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The Journal of Immunology, 2004, 173: 5786-5793.
Copyright © 2004 by The American Association of Immunologists

A Role for NF-{kappa}B Subunits p50 and p65 in the Inhibition of Lipopolysaccharide-Induced Shock1

Mihaela Gadjeva2,3,*, Michal F. Tomczak2,{dagger}, Ming Zhang*, Yan Yan Wang{dagger}, Karen Dull{ddagger}, Arlin B. Rogers§, Susan E. Erdman§, James G. Fox§, Michael Carroll* and Bruce H. Horwitz4,{dagger},{ddagger}

* Center for Blood Research, Harvard Medical School, {dagger} Department of Pathology, Immunology Research Division, Brigham and Women’s Hospital, and {ddagger} Division of Emergency Medicine, Children’s Hospital, Boston, MA 02115; and § Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To evaluate the possibility that NF-{kappa}B subunits p50 and p65 have a role in limiting the systemic inflammatory response induced by endotoxin, we compared the susceptibility of wild-type (WT), p65+/–, p50–/–, and p50–/–p65+/– (3X) mice to LPS-induced shock. Interestingly, whereas p65+/– mice were no more sensitive than WT mice to LPS-induced shock, 3X mice were exquisitely sensitive to the toxic effects of LPS. Mice lacking p50 alone displayed an intermediate phenotype. Sensitivity to LPS was a property of the innate immune system and was characterized by elevated circulating levels of TNF in both p50–/– and 3X mice. The ability of LPS to induce shock depended upon TNF, and 3X mice were significantly more sensitive to the toxic effects of TNF than were p50-deficient mice. The expression of several LPS-inducible proinflammatory genes, including IFN-{gamma}, was significantly higher within the spleens of p50–/– mice than in the spleens of WT mice, and interestingly, the expression of IFN-{gamma} was augmented still further within the spleens of 3X mice. These results demonstrate that NF-{kappa}B subunits p50 and p65 have critical inhibitory functions during the systemic response to LPS and raise the possibility that these functions could be essential in preventing mortality associated with systemic inflammatory response syndromes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The induction of proinflammatory gene expression by bacterial LPS plays a crucial role in inducing cardiovascular collapse experienced by patients with systemic Gram-negative bacterial infections, a major cause of morbidity and mortality (1). The transcription factor NF-{kappa}B is thought to be one of the central mediators of this process. NF-{kappa}B is composed of homo- and heterodimers of Rel family proteins (p65, RelB, c-Rel, p52, and p50) (2, 3). NF-{kappa}B is held in an inactive form in the cytoplasm by association with members of the I{kappa}B family (4). Phosphorylation and degradation of I{kappa}B in response to LPS lead to NF-{kappa}B translocation to the nucleus. This event is associated with the activation of a wide range of NF-{kappa}B-responsive proinflammatory genes.

Although there is little doubt that NF-{kappa}B has a critical function in mediating LPS-induced inflammatory gene expression, the role of individual NF-{kappa}B subunits in this process has not been clearly elucidated. In fact, it has been suggested that individual NF-{kappa}B subunits, including p50, p65, RelB, and p52, may have inhibitory activity in some situations. For instance, homodimers of the p50 subunit, which lack a transactivation domain, can inhibit the expression of NF-{kappa}B-dependent genes (5), and increased expression of p50 may mediate the tolerance to TNF induction observed after repetitive stimulation with LPS (6, 7, 8). It has also been suggested that RelB limits prolonged chemokine expression after stimulation of fibroblasts with LPS (9). In addition, it has been demonstrated that p50, p52, and p65 can interact with histone deacetylase 1 (10, 11, 12), an enzyme that has been shown to prevent the acetylation and transcriptional activation of several NF-{kappa}B-dependent genes (10, 11, 12). Finally, it has been suggested that late NF-{kappa}B activation is involved in limiting inflammation in a model of irritant-induced pleurisy (13). Thus, in addition to proinflammatory roles, it is possible that in some situations, NF-{kappa}B subunits have inhibitory functions. However, the importance of these putative inhibitory functions during systemic inflammatory responses in vivo has not been elaborated in detail.

The ability to study the roles of NF-{kappa}B subunits in mediating septic shock syndromes in vivo has been limited by the embryonic lethality of mice lacking the p65 subunit of NF-{kappa}B (14). However, we have recently described a hypozygous mouse model of NF-{kappa}B-deficiency in which mice lack p50 and are heterozygous for p65 (p50–/–p65+/–; 3X)5 (15). These 3X mice are viable, but cells derived from these animals express considerably lower levels of p65 protein than wild-type (WT) cells and have markedly lower levels of NF-{kappa}B binding activity detected by gel shift assay (16). Interestingly, although we have shown that p50-deficient and 3X mice are both susceptible to colitis induced by the inflammatory enterohepatic Helicobacter species H. hepaticus, colitis is significantly more severe in 3X mice than in mice that lack p50 alone (15). Furthermore, although macrophages that lack p50 express elevated levels of IL-12 p40 after bacterial challenge, levels of IL-12 p40 are notably higher after challenge of 3X macrophages (16). Animals and cells that are heterozygous for p65 alone behave no differently than WT controls. These data suggest that both p50 and p65 may have critical inhibitory functions, and that the inhibitory functions of p65 are more clearly demonstrated in the absence of p50. However, whether these inhibitory functions are important during systemic challenge with bacterial products has not been reported. Thus, we compared the susceptibility of WT and several strains of NF-{kappa}B-deficient mice to systemic administration of LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

WT, p50–/–, p65+/–, and p50–/–p65+/– (129/C57BL6) animals were maintained in a facility in which spontaneous colitis has not been observed. The p50–/–p65+/– mice were backcrossed onto the 129S6/SvEvTac-Rag2tm1 background for six generations and then intercrossed to achieve RAG2–/–, p50–/–RAG2–/–, p65+/–RAG2–/–, and p50–/–p65+/–RAG2–/– mice. Gender-matched groups, including predominantly male mice, were used for the experiments. All mice were housed in Association for the Assessment and Accreditation of Laboratory Animal Care-approved facilities. All experiments were approved by the Harvard Medical Area standing committee on animals.

In vivo treatment of mice with LPS or recombinant murine TNF

To assess LPS toxicity, mice were injected i.p. with doses of Escherichia coli LPS serotype 0127:B8 ranging from 0.01–10 mg/kg body weight; mice were monitored continuously for the first 24 h, then every 8 h thereafter for a 90-h period. To assess TNF toxicity, mice were challenged with 100 ng of murine recombinant TNF i.p. (Roche, Ingelheim, Germany). For in vivo TNF blocking experiments, mice received 100 µg of a murine TNF receptor 1 (TNFR1)-human IgG1 (hIgG1) fusion protein (17) or 100 µg of pooled human Ig as a control. For the purposes of this study we considered mice to be in irreversible shock (IS) when they were unable to drink, move about, or right themselves when rolled onto their side. Such mice were immediately euthanized by CO2 inhalation.

Immunoblotting

For immunoblotting, tissue was homogenized in 50 mM Tris (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and proteinase inhibitors. After homogenization, SDS was added to a final concentration of 2%, and DTT was added to a final concentration of 100 µM. Protein concentrations within the extracts were normalized using the Lowry method. Fifteen micrograms of protein from each sample was subjected to SDS-PAGE, followed by transfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were probed with Abs to p50 (SC-1190), p65 (SC-372; Santa Cruz Biotechnologies, Santa Cruz, CA), or ERK (Cell Signaling, Beverley, MA).

Radiation chimeras

RAG and 3X/RAG hosts were irradiated with doses of 800 and 400 rad, separated by 3 h, using a 137Cs source. Irradiated mice received 1 x 106 bone marrow cells harvested from RAG or 3X/RAG mice by retro-orbital injection after lysis of RBC. Host mice were maintained on water containing trimethoprim-sulfamethoxazole for 1 mo. Six weeks after transplantation, mice were challenged with 2 mg/kg LPS and monitored for signs of shock as described above.

Histopathology

Tissues collected at necropsy were fixed overnight in 10% neutral-buffered formalin, processed routinely, and stained with H&E. Tissues were assessed microscopically by a veterinary pathologist (A. B. Rogers).

ELISA

TNF ELISA was performed following the instructions of the manufacturer (eBioscience, San Diego, CA). IFN-{gamma} ELISA was performed using Ab clone R4-6A2 for capture and clone XMG1.2 for detection (BD Pharmingen, San Diego, CA).

Gene expression analysis

Spleens and livers were harvested immediately upon euthanasia and snap-frozen in liquid nitrogen. Frozen specimens were homogenized into Tri-Reagent (Molecular Research Center, Cincinnati, OH), and RNA was isolated according to the manufacturer’s instructions. RNase protection analyses were performed on 5–10 µg of total RNA using RiboQuant Multi Probe Template Sets (BD Pharmingen). The intensities of the protected fragments were quantified by phosphorimager analysis and normalized to internal controls (GAPDH).

Statistical analyses

Kaplan-Meier survival curves were compared using log-rank {chi}2 test. Survival at 24 h was compared using {chi}2 analysis. Cytokine secretion and inflammatory gene expression were compared using t tests. All values are given as a mean, with error bars showing the SEM. A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-{kappa}B-deficient mice exhibit increased susceptibility to LPS-induced shock

To evaluate the susceptibility of NF-{kappa}B-deficient mice to LPS-induced shock, we challenged WT, p50–/–, p65+/–, and 3X mice with 10 mg/kg E. coli LPS serotype 0127:B8, administered by i.p. injection. We considered mice to be suffering from IS when they were unable to drink, move about, or right themselves when rolled onto their sides. We immediately euthanized such mice by CO2 inhalation. WT and p65+/– mice challenged with LPS did not develop IS. In contrast, IS developed in 10 of 12 (83%) p50–/– mice and 11 of 11 (100%) 3X mice (Fig. 1a). However, IS developed significantly more quickly in 3X mice than in p50–/– mice (p = 0.0014; Fig. 1a).



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  		FIGURE 1. 3X mice are highly susceptible to LPS-induced shock. a, Cumulative survival after challenge with LPS is plotted as a function of time (hours after challenge) for WT ({square}; n = 12), p65+/– ({circ}; n = 4), p50–/– ({triangleup}; n = 12), and 3X mice (•; n = 11). *, p = 0.0014, comparing survival rates of 3X and p50–/– mice. b, Cumulative survival after LPS challenge for RAG ({square}; n = 10), p65+/–/RAG ({circ}; n = 2), p50/RAG ({triangleup}; n = 6), and 3X/RAG mice (•; n = 6). *, p = 0.0011 comparing survival rates of p50/RAG and 3X/RAG mice.

 
To further clarify the susceptibility of 3X mice to LPS-induced shock, we challenged WT, p65+/–, p50–/–, and 3X mice with decreasing doses of LPS and assessed the proportion of mice that developed IS within 24 h (Table I). We observed that 75% of 3X mice challenged with doses as low as 0.1 mg/kg developed IS within 24 h, whereas at this time point, none of the WT, p65+/–, or p50–/– mice had developed IS at any dose tested (Table I). We did not observe IS in any group challenged with 0.01 mg/kg LPS (data not shown). These data suggest that the absence of p50 sensitizes mice to LPS-induced shock, and that sensitivity is markedly increased by heterozygosity at the p65 locus. Compared with p50-deficient mice, 3X mice demonstrated lower levels of p65 protein within the spleen and liver (Fig. 2), suggesting that the p65 protein has a critical role in suppressing the ability of LPS to induce shock in animals that lack p50.


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  		Table I. Sensitivity of NF-{kappa}B-deficient mice to LPS-induced shock

 


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  		FIGURE 2. Decreased expression of p65 in 3X mice. Western blots comparing the expressions of p105, p50, p65, and ERK (as loading control) in the spleens (a) or comparing the expressions of p65 and ERK in the livers (b) of WT, p50–/–, and 3X mice.

 
Susceptibility to LPS-induced shock is lymphocyte-independent

NF-{kappa}B proteins play an important role in B lymphocyte development and function (18, 19). To determine whether alterations in B cell populations, serum Ab concentrations, or other lymphocyte-dependent functions could be responsible for increased sensitivity of NF-{kappa}B-deficient animals to LPS-induced shock, we backcrossed 3X mice onto the 129SvEv-RAG2–/– background for six generations (>98% 129SvEv) and then intercrossed these mice to generate RAG2–/– (RAG), p65+/–RAG2–/– (p65+/–/RAG), p50–/–RAG2–/– (p50/RAG), and p50–/–p65+/–RAG2–/– (3X/RAG) mice. We challenged these mice with 10 mg/kg E. coli LPS i.p. We did not observe the development of IS within groups of challenged RAG or p65+/–/RAG animals (Fig. 1b). Although we observed IS in both challenged p50/RAG and 3X/RAG mice, 3X/RAG mice developed IS significantly more quickly than p50/RAG mice (p = 0.0011) (Fig. 1b). In addition, when we assessed the proportion of mice that had developed IS by 24 h, 3X/RAG mice were susceptible to lower does of LPS than p50/RAG mice (Table II). These results indicate that increased susceptibility of p50–/– and 3X mice to LPS-induced shock is not secondary to alterations in lymphocyte functions, such as production of natural Abs, because the dramatic differences in the susceptibility of these strains to LPS-induced shock persists even in the absence of lymphocytes. Rather, it appears that increased susceptibility of NF-{kappa}B-deficient mouse strains to LPS is the result of defects within the innate immune system (defined in this report as components of the immune system present within RAG-2–/– mice).


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  		Table II. Sensitivity of NF-{kappa}B-deficient RAG mice to LPS-induced shock

 
Both hemopoietic and nonhemopoietic components of the innate immune system are involved in susceptibility to LPS-induced shock

To evaluate the contribution of hemopoietic and nonhemopoietic components of the innate immune system to the susceptibility of 3X/RAG mice to LPS-induced shock, we produced radiation chimeras in which RAG and 3X/RAG mice were reconstituted with either RAG or 3X/RAG bone marrow cells. Six weeks after reconstitution, the mice were challenged with 2 mg/kg LPS and monitored for the development of IS (Fig. 3). As expected, all the 3X/RAG animals that received 3X/RAG bone marrow cells (3X/RAG->3X/RAG) and none of the RAG animals that received RAG bone marrow cells (RAG->RAG) developed shock. Interestingly, although none of the RAG animals that received 3X/RAG bone marrow (3X/RAG->RAG) developed shock, five of the nine 3X/RAG animals that received RAG bone marrow (RAG->3X/RAG) did. Statistical comparison of the time to the development of IS between groups confirmed that RAG->3X/RAG animals were more sensitive to LPS-induced shock than RAG->RAG animals (p = 0.02) or 3X/RAG->RAG animals (p = 0.01), but were less sensitive than 3X/RAG->3X/RAG animals (p = 0.01). These results suggest that at least part of the increased sensitivity to LPS exhibited by 3X/RAG animals is due to a defect within the nonhemopoietic compartment of the innate immune system. However, the observation that 3X/RAG->3X/RAG animals were significantly more sensitive to shock after LPS challenge than were RAG->3X/RAG animals indicates that a defect within the hemopoietic compartment of the innate immune system also contributes to the sensitivity of 3X/RAG mice to LPS-induced shock. These results suggest that there are at least two defects within the innate immune system that contribute to the sensitivity of 3X mice to LPS-induced shock.



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  		FIGURE 3. Susceptibility of radiation chimeras to LPS-induced shock. Cumulative survival after challenge with LPS (2 mg/kg) is plotted as a function of time (hours after challenge) for RAG->RAG ({circ}; n = 7), 3X/RAG->RAG ({triangleup}; n = 9), RAG->3X/RAG (•; n = 9), and 3X/RAG->3X/RAG ({blacktriangleup}; n = 11) chimeric mice. *, p = 0.02 and 0.01, comparing survival rates of RAG->3X/RAG and RAG->RAG or 3X/RAG->RAG mice, respectively. **, p = 0.01, comparing RAG->3X/RAG and 3X/RAG->3X/RAG mice.

 
LPS induces signs of vascular congestion and edema, but not of hepatocyte apoptosis

Although the systemic inflammatory response to LPS is generally characterized by vascular congestion and edema, administration of LPS after treatment with N-galactosamine leads to rapid mortality associated with fulminant hepatocellular apoptosis (20). Because it has been demonstrated that mice that completely lack p65 are vulnerable to TNF-mediated hepatocyte apoptosis during embryogenesis (14, 21), we considered the possibility that the development of shock in the NF-{kappa}B-deficient mice evaluated in this study might also be associated with hepatocellular apoptosis. To address this possibility, we performed histological assessment of tissues harvested from RAG, p50/RAG, and 3X/RAG mice 6 h after treatment with 2 mg/kg LPS (Fig. 4), a dose and time point at which the majority of 3X/RAG mice demonstrate signs of IS. Although there were no histological differences between tissues harvested from untreated mice of any genotype (Fig. 4, a, e, and i, and data not shown), LPS induced mild to moderate submucosal edema of the colon, mild pulmonary congestions, and minimal hepatic congestion in RAG (Fig. 4, b, f, and j) and p50/RAG (Fig. 4c, g, and k) mice. In contrast, LPS-treated 3X/RAG mice demonstrated severe submucosal edema, vascular congestion, and lymphedema of the colon; marked congestion and focal perivascular edema of the lungs; and severe congestion, but not hepatocellular apoptosis, of the liver (Fig. 4, d, h, and l). These results confirm that the development of LPS-induced IS in 3X/RAG mice is characterized by signs of vascular collapse and also indicate that hepatocellular apoptosis is unlikely to be involved in this process.



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  		FIGURE 4. Histopathology of LPS-challenged, NF-{kappa}B-deficient RAG mice. Top row, Proximal colon; middle row, lung; bottom row, liver from untreated RAG mice (first column) or RAG, p50/RAG, and 3X/RAG mice treated with LPS for 6 h (second, third, and fourth columns, respectively). Compared with control RAG mice (a, e, and i), treated RAG (b, f, and j) and p50/RAG (c, g, and k) mice demonstrate mild to moderate submucosal edema of the colon (b and c), mild pulmonary congestion (f and g), and minimal hepatic congestion (j and k). In contrast, treated 3X/RAG mice exhibit severe colonic submucosal edema, vascular congestion, and lymphedema (arrow, d), marked pulmonary congestion and focal perivascular edema (arrow, h), and severe congestion, but not hepatocellular apoptosis, of the liver (l). Untreated p50/RAG and 3X/RAG mice were histologically indistinguishable from untreated RAG mice (data not shown). H&E stain; bar = 160 µm (a–d), 450 µm (e–h), and 200 µm (i–l).

 
LPS induces elevated levels of TNF in the serum of NF-{kappa}B-deficient mice

Previous data have demonstrated that TNF is induced rapidly in response to LPS and is a central mediator of LPS-induced shock (22). To determine whether LPS induces higher levels of TNF in p50–/– and 3X mice than in WT mice, we measured TNF levels in serum samples 1 h after challenge with 2 mg/kg LPS. We observed higher levels of TNF in the serum of both p50-deficient and 3X animals compared with levels observed in the serum of WT animals (p < 0.05; Fig. 5a). However, despite the observation that 3X animals were dramatically more sensitive than p50-deficient animals to LPS-induced shock, serum TNF levels were not significantly higher in 3X animals than in p50-deficient animals (p = 0.59; Fig. 5a). None of the groups had detectable TNF in the serum without LPS challenge (data not shown). Compared with RAG mice, p50/RAG and 3X/RAG mice also displayed higher levels of TNF in the serum after LPS challenge (p < 0.05; Fig. 5b). However, as in the lymphocyte sufficient animals, 3X/RAG and p50/RAG mice had similar levels of circulating TNF. It is likely that this rapid increase in circulating TNF observed after LPS challenge of NF-{kappa}B-deficient mice is mediated at the post-transcriptional level, because we observed similar levels of TNF mRNA in both the spleens and colons of WT and NF-{kappa}B-deficient animals 1 h after LPS challenge (Fig. 5c).



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  		FIGURE 5. LPS induces elevated levels of TNF in the serum of NF-{kappa}B-deficient mice. a, Serum TNF levels in WT (n = 6), p50–/– (n = 6), and 3X (n = 6) mice 1 h after challenge with LPS. *, p < 0.05 compared with levels observed in WT mice. b, Serum TNF levels in RAG (n = 5), p50/RAG (n = 5), and 3X/RAG (n = 5) animals 1 h after LPS challenge. c, Relative expression levels of TNF mRNA compared with GAPDH mRNA within the colons (left) and spleens (right) of RAG and 3X/RAG mice 1 h after LPS challenge. There were four animals per group. No statistically significant differences between groups were found.

 
LPS-induced TNF is necessary and sufficient for the development of shock in 3X/RAG mice

To determine whether elevated expression of TNF was required for LPS to induce shock in 3X/RAG mice, we treated 3X/RAG mice with 100 µg of a murine TNFR1-hIgG1 fusion protein (TNFR55-Ig) (17) to deplete TNF or with 100 µg of hIg as a control. The following day, mice were challenged with 2 mg/kg LPS. Although, as expected, all control 3X/RAG mice developed IS within 24 h of challenge, none of the 3X/RAG mice treated with TNFR55-Ig developed IS within the time frame of the experiment (4 days; Fig. 6). This indicates that production of TNF is necessary to sensitize 3X/RAG mice to LPS-induced shock. However, elevated levels of TNF cannot in and of themselves explain why 3X/RAG mice are more sensitive than p50/RAG mice to LPS, because LPS challenge induced similar levels of TNF in these two strains. These observations raised the possibility that in addition to increased circulating levels of TNF, 3X/RAG mice might also exhibit increased sensitivity to the toxic effects of TNF. To examine this directly, we challenged RAG, p65+/–/RAG, p50/RAG, and 3X/RAG mice with 100 or 300 ng of recombinant murine TNF i.p. (Table III and data not shown). We monitored the challenged animals for 90 h. Interestingly, although none of the animals in the RAG, p65+/–/RAG, or p50/RAG group developed IS, 6 of 10 animals in the 3X/RAG group challenged with 100 ng of TNF (p < 0.013) and 2 of 3 3X/RAG animals in the group challenged with 300 ng of TNF developed IS. The development of IS was rapid, occurring 12–24 h after challenge. As was the case after LPS challenge, we observed no evidence of hepatocyte apoptosis by histological assessment, but we did document moderate venous congestion and edema formation, especially in the proximal lower bowel, consistent with a severe shock-like syndrome (data not shown). It is noteworthy that this dose of TNF is at least 10-fold lower than doses previously reported to cause shock in WT mice on several genetic backgrounds, including 129 and C57BL/6 (23, 24). These results suggest that 3X/RAG mice exhibit profound sensitivity to the toxic effects of TNF and are significantly more sensitive than p50/RAG mice.



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  		FIGURE 6. Survival of 3X/RAG mice treated with TNFR55-Ig. Cumulative survival of 3X/RAG mice treated with 100 µg of TNFR55-Ig ({square}; n = 8) or 100 µg of hIg (•; n = 8), before challenge with LPS.

 

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  		Table III. Sensitivity of NF-{kappa}B-deficient RAG mice to TNF

 
Alterations of proinflammatory gene expression in NF-{kappa}B-deficient mice

We hypothesized that alterations in NF-{kappa}B-dependent gene expression could be responsible for the increased sensitivity of NF-{kappa}B-deficient mice to LPS-induced shock. Furthermore, we speculated that despite similar levels of circulating TNF, differences in the transcriptional responses of p50/RAG and 3X/RAG mice to these elevated levels of TNF could explain the increased sensitivity of the latter strain to LPS. To address these issues, we profiled the expression of a group of proinflammatory genes within the spleen of LPS-challenged RAG, p50/RAG, and 3X/RAG mice. Three hours after LPS challenge (2 mg/kg), 8 of 10 proinflammatory genes examined were induced in the spleens of all three strains (Fig. 7). Four of these genes, IFN-{gamma}, TNF, MIP-1{beta}, and MIP-2, were expressed at significantly higher levels in the spleens of p50/RAG mice than in the spleens of RAG mice (Fig. 7). (Although the mean expression of inducible NO synthase and E-selectin was also higher in p50/RAG mice than in RAG mice, these differences were not statistically significant.). However, of the genes examined, only IFN-{gamma} was expressed at significantly higher levels in the spleens of 3X/RAG mice than in the spleens of p50/RAG mice (p = 0.004; Fig. 7). As in the spleen, we observed elevated expression of TNF, MIP-1{beta}, and MIP-2 in the livers of p50/RAG compared with RAG mice (p < 0.05 in all cases; data not shown). However, of the genes examined in the liver, only IFN-{gamma} was expressed at higher levels in 3X/RAG mice than in either RAG or p50/RAG mice (p = 0.01 and p = 0.02, respectively; data not shown). Elevated IFN-{gamma} mRNA expression within organs was reflected by higher IFN-{gamma} protein levels within the serum of 3X/RAG mice than within the serum of RAG or p50/RAG mice 3 h after LPS challenge (Fig. 8), suggesting that higher mRNA expression leads to elevated protein levels in the serum.



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  		FIGURE 7. Alterations of proinflammatory gene expression in NF-{kappa}B-deficient mice. Relative levels of proinflammatory mRNA expression compared with GAPDH mRNA expression within the spleens of RAG ({square}), p50/RAG ({cjs2108}), and 3X/RAG mice ({blacksquare}) before (control) or 3 h after (LPS) LPS challenge. Eight animals were included in each group. *, p < 0.01 compared with relative expression observed in RAG mice. **, p = 0.0006 and 0.004 compared with relative expression of IFN-{gamma} observed in RAG and p50/RAG mice, respectively.

 


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  		FIGURE 8. Elevated expression of IFN-{gamma} in the serum of 3X/RAG mice after LPS challenge. Levels of IFN-{gamma} were measured by ELISA in the serum of RAG ({square}; n = 8), p50/RAG ({cjs2108}; n = 8), and 3X/RAG ({blacksquare}; n = 9) mice 3 h after challenge with LPS (2 mg/kg). *, p < 0.004 and <0.01 compared with levels observed in RAG and p50/RAG mice, respectively. The difference between levels observed in RAG and p50/RAG mice was not statistically significant.

 
To determine the role of TNF in modulating IFN-{gamma} expression after LPS challenge of 3X/RAG mice, we compared IFN-{gamma} expression in the spleens of mice treated with TNFR55-Ig or control serum. TNFR55-Ig inhibited the induction of IFN-{gamma} (p < 0.0001; Fig. 9a) as well as a subset of other proinflammatory genes evaluated (IL-12 p40, MIP-2, and IFN-inducible protein-10; data not shown). Furthermore, challenge of mice with TNF alone induced higher levels of IFN-{gamma} expression in the spleens of 3X/RAG mice than in the spleens of RAG mice (p = 0.035; Fig. 9b). Higher expression of IFN-{gamma} was also observed in the spleens of TNF-treated p50/RAG mice, although this difference did not reach statistical significance. These results indicate that TNF is both necessary and sufficient for the induction of IFN-{gamma} expression observed after LPS challenge of 3X/RAG mice and suggest that in the absence of p50, p65 has a critical role in suppressing TNF-dependent IFN-{gamma} expression.



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  		FIGURE 9. Elevated expression of IFN-{gamma} in LPS-challenged 3X/RAG mice depends upon TNF. a, Percent inhibition of LPS-induced IFN-{gamma} gene expression within the spleens of 3X/RAG mice treated with TNFR55-Ig ({square}) compared with mice treated with hIg ({blacksquare}). Eight animals were included in each group. p < 0.0001 between the two groups. b, IFN-{gamma} mRNA levels in the spleens of RAG ({square}), p50/RAG ({cjs2108}), and 3X/RAG ({blacksquare}) mice 2.5 h after challenge with TNF. Eight animals were included in each group. *, p = 0.035 compared with levels observed in RAG mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated an unexpected role for the NF-{kappa}B subunits p50 and p65 in the inhibition of LPS-induced shock. Although p50-deficient mice exhibited a modest increase in susceptibility to LPS-induced shock, p50–/–p65+/– (3X) mice were exquisitely sensitive to LPS. Susceptibility to shock is a property of the innate immune system, because 3X/RAG mice were also considerably more susceptible to LPS-induced shock than RAG or p50/RAG mice. Furthermore, radiation chimeras suggest that there are defects within both the hemopoietic and nonhemopoietic components of the innate immune system that contribute to susceptibility of 3X/RAG mice to shock.

LPS induced significantly higher levels of TNF in the serum of both p50–/– and 3X mice. However, because TNF levels were comparable in 3X and p50–/– mice and in 3X/RAG and p50/RAG mice, respectively, higher TNF levels cannot in and of themselves explain why heterozygosity at the p65 locus strongly exacerbates sensitivity to LPS. LPS-induced shock in 3X/RAG mice depended upon the expression of TNF, and remarkably, 3X/RAG mice were significantly more sensitive than either RAG or p50/RAG mice to shock induced by exogenous TNF alone, suggesting that heterozygosity at the p65 locus significantly sensitizes mice lacking p50 to the toxic effects of TNF. This increased sensitivity to the toxic effects of TNF did not manifest as increased susceptibility to TNF-induced apoptosis, because histopathological assessment of livers and other organs from LPS- and TNF-treated 3X/RAG animals did not demonstrate lesions consistent with apoptotic cell death. Rather, increased sensitivity to TNF-dependent LPS-induced shock in 3X/RAG mice was associated with higher expression of IFN-{gamma} compared with levels observed in either RAG or p50/RAG mice. Thus, a plausible model to describe the increased sensitivity of 3X mice to LPS includes at least a two-step mechanism. The first step is characterized by increased induction of circulating TNF levels after LPS challenge and occurs in both p50-deficient and 3X mice. The second step, which occurs specifically in 3X mice, is characterized by a dramatic increase in the sensitivity of challenged mice to these elevated levels of circulating TNF and is associated with elevated expression of IFN-{gamma} mRNA.

What is the basis of these two steps? With regard to the first step (elevated expression of TNF), it has been previously reported that p50 overexpression leads to inhibition of TNF gene expression, and that p50–/– splenocytes produce higher amounts of TNF after LPS challenge (25, 26, 27). Our results demonstrating increased levels of circulating TNF after LPS challenge are consistent with these observations. In addition, it has previously been observed that serum TNF levels peak rapidly, within 1–2 h after LPS administration, suggesting that LPS may lead to the release of preformed TNF (28). However, the cellular source of TNF found in the serum after LPS challenge has not been clearly identified. In concert with these previous observations, TNF levels in serum also peak 1 h after administration of LPS to p50 and 3X mice (data not shown), and we were unable to detect increases in TNF mRNA in several organs at the 1 h point, suggesting that the elevated serum levels of TNF observed in NF-{kappa}B-deficient mice may also be the result of the release of preformed TNF. Although 3 h after LPS challenge, higher levels of TNF mRNA were found in the spleens of p50/RAG mice than in the spleens of RAG mice, this is several hours past the peak levels observed in serum, suggesting that these differences in mRNA expression are unlikely to be responsible for the differences in protein levels observed in the serum 1 h after challenge. These observations suggest that it is possible that p50 prevents the accumulation of stored TNF.

In addition to elevated levels of circulating TNF, p50/RAG mice exhibited higher levels of several proinflammatory genes within the spleen 3 h after challenge. These results suggest that p50 has broad inhibitory properties on LPS-induced proinflammatory gene expression in vivo. Although increased proinflammatory gene expression in p50-deficient mice is not unexpected given the large increase in the serum levels of TNF observed after 1 h, the observation that the expression of several of these genes is not effected by TNF depletion (data not shown), indicates that some of the increased gene expression observed in mice lacking p50 is independent of elevated levels of TNF.

With regard to the second step (increased sensitivity of 3X/RAG mice to the toxic effects of TNF), although it is clear that TNF is a central mediator of LPS-induced shock, there are conflicting reports regarding whether administration of TNF alone can induce lethal shock. Prior studies suggest an LD50 of at least 1.5 µg/mouse or higher, although some studies report the absence of mortality at doses as high as 10 µg/mouse (20, 29). Differences in these studies may reflect differences in the mouse strains used and the sources of TNF. Susceptibility to TNF-induced shock can be increased by concomitant administration of cytokines such as IL-1 or IFN-{gamma}, an effect that may be enhanced by priming mice with LPS, IFN-{gamma}, or IL-12 (29, 30). Nonetheless, susceptibility of mice to TNF alone in the range of 100 ng, as observed in 3X/RAG mice, is, to our knowledge, unprecedented. Increased sensitivity to TNF-induced mortality has been observed in mice pretreated with N-galactosamine (20), but mortality in these models is characterized by massive apoptosis of hepatocytes (31), an event that was not observed in 3X/RAG mice challenged with TNF, suggesting that TNF-induced shock in 3X/RAG mice occurs independently of effects on hepatocyte survival. Thus, the observation that 3X mice are highly susceptible to the toxic effects of TNF in the absence of hepatocyte apoptosis suggests that the p50 and p65 subunits of NF-{kappa}B have a previously unappreciated, but essential, role in preventing the ability of TNF to induce shock after LPS challenge.

Our observations that there are defects within both the hemopoietic and nonhemopoietic components of the innate immune system that contribute to the sensitivity of 3X mice to LPS-induced shock may be supportive of the proposed two-step model. It is possible that one of the steps (perhaps increased secretion of TNF by hemopoietic cells) is the result of the absence of p50, whereas the second step (perhaps increased sensitivity to TNF by nonhemopoietic cells) is the result of the absence of p50 in addition to heterozygosity at the p65 locus. Clarifying these issues will require additional experimentation.

The molecular basis for the observation that 3X/RAG mice are dramatically more sensitive than p50/RAG mice to LPS-induced shock has not yet been completely defined. Although it might be expected that the increased sensitivity of 3X/RAG mice would be accompanied by broad increases in proinflammatory gene expression compared with levels detected in p50/RAG mice, surprisingly, this was not observed. However, we did observe increases in the expression of IFN-{gamma} mRNA in both spleen and liver of challenged 3X/RAG mice, and this was reflected in higher circulating levels of IFN-{gamma}. Furthermore, our data demonstrate that TNF is both necessary and sufficient for induction of elevated levels of IFN-{gamma}. Thus, although increased sensitivity of 3X mice to LPS challenge is not associated with a broad increase in proinflammatory gene expression, it is associated with elevated expression of IFN-{gamma}. These data suggest that the ability of LPS to induce shock does not necessarily correlate with massive induction of a broad array of inflammatory factors, but rather, correlates with the focal induction of IFN-{gamma}.

Previous results have suggested a significant role for IFN-{gamma} in the pathophysiology of LPS-induced shock. Anti-IFN-{gamma} Abs given before or after LPS challenge protected mice from the development of shock (32, 33). Furthermore, IFN-{gamma} increased LPS-induced mortality when administered before or up to 4 h after LPS challenge (33), and IFN-{gamma} has been shown to synergize with TNF to produce shock (32). As in our model, previous results have demonstrated that neutralizing Abs to TNF prevent subsequent IFN-{gamma} expression and protect from the development of shock (32). Thus, the increased expression of IFN-{gamma} observed in LPS-challenged 3X/RAG mice could be an important factor that sensitizes these mice to the development of shock. However, whether IFN-{gamma} is, in fact, required for the development of shock in LPS-challenged 3X/RAG mice, and whether there are other factors induced specifically in 3X/RAG mice that contribute to sensitizing these mice to shock have not been evaluated during this study and therefore will require additional clarification. Although we have suggested that increased expression of proinflammatory mediators may be responsible for the increased susceptibility of 3X mice to LPS, it is also possible that the susceptibility of 3X mice is caused by a defect in the induction of a key inhibitor of inflammation. However, as yet we have no direct evidence for such a mechanism.

An important question raised by these studies is whether NF-{kappa}B subunits directly inhibit the transcription of the set of four genes (IFN-{gamma}, TNF, MIP-1{beta}, and MIP-2) whose expression in vivo was increased after LPS challenge of NF-{kappa}B-deficient mice. We and others have previously demonstrated that both p50 and p65 can have direct inhibitory activity on gene expression. However, we were unable to detect elevated expression of any of these four genes after either LPS or TNF stimulation of p50- or 3X-cultured bone marrow-derived macrophages (BMDM) (data not shown). This suggests that either the subunits are acting indirectly to inhibit the expression of these proinflammatory genes in vivo, or that stimulation of BMDM in vitro does not accurately represent the cell type or the nature of the stimulation occurring in vivo. Interestingly, although we have not observed increased expression of IFN-{gamma} in LPS- or TNF-challenged 3X BMDM, we have observed striking elevations in IL-12 p40 after TNF challenge of p50 and 3X BMDM (data not shown), reminiscent of the elevations in IL-12 p40 expression observed after stimulation of p50 and 3X BMDM with H. hepaticus (16). Previous studies have shown that IL-12 is a potent inducer of IFN-{gamma} expression, and it is possible that elevated expression of IL-12 is involved in the elevated levels of IFN-{gamma} observed after in vivo challenge (34). Although acute levels of IL-12 p40 mRNA within the spleen after LPS challenge are similar in RAG, p50/RAG, and 3X/RAG mice, it is possible that subtle differences in the expression of IL-12 p40 before challenge may have great effects on the subsequent expression of IFN-{gamma}. This would be consistent with previous studies suggesting that the expression of IL-12 regulates IFN-{gamma} expression after endotoxin challenge (30, 35).

These studies clearly suggest that NF-{kappa}B subunits p50 and p65 have critical in vivo functions necessary to inhibit mortality in response to septic challenge. Understanding the molecular basis for this phenomenon could lead to the identification of novel inhibitory pathways and define new targets for therapeutic approaches to treat systemic inflammatory syndromes. However, these studies also suggest caution in the application of systemic NF-{kappa}B inhibitors to clinical practice until the basis of these inhibitory phenomena is more thoroughly understood.


   Acknowledgments
 
We thank Paul Rennert (Biogen Idec, Cambridge, MA) for providing TNFR55-Ig.


   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 in part by National Institutes of Health Grants CA67529 and AI50952 (to J.G.F.) and AI052267 (to B.H.H. and S.E.E.). Back

2 M.G. and M.F.T. made equal contributions to this work. Back

3 Current address: Department of Medical Microbiology, Aarhus University, Aarhus, Denmark. Back

4 Address correspondence and reprint requests to Dr. Bruce H. Horwitz, Department of Pathology, Brigham and Women’s Hospital, HNRB 630E, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: bhorwitz{at}rics.bwh.harvard.edu Back

5 Abbreviations used in this paper: 3X, p50–/–p65+/–; BMDM, bone marrow-derived macrophage; hIgG1, human IgG1; HDAC-1, histone deacetylase-1; IS, irreversible shock; RAG, RAG2–/–; p65+/–/RAG, p65+/–RAG2–/–; p50/RAG, p50–/– RAG2–/–; 3X/RAG, p50–/–p65+/–RAG2–/–; TNFR1, TNF receptor 1; WT, wild type. Back

Received for publication March 3, 2004. Accepted for publication August 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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