expression (Figure 4B). Furthermore, cortisol levels correlated in a similar relationship with HLA-DR mRNA (Figure 4C) and CIITA mRNA (Figure 4D). Consequently, we deemed that the increased levels in endogenous cortisol observed during sepsis could be involved in the loss of expression of HLA-DR on circulating monocytes.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 4. Role of the high circulating cortisol levels in HLA-DR transcription and expression on monocytes. A significant increase in cortisol levels was observed in SS (black circles) patients (n = 20) and sepsis (gray circles) patients (n = 24) when compared with control subjects (open circles) (n = 20) (A) (*p < 0.05, **p < 0.01, Fischer's test), and a negative logarithmic relationship was observed between cortisol levels and HLA-DR expression on patient's monocytes (B) (r = 0.403, p = 0.001). Similar negative logarithmic relationships were also observed between cortisol levels and HLA-DR mRNA (C) (r = 0.596, p = 0.0034) and CIITA mRNA levels (D) (r = 0.491, p = 0.02).

 
Effect of Corticosteroids on HLA-DR Expression and on Its Transcriptional Regulation
To confirm the in vivo data previously described, we performed in vitro experiments on purified normal monocytes. We tested whether the glucocorticoid dexamethasone regulates HLA-DR expression on purified monocytes in vitro. Dexamethasone induced a decreased membrane expression of HLA-DR on cultured monocytes when compared with control monocytes (52 ± 7 vs. 41 ± 6 mean fluorescence index, respectively, n = 14, p < 0.01, Wilcoxon's test) (Figure 5A) . Moreover, dexamethasone decreased HLA-DR mRNA when compared with nonstimulated monocytes (–66%, n = 13, p < 0.01, Wilcoxon's test) (Figure 5B). This effect on the transcriptional regulation of HLA-DR was confirmed by the down-modulating effect of dexamethasone observed on its transactivator CIITA (–60%, n = 13, p < 0.01, Wilcoxon's test), mainly by an effect on the CIITA forms I and III expression (–85% and –57%, respectively, p < 0.01, Wilcoxon's test), whereas CIITA form IV was slightly decreased (–30%, p < 0.01, Wilcoxon's test) (Figure 5C). Furthermore, the specificity of this downmodulation by dexamethasone was evidenced in six experiments that demonstrated the blockade of the effects of dexamethasone on HLA-DR and CIITA transcription by pretreatment with the inhibitor of the glucocorticoid receptor RU486 (Figure 6) . Moreover, we observed a similar effect of the blockade of the glucocorticoid receptor on the downmodulation of CIITA isoform transcription induced by dexamethasone (data not shown). Contrasting with this result, we found no in vitro effect of norepinephrine or of IL-10 on the expression of HLA-DR (data not shown).



View larger version (13K):
[in this window]
[in a new window]
  		Figure 5. In vitro effects of the glucocorticoid dexamethasone on the regulation of HLA-DR expression in monocytes. Effect on HLA-DR cell surface expression (A): compared with control cultures (unstimulated monocytes), monocytes cultured 24 hours in the presence of the glucocorticoid dexamethasone exhibited a decrease in HLA-DR expression (n = 14, –21%) (*p < 0.01, Wilcoxon's test). Effect on HLA-DR transcription (B): compared with control cultures, monocytes cultured 24 hours in presence of dexamethasone exhibited a decrease in HLA-DR mRNA levels (n = 13, –66%, p < 0.01, Wilcoxon's test). Effect on CIITA transcription (C): compared with control cultures (dark line), monocytes cultured 24 hours in presence of dexamethasone exhibited a decrease in total CIITA mRNA levels (–60%, n = 13, p < 0.01, Wilcoxon's test). This down-modulating effect on CIITA transcription was observed mainly on the CIITA isoforms I and III (–85% and –57%, respectively, p < 0.01, Wilcoxon's test), whereas CIITA isoform IV was slightly altered (–30%, p < 0.01, Wilcoxon's test).

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. Blocking effect of the inhibitor of the glucocorticoid receptor RU 486 (mifepristone) on the dexamethasone-induced alterations of HLA-DR and CIITA transcription. The decreased HLA-DR expression (A) and transcription (B) levels induced by dexamethasone were significantly different than those observed when monocytes were pretreated by RU486 (n = 6, *p < 0.05, Wilcoxon's test). This inhibitor effect was also observed on the dexamethasone-induced alterations of CIITA (C) (Dex = dexamethasone; Dex-RU = dexamethasone + RU486; RU = RU486 alone).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The loss of HLA-DR expression on monocytes indicates their functional deactivation and is supposed to be a determining factor of the immunodeficiency observed in SS (6, 11, 20), but until now, the prognostic value of this alteration was not well established and remained controversial (21, 22). Here, we demonstrate that HLA-DR expression on monocyte decreases early in the course of infection, tends to normalize in patients who survived, and remains deeply lowered in those who acquired secondary infections or who ultimately died. As in other studies (21–23), we did not find relationship between HLA-DR downregulation at Day 1 and patient outcome, and as others (6, 24), we evidenced a prognostic value of persistence of the downregulation 6 days later. Interestingly, we observed a comparable prognostic value of persistence of the lymphopenia in a previous study investigating the role of lymphocyte apoptosis in sepsis (4). Taking together these findings could suggest that the inability to restore their immunologic status is a key point of the immunodepression observed in septic patients, perhaps more important than the severity of the triggering event. Consequently, one can expect a beneficial effect of immunotherapy targeted on immune restoration such as granulocyte-macrophage colony-stimulating factor or granulocyte-colony stimulating factor and IFN-{gamma}. In line with this hypothesis are the recent studies showing an improvement of monocyte function in patients receiving granulocyte-macrophage colony-stimulating factor treatment (25–27) or IFN-{gamma} (28) or even a reduction in the mortality rate (29). Although interesting, these data are preliminary, and one must be cautious about interpreting the results of these studies because the effect on monocyte function could not be sufficient to affect mortality in large series. Indeed, other clinical studies or meta-analysis evidenced no beneficial effect of these strategies on mortality (30, 31). Significance of the loss of HLA-DR expression on monocytes remained questionable; thus, this abnormality could be seen either as an immune defect or as an adaptive mechanism preventing monocyte activation in circulating blood. These data argue for the first hypothesis and attribute to the loss of HLA-DR expression on circulating monocytes a major role in the immunosuppression observed in sepsis. Of course, a major point will be to know whether such a down modulation also occurs in other professional antigen-presenting cells and if such alterations in the ability to present foreign antigens lead to an abnormal T-cell response in vivo.

In this study, we demonstrate a decrease in circulating HLA-DR mRNA levels in septic patients and observe a relationship between mRNA and expression of HLA-DR in vivo. This phenomenon is regulated by the transactivator factor CIITA, as evidenced by its decrease observed in all infected patients and by the observed relationships between CIITA mRNA and HLA-DR mRNA and cell surface expression. These in vivo results are in accordance with previous in vitro studies that evidenced the transcriptional control of constitutive and IFN-{gamma}–inducible expression of HLA-DR, mainly by the class II transactivator CIITA (13–15). Another mechanism of HLA-DR down modulation recently described in SS involves an intracellular sequestration of the molecule. This mechanism, partly induced by IL-10, is not exclusive because its blockade restores modestly HLA-DR expression (11). Furthermore, both intracellular sequestration and transcriptional regulation have been recently observed in in vitro studies describing the mechanisms of endotoxin tolerance (12). Consequently, in vivo regulation of HLA-DR expression in monocytes could involve transcriptional and post-translational mechanisms.

Several elements released in high concentrations during SS such as prostaglandins, transforming growth factor-ß1, catecholamines, IL-10, and cortisol can modulate the immune response. Catecholamines stimulate {alpha}- and ß-adrenergic pathways with opposite effects on cytokine transcription (8), but their effects on HLA-DR expression in SS remained unknown. The role of IL-10 has been proved (6, 11); however, no relationship was found between IL-10 and HLA-DR expression, and its neutralization did not totally restore HLA-DR expression on monocytes (11). According to previous reports, we observed high levels of catecholamines and IL-10 in SS but found no relationship with HLA-DR expression on circulating monocytes. These negative data are interesting because even if they did not exclude the role of catecholamines and IL-10 in HLA-DR regulation they argue for the role of other mediators. Actually, endogenous cortisol is one of the main components of the antiinflammatory response induced by the central nervous system during SS. Its down-modulating effect on monocyte HLA-DR expression has been described in other clinical settings and in vitro observations (32–36), and basal plasma cortisol levels are higher in the patients who have the highest risk of mortality (37). Paradoxically, a beneficial effect of supplementation by low doses of corticosteroids has been demonstrated in catecholamine-dependent SS (38). Actually, in the study of Annane and colleagues, survival improvement was mainly due to a rapid stabilization of hemodynamic parameters in patients with relative adrenal insufficiency but the immunologic effects of such treatment were not evaluated. Interestingly, a recent report (39) confirmed hemodynamic effects of a low dose of hydrocortisone in SS patients, probably via a reduction in nitric oxide formation but evidenced no major immunosuppressive effects. In particular, monocyte HLA-DR expression, which was already low in septic patients was slightly more depressed by the adjunction of hydrocortisone. Consequently, low-dose steroid supplementation has a moderate impact on the underlying immunosuppression observed in SS and particularly on HLA-DR down modulation. Our results argue for a role of endogenous cortisol in the HLA-DR down modulation observed on monocyte during SS: circulating cortisol levels significantly increased in SS patients and correlated with low monocyte HLA-DR expression. We observed similar relationships between circulating cortisol levels and mRNA levels for HLA-DR and for its transactivator CIITA. Even if these relationships do not establish the direct responsibility of the high level of endogenous cortisol in HLA-DR regulation, they argue for a transcriptional control of HLA-DR expression and for the role of cortisol in this process. In line with this hypothesis, this effect observed in vivo was confirmed by our in vitro experiments, which demonstrate the down-modulating effect of dexamethasone on HLA-DR expression and, as observed in vivo, evidenced that regulation takes place at a transcriptional level. According to reports showing that glucocorticoids could lower HLA-DR transcription not only by a competitive use of a common coactivator cAMP response element-binding–binding protein (34) but also by a direct mechanism on CIITA transcription, we observed a direct down-modulating effect of dexamethasone on CIITA. CIITA expression and consequently major histocompatibility complex class II molecule expression are tightly regulated. Thus, a complex regulatory region containing at least three independent promoters (pI, pIII, and pIV) induces the transcription of three different isoforms, which exhibit different activities. The respective levels of transcription of each isoform vary according to the cell type and drive different levels of major histocompatibility complex class II expression. Thus, type I and type III CIITA isoforms drive basal HLA-DR expression mainly in professional bone marrow–derived antigen-presenting cells such as dendritic cells and B lymphocytes, whereas type IV isoform is activated by IFN-{gamma} through signal transducer and activator of transcription 1 phosphorylation also in other cell types such as epithelial and endothelial cells (13–15). Actually, dexamethasone lowered the two constitutive transducers CIITA-I and CIITA-III and had a slight effect on the signal transducer and activator of transcription 1–sensitive CIITA-IV transducer (40). CIITA-III is the major transcript expressed in monocytes and is moderately decrease by dexamethasone. On the other hand, CIITA-I is expressed at a low level but is a more potent transactivator of the HLA-DR gene than CIITA-III (41) and is deeply reduced by dexamethasone. Consequently, because different promoters could interact with each other, their respective roles in HLA-DR expression remained to be clarified, but the almost complete disappearance of CIITA-I induced by dexamethasone is probably a major event in its down-modulating effect on HLA-DR expression.

In summary, the loss of HLA-DR molecules on monocytes appears as an early event, encountered even in mild sepsis. The persistence of HLA-DR downregulation, and not the initial value, is associated with severity of the disease and constitutes a prognosis marker. Furthermore, we provide new insights in the understanding of this phenomenon: besides the post-translational effect of IL-10 on HLA-DR expression described by Fumeaux and Pugin (11), we observed a transcriptional regulatory effect of cortisol through a mechanism involving the main major histocompatibility complex type II transactivator CIITA and particularly the isoforms I and III.


   Acknowledgments
 
The authors thank Dr. Catherine Massart from the Department of Molecular Genetics and Hormonology, C.H.U. Rennes, for her assistance, and Dr. Catherine Alcaide-Loridan from Unité d'Immunogénétique Humaine, INSERM U396, Paris, for providing CIITA cDNA control subjects and for her helpful suggestions regarding the HLA-DR regulation analysis.


   FOOTNOTES
 
Supported by grants from Société de Réanimation de Langue Française and Medical University of Rennes.

Yves Le Tulzo and Celine Pangault contributed equally to this study.

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

Conflict of Interest Statement: Y.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; L.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; V.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; O.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; B.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form September 25, 2003; accepted in final form March 17, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Friedman G, Silva E, Vincent JL. Has the mortality of septic shock changed with time. Crit Care Med 1998;26:2078–2086.[CrossRef][Medline]
  2. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992;20:864–874.
  3. Hotchkiss RS, Tinsley KW, Swanson PE, Schmieg RE Jr, Hui JJ, Chang KC, Osborne DF, Freeman BD, Cobb JP, Buchman TG, et al. Sepsis-induced apoptosis causes progressive profound depletion of B and CD4+T lymphocytes in humans. J Immunol 2001;166:6952–6963.[Abstract/Free Full Text]
  4. Le Tulzo Y, Pangault C, Gacouin A, Guilloux V, Tribut O, Amiot L, Tattevin P, Thomas R, Fauchet R, Drenou B. Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome. Shock 2002;18:487–494.[CrossRef][Medline]
  5. Munoz C, Carlet J, Fitting C, Misset B, Bleriot JP, Cavaillon JM. Dysregulation of in vitro cytokine production by monocytes during sepsis. J Clin Invest 1991;88:1747–1754.
  6. Docke WD, Randow F, Syrbe U, Krausch D, Asadullah K, Reinke P, Volk HD, Kox W. Monocyte deactivation in septic patients: restoration by IFN-gamma treatment. Nat Med 1997;3:678–681.[CrossRef][Medline]
  7. DeRijk R, Michelson D, Karp B, Petrides J, Galliven E, Deuster P, Paciotti G, Gold PW, Sternberg EM. Exercise and circadian rhythm-induced variations in plasma cortisol differentially regulate interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha (TNF alpha) production in humans: high sensitivity of TNF alpha and resistance of IL-6. J Clin Endocrinol Metab 1997;82:2182–2191.[Abstract/Free Full Text]
  8. Le Tulzo Y, Shenkar R, Kaneko D, Moine P, Fantuzzi G, Dinarello CA, Abraham E. Hemorrhage increases cytokine expression in lung mononuclear cells in mice: involvement of catecholamines in nuclear factor-kappaB regulation and cytokine expression. J Clin Invest 1997;99:1516–1524.[Medline]
  9. Wang P, Wu P, Siegel MI, Egan RW, Billah MM. Interleukin (IL)-10 inhibits nuclear factor kappa B (NF kappa B) activation in human monocytes: IL-10 and IL-4 suppress cytokine synthesis by different mechanisms. J Biol Chem 1995;270:9558–9563.[Abstract/Free Full Text]
  10. Reddy RC, Chen GH, Newstead MW, Moore T, Zeng X, Tateda K, Standiford TJ. Alveolar macrophage deactivation in murine septic peritonitis: role of interleukin 10. Infect Immun 2001;69:1394–1401.[Abstract/Free Full Text]
  11. Fumeaux T, Pugin J. Role of interleukin-10 in the intracellular sequestration of human leukocyte antigen-DR in monocytes during septic shock. Am J Respir Crit Care Med 2002;166:1475–1482.[Abstract/Free Full Text]
  12. Wolk K, Kunz S, Crompton NE, Volk HD, Sabat R. Multiple mechanisms of reduced major histocompatibility complex class II expression in endotoxin tolerance. J Biol Chem 2003;278:18030–18036.[Abstract/Free Full Text]
  13. Nagarajan UM, Bushey A, Boss JM. Modulation of gene expression by the MHC class II transactivator. J Immunol 2002;169:5078–5088.[Abstract/Free Full Text]
  14. Boss JM. Regulation of transcription of MHC class II genes. Curr Opin Immunol 1997;9:107–113.[CrossRef][Medline]
  15. Muhlethaler-Mottet A, Otten LA, Steimle V, Mach B. Expression of MHC class II molecules in different cellular and functional compartments is controlled by differential usage of multiple promoters of the transactivator CIITA. EMBO J 1997;16:2851–2860.[CrossRef][Medline]
  16. Le Tulzo YP, Le Friec C, Lavoué G, Guilloux S, Tribut V, Amiot O, Tattevin L, Fauchet P, Thomas R, Drénou RB. Septic shock monocyte HLA-DR and GM-CSF receptor (CD116) low expression is related to severity [abstract]. Am J Respir Cell Mol Biol 2002;165:B31.
  17. Le Gall JR, Lemeshow S, Saulnier F. A new Simplified Acute Physiology Score (SAPS II) based on a European/North American multicenter study. JAMA 1993;270:2957–2963.[Abstract]
  18. Le Gall JR, Klar J, Lemeshow S, Saulnier F, Alberti C, Artigas A, Teres D. The logistic organ dysfunction system: a new way to assess organ dysfunction in the intensive care unit: ICU Scoring Group. JAMA 1996;276:802–810.[Abstract]
  19. Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM. CDC definitions for nosocomial infections, 1988. Am J Infect Control 1988;16:128–140.[CrossRef][Medline]
  20. Lin RY, Astiz ME, Saxon JC, Rackow EC. Altered leukocyte immunophenotypes in septic shock: studies of HLA-DR, CD11b, CD14, and IL-2R expression. Chest 1993;104:847–853.[Abstract/Free Full Text]
  21. Oczenski W, Krenn H, Jilch R, Watzka H, Waldenberger F, Koller U, Schwarz S, Fitzgerald RD. HLA-DR as a marker for increased risk for systemic inflammation and septic complications after cardiac surgery. Intensive Care Med 2003;29:1253–1257.[CrossRef][Medline]
  22. Perry SE, Mostafa SM, Wenstone R, Shenkin A, McLaughlin PJ. Is low monocyte HLA-DR expression helpful to predict outcome in severe sepsis? Intensive Care Med 2003;29:1245–1252.[CrossRef][Medline]
  23. Muller Kobold AC, Tulleken JE, Zijlstra JG, Sluiter W, Hermans J, Kallenberg CG, Cohen Tervaert JW. Leukocyte activation in sepsis: correlations with disease state and mortality. Intensive Care Med 2000;26:883–892.[CrossRef][Medline]
  24. Monneret G, Elmenkouri N, Bohe J, Debard AL, Gutowski MC, Bienvenu J, Lepape A. Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: application to the monitoring of patients with septic shock. Clin Chem 2002;48:1589–1592.[Free Full Text]
  25. Drossou-Agakidou V, Kanakoudi-Tsakalidou F, Sarafidis K, Tzimouli V, Taparkou A, Kremenopoulos G, Germenis A. In vivo effect of rhGM-CSF and rhG-CSF on monocyte HLA-DR expression of septic neonates. Cytokine 2002;18:260–265.[CrossRef][Medline]
  26. Flohe S, Lendemans S, Selbach C, Waydhas C, Ackermann M, Schade FU, Kreuzfelder E. Effect of granulocyte-macrophage colony-stimulating factor on the immune response of circulating monocytes after severe trauma. Crit Care Med 2003;31:2462–2469.[CrossRef][Medline]
  27. Presneill JJ, Harris T, Stewart AG, Cade JF, Wilson JW. A randomized phase II trial of granulocyte-macrophage colony-stimulating factor therapy in severe sepsis with respiratory dysfunction. Am J Respir Crit Care Med 2002;166:138–143.[Abstract/Free Full Text]
  28. Kox WJ, Bone RC, Krausch D, Docke WD, Kox SN, Wauer H, Egerer K, Querner S, Asadullah K, von Baehr R, et al. Interferon gamma-1b in the treatment of compensatory anti-inflammatory response syndrome: a new approach: proof of principle. Arch Intern Med 1997;157:389–393.[CrossRef][Medline]
  29. Cheng AC, Stephens DP, Anstey NM, Currie BJ. Adjunctive granulocyte colony-stimulating factor for treatment of septic shock due to melioidosis. Clin Infect Dis 2004;38:32–37.[CrossRef][Medline]
  30. Carr R, Modi N, Dore C. G-CSF and GM-CSF for treating or preventing neonatal infections. Cochrane Database Syst Rev 2003;CD003066.
  31. Root RK, Lodato RF, Patrick W, Cade JF, Fotheringham N, Milwee S, Vincent JL, Torres A, Rello J, Nelson S. Multicenter, double-blind, placebo-controlled study of the use of filgrastim in patients hospitalized with pneumonia and severe sepsis. Crit Care Med 2003;31:367–373.[CrossRef][Medline]
  32. Watanabe Y, Lee S, Allison AC. Control of the expression of a class II major histocompatibility gene (HLA-DR) in various human cell types: down-regulation by IL-1 but not by IL-6, prostaglandin E2, or glucocorticoids. Scand J Immunol 1990;32:601–609.[CrossRef][Medline]
  33. Asadullah K, Woiciechowsky C, Docke WD, Egerer K, Kox WJ, Vogel S, Sterry W, Volk HD. Very low monocytic HLA-DR expression indicates high risk of infection: immunomonitoring for patients after neurosurgery and patients during high dose steroid therapy. Eur J Emerg Med 1995;2:184–190.[CrossRef][Medline]
  34. Fontes JD, Kanazawa S, Jean D, Peterlin BM. Interactions between the class II transactivator and CREB binding protein increase transcription of major histocompatibility complex class II genes. Mol Cell Biol 1999;19:941–947.[Abstract/Free Full Text]
  35. Schwiebert LM, Schleimer RP, Radka SF, Ono SJ. Modulation of MHC class II expression in human cells by dexamethasone. Cell Immunol 1995;165:12–19.[CrossRef][Medline]
  36. Haveman JW, van den Berg AP, van den Berk JM, Mesander G, Slooff MJ, de Leij LH, The TH. Low HLA-DR expression on peripheral blood monocytes predicts bacterial sepsis after liver transplantation: relation with prednisolone intake. Transpl Infect Dis 1999;1:146–152.[CrossRef][Medline]
  37. Annane D, Sebille V, Troche G, Raphael JC, Gajdos P, Bellissant E. A 3-level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin. JAMA 2000;283:1038–1045.[Abstract/Free Full Text]
  38. Annane D, Sebille V, Charpentier C, Bollaert PE, Francois B, Korach JM, Capellier G, Cohen Y, Azoulay E, Troche G, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock. JAMA 2002;288:862–871.[Abstract/Free Full Text]
  39. Keh D, Boehnke T, Weber-Cartens S, Schulz C, Ahlers O, Bercker S, Volk HD, Doecke WD, Falke KJ, Gerlach H. Immunologic and hemodynamic effects of "low-dose" hydrocortisone in septic shock: a double-blind, randomized, placebo-controlled, crossover study. Am J Respir Crit Care Med 2003;167:512–520.[Abstract/Free Full Text]
  40. Schreiber S, Rosenstiel P, Hampe J, Nikolaus S, Groessner B, Schottelius A, Kuhbacher T, Hamling J, Folsch UR, Seegert D. Activation of signal transducer and activator of transcription (STAT) 1 in human chronic inflammatory bowel disease. Gut 2002;51:379–385.[Abstract/Free Full Text]
  41. Nickerson K, Sisk TJ, Inohara N, Yee CS, Kennell J, Cho MC, Yannie PJ II, Nunez G, Chang CH. Dendritic cell-specific MHC class II transactivator contains a caspase recruitment domain that confers potent transactivation activity. J Biol Chem 2001;276:19089–19093.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Pachot, M.-A. Cazalis, F. Venet, F. Turrel, C. Faudot, N. Voirin, J. Diasparra, N. Bourgoin, F. Poitevin, B. Mougin, et al.
Decreased Expression of the Fractalkine Receptor CX3CR1 on Circulating Monocytes as New Feature of Sepsis-Induced Immunosuppression
J. Immunol., May 1, 2008; 180(9): 6421 - 6429.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
R. G. Wunderink
Nosocomial Pneumonia, Including Ventilator-associatedPneumonia
Proceedings of the ATS, December 1, 2005; 2(5): 440 - 444.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
P. Gessler, R. Pretre, C. Burki, V. Rousson, B. Frey, and D. Nadal
Monocyte function-associated antigen expression during and after pediatric cardiac surgery
J. Thorac. Cardiovasc. Surg., July 1, 2005; 130(1): 54 - 60.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
D. Angus, A. Ishizaka, M. Matthay, F. Lemaire, W. MacNee, and E. Abraham
Critical Care in AJRCCM 2004
Am. J. Respir. Crit. Care Med., March 15, 2005; 171(6): 537 - 544.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200309-1329OCv1
169/10/1144    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Le Tulzo, Y.
Right arrow Articles by Drénou, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Le Tulzo, Y.
Right arrow Articles by Drénou, B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society