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The Journal of Immunology, 2006, 177: 4080-4085.
Copyright © 2006 by The American Association of Immunologists, Inc.

Induction of RelB Participates in Endotoxin Tolerance1

Barbara K. Yoza2,*,{dagger}, Jean Y.-Q. Hu{dagger}, Sue L. Cousart{dagger}, Lolita M. Forrest{dagger} and Charles E. McCall{dagger}

* Department of General Surgery, Wake Forest University School of Medicine, Winston-Salem, NC 27157; and {dagger} Department of Internal Medicine, Section on Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Using a THP-1 human promonocyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype, we previously showed that tolerant cells remain responsive to LPS endotoxin with degradation of I{kappa}B in the cytosol and nuclear translocation and accumulation of p50 and p65 NF-{kappa}B transcription factors. Despite this, endotoxin-inducible NF-{kappa}B-dependent innate immunity genes, like IL-1beta, remained transcriptionally unresponsive in the tolerant phenotype, similar to the endotoxin tolerance observed in sepsis patients. In this study, we examined this paradox and found that RelB, another member of the NF-{kappa}B family, is induced during the establishment of tolerance. RelB expression correlated with IL-1beta repression, and sepsis patients showed increased RelB when compared with normal controls. Transient expression of RelB inhibited IL-1beta in endotoxin-responsive cells. In the inverse experiment, small inhibitory RNAs decreased RelB expression in tolerant cells and restored endotoxin induction of IL-1beta. When we examined tolerant cell extracts, we found transcriptionally inactive NF-{kappa}B p65/RelB heterodimers. Taken together, our findings demonstrate that RelB can repress proinflammatory gene expression, and suggest that RelB expression in sepsis patient blood leukocytes may play a role in the endotoxin-tolerant phenotype.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human sepsis is associated with an immunosuppressed phenotype that involves both innate and adaptive immunity and that is associated with a poor clinical outcome (1). NF-{kappa}B plays an essential, albeit controversial role in sepsis and in the LPS-tolerant phenotype that occurs during sepsis (2, 3, 4, 5, 6). Although the expression of many NF-{kappa}B-dependent genes, like IL-1beta and TNF-{alpha}, are repressed in leukocytes from patients with septic shock (1), several studies have demonstrated elevated levels of p65 in sepsis nuclei (6, 7). Indeed, elevated levels of p65, the transcription-activating subunit of NF-{kappa}B, correlate with poor outcomes in these patients (2, 3). Animal and tissue culture models also show increased nuclear p50 levels, and formation of p50 homodimers is promoted by some investigators as a mechanism for transcription repression during endotoxin tolerance (8, 9, 10). Other studies (11) find no such connection.

Several lines of evidence also revealed that down-regulation of NF-{kappa}B signaling pathways, for example IL-1R-associated kinase (IRAK)3 (12, 13, 14, 15, 16) and I{kappa}B kinase, may significantly impact LPS response in the tolerant phenotype (17, 18). These dysfunctions, characteristic of LPS tolerance, may have developed as a mechanism to fine-tune the balance between a pro- and anti-inflammatory response that would ensue from the initial encounter with an infectious agent (19, 20). Many studies have found that expression of some anti-inflammatory genes is sustained during sepsis and LPS tolerance, and the physiological consequence of this immunosuppressed state continues to be explored (21, 22, 23, 24, 25, 26).

We reported that cytosolic NF-{kappa}B activation occurs in the human LPS-tolerant phenotype, as evidenced by LPS-mediated I{kappa}B degradation (27), nuclear translocation of NF-{kappa}B p50 and p65, and formation of DNA-binding competent NF-{kappa}B complexes, as assessed by EMSAs (28). Two notable defects in LPS-mediated response were observed in tolerance when compared with normal cells. In examining LPS-dependent TLR4 signaling pathways, we found that IRAK1 was inactivated, degraded, and absent in tolerant cells (29, 30, 31). In examining LPS repression of the IL-1beta gene, chromatin immunoprecipitation (ChIP) studies revealed that the chromatin structure at or near the IL-1beta TATA box was altered and in a transcriptionally inactive state (32, 33). To further understand this latter phenomenon, we also found that p65 did not bind to the NF-{kappa}B site within the IL-1beta promoter, although inducible and present in tolerant nuclei (32). This finding was in contrast to our findings that p65 NF-{kappa}B complexes could bind in EMSA (28) and highlights the differences in in vitro (EMSA) and in vivo (ChIP) DNA-binding assays.

In this study, we sought an explanation to the increased levels of p65 in the nucleus of tolerant cells and the inability of p65 to access the IL-1beta promoter in vivo and activate transcription. We tested the hypothesis that RelB may be induced as a negative feedback mechanism for repressing NF-{kappa}B transcription during LPS tolerance. We find increased expression of RelB that is coincident with the establishment of LPS tolerance. Overexpression of RelB significantly reduces LPS-induced IL-1beta expression in naive cells. We also show that decreased expression of RelB by RelB small interfering RNA (siRNA) can restore endotoxin induction of IL-1beta expression in tolerant cells. Finally, we find that blood leukocytes from sepsis patients expressed RelB, whereas normal healthy controls did not. Taken together, our findings suggest that RelB expression, as a negative feedback, may provide a necessary and sufficient molecular mechanism involved in the repression of NF-{kappa}B-dependent innate immunity genes observed in LPS tolerance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and stimulation

THP-1 cells obtained from the American Type Culture Collection were maintained in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10 U/ml penicillin G, 10 µg/ml streptomycin, 2 mM L-glutamine, and 10% FCS (low LPS FCS; Sigma-Aldrich) at 37°C and 5% CO2 in a humidified incubator. LPS-tolerant THP-1 cells were made and are characterized in detail, as described previously (27). Briefly, THP-1 cells were rendered LPS tolerant by preincubation for 16 h with LPS (1.0 µg/ml Gram-negative LPS (Escherichia coli 0111:B4; Sigma-Aldrich)). Normal (no preincubation) and tolerant THP-1 cells were washed with FCS-free RPMI 1640 medium, then resuspended in FCS-supplemented RPMI 1640 medium at 1 x 106 cells/ml, and stimulated with LPS (1.0 µg/ml) for the times as indicated in the figure legends. Nonspecific and RelB siRNAs (Santa Cruz Biotechnology) were transfected using an AMAXA THP-1 transfection kit, according to the manufacturer’s protocol. pcDNA3-HA and HA-RelB (34) were gifts from G. Bren (Mayo Clinic, Rochester, MN). NF-{kappa}B p65 was provided by A. Baldwin (University of North Carolina, Chapel Hill, NC). Low passage number and log-phase cells were used for all experiments. All solutions were monitored routinely for LPS contamination by the Limulus amebocyte lysate assay (sensitivity <1 pg/ml).

Peripheral blood leukocyte, polymorphonuclear blood leukocyte (PMN), and PBMC isolation

The selection of patients was based on a modified criteria of Bone (35), as previously described (36). Using these criteria, 95% of patients reproducibly have reduced IL-1beta production in response to LPS (23, 36). Patients enrolled in this study were positive for Gram-positive and/or Gram-negative sepsis. The patient without sepsis was a trauma patient with pulmonary contusion. Patients with known HIV infection, hematological malignancies affecting leukocyte counts, current cytotoxic chemotherapy, and high-dose glucocorticoid treatment were excluded. The Internal Review Board endorsed the study for Clinical Research associated with the General Clinical Research Center of the Medical Center. A venous or arterial heparinized blood sample (up to 30 ml) was drawn from patients within 1–4 days after the onset of sepsis or from normal healthy controls and immediately processed. PBL were isolated by Isolymph sedimentation (Gallard-Schlesinger), followed by centrifugation for 5 min at 200 x g at room temperature. The remaining erythrocytes were lysed in LPS-free H2O for 20 s before isotonicity was restored with 3.6% NaCl. Isolated PBL were washed and counted in PBS. PBL whole cell lysates were immediately prepared in SDS-Tris sample solubilization/loading buffer (reducing) and stored at –20°C until further use. PMN and PBMC were isolated, as previously described (23, 27). The proportions of PMN in PBL vary in sepsis (85–95%) when compared with normal (60–80%). In two patients, one without sepsis and one with sepsis, PBL were further separated into PMN and PBMC by a second centrifugation over 3 ml of Isolymph for 30 min at 400 x g. The PMN pellet and PBMC interface were washed and counted in PBS, and lysates were immediately prepared in SDS-Tris sample solubilization/loading buffer (reducing) and stored at –20°C until further use in Western blot analysis, as described below.

Immunoprecipitation and Western blot analysis

Whole cell and nuclear extracts (28) were prepared, using the protocols described previously. Whole cell and nuclear extract proteins (50 µg) were separated by SDS-PAGE and transferred to an Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad). RelB, B cell lymphoma protein 2 (Bcl-2), beta-actin, I{kappa}B ({alpha}), agarose-conjugated p50 and p65 NF-{kappa}B Abs, and appropriate HRP-conjugated secondary Abs (Santa Cruz Biotechnology) were used to immunoprecipitate and/or visualize proteins using Western blot protocols, as specified by the manufacturer. Blots were stripped and reprobed with control Abs (Bcl-2, beta-actin), as indicated. Protein levels on the blots were quantitated using ImageQuant software (Amersham Biosciences).

Real-time PCR analysis

Total RNA was isolated from 1 x 107 cells/condition using RNA STAT-60 (Tel-Test), according to the manufacturer’s instructions. cDNA was reverse transcribed in a final volume of 20 µl containing 1 µg of total RNA, 10x PCR buffer, 200 µM deoxynucleotides, 1 µM oligo(dT), 2 mM MgCl2, and 2.5 U of murine leukemia virus reverse transcriptase (Applied Biosystems) for 1 h at 42°C. IL-beta mRNA was quantitated by real-time PCR using TaqMan methodology and an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Real-time PCR were performed in 25 µl of a mixture containing 1 µl of cDNA (1 µg), 300 nM IL-1beta primers, 100 nM FAM/TAMRA TaqMan probe, and 12.5 µl of TaqMan Universal PCR Master Mix, containing AmpliTaq Gold DNA polymerase, reaction buffer, and dNTP mix (Applied Biosystems). Results from at least three independent experiments were quantitated by continuous measurement during 40 cycles. All data were normalized to GAPDH quantitated in parallel samples (Applied Biosystems).

Statistical analysis

Statistical analysis of data is presented as the average of at least three independent experiments ± SEM. Analyses were performed after consultation with a statistician using Student’s t test to determine significant changes (p ≤ 0.05) using Microsoft Excel 2002 statistical analysis software, as indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RelB is expressed in LPS-tolerant cells

We first determined the time course of RelB expression in the THP-1 human promonocyte model of LPS tolerance. We found that LPS-tolerant cells express increased levels of RelB, as shown in Fig. 1A. We next examined nuclear extracts for RelB expression and found low levels of RelB in normal cells that is increased in tolerant cell nuclei. Thus, the appearance and localization of RelB are consistent with a potential role in LPS tolerance. We also compared sepsis PBL with normal PBL (Fig. 1B) and also found increased RelB in sepsis patients’ PBL, further supporting our hypothesis that RelB may mediate LPS tolerance. To further associate RelB expression with LPS tolerance, PMN and PBMC were isolated from two patients, without (Fig. 1C, P4) and with sepsis (Fig. 1C, P5). Consistent with our previous results, RelB was found only in the patient with sepsis.


Figure 1
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  		FIGURE 1. RelB is expressed in LPS-tolerant cells. A, Normal and tolerant THP-1 cells were stimulated with LPS (1 µg/ml), and levels of RelB were determined in whole cell (WCE) and nuclear extracts (NE) by Western blot analysis, as described in Materials and Methods. B, PBL were isolated from normal healthy controls (C1, C2, C3) and sepsis patients (P1, P2, P3), and RelB levels were determined by Western blot. Bcl-2 and beta-actin levels were used as immunoblot control Abs. Samples shown are representative of at least five independent observations. C, PBMC and PMN were isolated from two patients, one without (P4) and one with (P5) sepsis. RelB and beta-actin levels were determined by Western blot.

 
Expression of RelB in normal cells mimics tolerance and inhibits IL-1beta expression

We and others had observed that tolerant cells do not respond to LPS with an immediate and robust induction of proinflammatory genes. Paradoxically, we found that LPS-tolerant cells still showed LPS-stimulated I{kappa}B degradation and p50/p65 nuclear translocation, despite the continued repression of IL-1beta expression. Our recent ChIP studies provided a partial explanation for this discrepancy, in which we found that p65 was unable to bind the IL-1beta promoter in tolerant cells (32). Given the correlation between the appearance of RelB and transcription inhibition, we next tested the hypothesis that RelB mediated decreased expression of IL-1beta. THP-1 cells were transfected with a human RelB expression vector (Bren and colleagues (34)) or empty vector (control). As shown in Fig. 2A, cells transfected with RelB showed increased levels of RelB protein compared with cells transfected with the empty vector. Levels of transfected RelB were comparable to RelB protein levels in tolerant cells (Fig. 2B). RelB-transfected or control cells were stimulated with LPS and IL-1beta mRNA measured by real-time PCR at 1 and 3 h after stimulation. In these experiments (Fig. 2C), we found that RelB expression decreased IL-1beta mRNA levels after LPS stimulation. Control cells (empty vector) showed the rapid and transient expression of IL-1beta mRNA that is typical of the immune response to LPS. In contrast, RelB-expressing cells showed a significant decrease in IL-1beta expression that is characteristic of LPS tolerance. Thus, this experiment supports the hypothesis that RelB can repress LPS-induced expression of IL-1beta and is consistent with a role in the LPS-tolerant phenotype. We further examined whether expression of NF-{kappa}B p65 might overcome the LPS tolerance induced by RelB. As shown in Fig. 2D, p65 was able to overcome RelB-mediated suppression.


Figure 2
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  		FIGURE 2. RelB expressed in normal cells mimics tolerance and inhibits IL-1beta expression. A, A RelB expression vector (HA-RelB) was transfected into THP-1 cells, as described in Materials and Methods. The empty vector was used as a control. RelB levels in expression vector- and empty vector-transfected cells were assessed by immunoblot, as shown on the right. RelB levels in tolerant THP-1 cells are shown on the left for comparison. B, RelB protein levels in transfected cells were quantitated on Western blot, as described in Materials and Methods. Increased RelB levels in transfected cells are the average of two experiments ± SEM; RelB levels in normal and tolerant cells (nontransfected) are shown for comparison. C, RelB ({blacksquare})- and empty vector ({square})-transfected cells were stimulated with LPS and total RNA isolated at various times after stimulation. IL-1beta and GAPDH mRNA were measured by real-time PCR, as described in Materials and Methods. Relative IL-1beta mRNA levels are normalized to GAPDH mRNA and are shown as the average (±SEM) for each of three independent experiments. D, RelB ({blacksquare})-, empty vector ({square})-, or RelB + NF-{kappa}B p65 (Figure 2)-transfected cells were stimulated with LPS and total RNA isolated at various times after stimulation. IL-1beta and GAPDH mRNA were measured by real-time PCR, as described in Materials and Methods. Relative IL-1beta mRNA levels are normalized to GAPDH mRNA in each sample. Data shown are representative of two independent experiments, with mRNA measured in duplicate for each experiment.

 
Gene silencing of RelB by siRNA can reverse LPS tolerance

The introduction of siRNA as a molecular tool has developed into a powerful technique for selective gene silencing (reviewed in Ref. 37). We used RelB-specific siRNA to test whether the knockdown of RelB protein levels in tolerant cells could restore LPS responsiveness and increase IL-1beta mRNA. As shown in Fig. 3, introduction of RelB siRNA significantly reduced RelB protein levels in tolerant cells. This decrease persisted even with LPS stimulation. In contrast, control siRNA still showed the high levels of RelB associated with LPS tolerance.


Figure 3
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  		FIGURE 3. Gene silencing of RelB by siRNA can significantly decrease RelB levels. RelB levels in tolerant cells in the presence of nonspecific or control (ctrl siRNA) or RelB-specific siRNAs (RelB siRNA) were assessed by Western blot. A, Shows that RelB levels are decreased in the tolerant cells by RelB siRNA. I{kappa}B and p65 protein levels in RelB knockdown cell are unchanged, as shown below. Western blots were stripped and reprobed with beta-actin as an immunoblot control. B, Quantitation of RelB levels from three separate experiments shows a significant decrease in RelB levels in tolerant cells by RelB siRNA (p = 0.003 for RelB siRNA compared with control siRNA; p = 0.02 for RelB siRNA + LPS compared with control siRNA + LPS).

 
Given the demonstration of significantly reduced RelB levels by RelB-specific siRNA, we further asked whether this gene silencing could restore LPS response and induce IL-1beta expression, i.e., reverse the LPS-tolerant phenotype. Fig. 4A shows the decreased LPS response to induction of IL-1beta that is a salient feature of LPS-tolerant cells (compare control ±LPS with tolerant ±LPS). We found that introduction of control siRNA still showed this repressed LPS response and decreased IL-1beta expression in tolerant cells (Fig. 4B). In contrast, RelB siRNA was able to reverse the repression and near normal levels of IL-1beta mRNA were induced by LPS in RelB knockdown tolerant cells (Fig. 4C). These data strongly support a role for RelB in mediating LPS tolerance.


Figure 4
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  		FIGURE 4. Gene silencing of RelB by siRNA can reverse LPS tolerance. Normal (N) and tolerant (T) THP-1 cells were stimulated with (+) or without (–) LPS, and IL-1beta mRNA levels were measured by real-time PCR, as described in Materials and Methods. A, Shows IL-1beta mRNA levels in cells not transfected for comparison; B and C, show IL-1beta mRNA levels in cells transfected with control siRNA (nonspecific) or RelB siRNA (specific), respectively. Data shown are the results of three independent experiments, with RNA levels measured in duplicate and relative to GAPDH mRNA for each sample. Significant differences (*) are observed between normal and tolerant stimulated IL-1beta mRNA levels in nontransfected cells (A, compare N+ with T+; p = 0.002) or control siRNA cells (B, compare N+ with T+; p = 0.03). Normal and tolerant cells transfected with RelB siRNA (C, compare N+ with T+) show comparable levels of IL-1beta in response to LPS.

 
RelB inhibition of LPS-stimulated immune response

Studies in relb–/– mice implicate an important role for RelB in limiting multiorgan inflammation and showed that RelB is a key transcriptional regulator in suppressing proinflammatory cytokine expression (38). In particular, RelB was implicated in delimiting the LPS response in relb–/– fibroblasts and, in the absence of RelB, IL-1beta was overexpressed (39). This study revealed that relb–/– mice have lowered levels of I{kappa}B and, consequently, augmented NF-{kappa}B activation and enhanced expression of a number of proinflammatory cytokines and chemokines. As we had previously implicated I{kappa}B as a labile transcription inhibitor in LPS tolerance (27), we first tested whether RelB knockdown (i.e., RelB siRNA-transfected) cells, like relb–/– cells, had lowered levels of I{kappa}B, and whether this decrease could provide a mechanism for enhanced IL-1beta and the reversal of tolerance in knockdown cells. However, as shown in Fig. 3A, we did not observe a decrease in I{kappa}B levels in RelB knockdown cells.

Alternatively, RelB has also been shown to have differential transcription activity dependent on interaction with variable cofactors. A number of NF-{kappa}B (34, 40) and non-NF-{kappa}B proteins (41, 42) have been implicated in this role. In particular, several studies suggest that p65/RelB heterodimers are unable to bind DNA and, as such, form transcriptionally inactive complexes (43, 44). As shown in Fig. 5, we examined immunoprecipitates from tolerant THP-1 cells for formation of p65 and RelB complexes. In these experiments, we found RelB associated with p65, suggesting that repressed transcription of IL-1beta observed in tolerant cells is due, at least in part, to inactive NF-{kappa}B.


Figure 5
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  		FIGURE 5. RelB inhibition of LPS-stimulated immune response. Normal (NOR) and tolerant (TOL) THP-1 cells were unstimulated (0) or stimulated with LPS (0.5 or 1 h), as indicated in the figure. Whole cell (WCE) and nuclear (NE) extracts were prepared, and p65 immunoprecipitates (IP) were examined by Western blot for the presence of RelB. Whole cell and nuclear extracts from tolerant cells show coprecipitation of inactive p65/RelB, consistent with the repressed NF-{kappa}B transcription observed in LPS tolerance.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
RelB as a mediator of immunosuppression

In this study, we show a novel role for RelB in the transcription-repressed phenotype of LPS-tolerant cells. We found that RelB expression is induced as part of a primary NF-{kappa}B response to inflammation, which includes I{kappa}B degradation, NF-{kappa}B nuclear translocation, and transcription of proinflammatory genes as immediate early responses. Increased levels of RelB were observed after this initial wave of proinflammatory response and are consistent with the observation that induction of RelB itself is NF-{kappa}B dependent (45). Subsequent to the initial response is the LPS-tolerant refractory state. We had shown previously that I{kappa}B degradation and NF-{kappa}B nuclear translocation remained viable in LPS tolerance, but that the transcription activation of proinflammatory genes, like IL-1beta, was repressed (28). Using ChIP assays, we further showed that LPS-tolerant cells do not bind p65 at the IL-1beta promoter despite increased p65 in the nucleus, in contrast to LPS-responsive cells that show a rapid and transient p65 binding to IL-1beta promoter (32). In the study presented in this work, we show that RelB inhibits IL-1beta expression, suggesting that this regulatory pathway may function as a negative feedback loop in the immunosuppression that is found in association with septic shock (19, 20), in which many acute proinflammatory genes become hyporesponsive to inflammatory stimuli. Other proteins, such as A20 and IRAK-M, are advocated as potential negative feedback loops in LPS tolerance (46), but these negative regulators are implicate in disrupting cytosolic activation of NF-{kappa}B. Tolerant cells do not show reduced I{kappa}Ba levels, consistent with a distinct role for RelB in tolerant cells that is independent of I{kappa}B.

Our present studies suggest that immunosuppression is a result, at least in part, of the sequestration of p65 by RelB and the formation of transcription inactive complexes that are unable to activate proinflammatory genes in LPS-tolerant cells. These inactive complexes are unable to bind the IL-1beta promoter and activate transcription, thus providing a reasonable explanation for our ChIP results that showed disruption of p65 binding in LPS-tolerant cells. RelB induction in tolerant cells may also suppress other acute proinflammatory genes, such as TNF-{alpha}, during sepsis and LPS tolerance; this hypothesis remains to be tested. However, the LPS-tolerant phenotype found in sepsis blood leukocytes and cell models is associated with the disruption of other transcription factors unrelated to the NF-{kappa}B family (28). Further studies will be needed to identify what, if any, role RelB may play in the suppression of other transcription factors and LPS-tolerant genes. The complex phenotype of sepsis and LPS tolerance is multifactorial and involves the interaction of many regulatory pathways (46, 47).

Regulation of RelB

The canonical NF-{kappa}B pathway is largely dependent on I{kappa}B kinase beta to promote phosphorylation and degradation of I{kappa}B and translocation of NF-{kappa}B to the nucleus (48). More recently, an alternative pathway to NF-{kappa}B activation has been proposed, in which I{kappa}B kinase {alpha} and NF-{kappa}B-inducing kinase play a more essential role and in which nuclear localization of RelB regulates the expression of a subset of chemokines involved in spleen development (49). In addition to nuclear localization, RelB is regulated by phosphorylation (50) and dimerization (49, 51). The roles that these pathways may play in the establishment of the tolerant phenotype remain to be elucidated.

The LPS-induced RelB, acting as a negative feedback loop to curb an overwhelming and potentially devastating inflammatory response, can be implicated in the sustained LPS-tolerant phenotype that is observed during the course of sepsis (3, 4, 5, 6, 7, 8). Consistent with this model, RelB was constitutively elevated in the sepsis patient leukocytes that we examined. More detailed temporal studies of sepsis patients will be required to assess whether RelB expression correlates with the onset of the immunosuppressed phenotype and whether RelB is lost when the disease state is resolved and immunoresponsiveness is restored. Sepsis is a complicated and not very well-understood syndrome that will require further investigations to examine the participation of RelB in this immune response.

RelB as a mediator of innate and adaptive immunity

Although mechanisms of NF-{kappa}B activation are relatively well understood, the specificity and diversity of transcription control imparted by the activity of this family of proteins continue to be discovered. Given the central role these proteins play in inflammatory processes, we and others (for a review, see Ref. 52) have focused on NF-{kappa}B proteins in immune response and disease. In particular, we have studied the immunosuppressed state associated with LPS tolerance that is related to clinical immunosuppressed state observed in sepsis. Our finding of a role for RelB in immunosuppression is consistent with the experimental findings in relB(–/–) mice that showed increased expression of proinflammatory cytokines and an increased susceptibility to opportunistic infection (53). Relb-deficient mice also show a generalized multiorgan inflammation (54, 55). More recently, it has been demonstrated that RelB plays a unique role in the transition from immediate early innate immune responses to that of more prolonged adaptive immune responses. In particular, RelB is thought to be a critical regulator of dendritic cell (DC) maturation that assures a functional transition from innate to adaptive immunity (56). RelB-deficient mice show a dramatic decrease in thymic and splenic DCs and are impaired in Ag-presenting function and cellular immunity (57). In this study, our results extend this regulatory role and suggest that RelB is essential to an immune response program. Within innate immunity, RelB may down-regulate acute inflammation; as an activator, RelB facilitates the maturation of DCs that are necessary in Ag presentation, T cell activation, and acquisition of adaptive immunity (58).


   Acknowledgments
 
We acknowledge the gifts of RelB (G. Bren) and p65 plasmids (A. Baldwin); the assistance of J. Smith, A. Howard, and D. Hite with patient samples; and the thoughtful discussions with A. Baldwin in the preparation of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI-09169 (to C.E.M.) and MO1-RR007122 to the Wake Forest University Medical Center General Clinical Research Center. Back

2 Address correspondence and reprint requests to Dr. Barbara K. Yoza, Wake Forest University School of Medicine, Department of Surgery, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: byoza{at}wfubmc.edu Back

3 Abbreviations used in this paper: IRAK, IL-1R-associated kinase; ChIP, chromatin immunoprecipitation; DC, dendritic cell; PMN, polymorphonuclear blood leukocyte; siRNA, small interfering RNA. Back

Received for publication March 22, 2006. Accepted for publication July 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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