Alert Cell Strategy in Severe Sepsis and Septic Shock PART 2
Prof. Naoyuki Matsuda M.D., Ph.D.
Department of Emergency & Critical Care Medicine
Nagoya University Graduate School of Medicine, Nagoya, Japan
E-mail: nmatsuda@med.nagoya-u.ac.jp
3. NF-κB activation
NF-κB is a collective term for the Rel/NF-κB family, which contains a domain homologous to the proto-oncogene c-rel. The Rel/NF-κB family comprises RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52) 27). These 5 subunits can homo or heterodimerize, and when inactive, they are prevented from binding to DNA through interactions with inhibitory κB (I-κB) proteins (i.e., I-κBα，I-κBβ，I-κBε，I-κBγ，I-κBζ，Bcl-3，p105，p100). NF-κB activation occurs through canonical and non-canonical pathways.
TLR signaling in response to infection or inflammation amplifying signals of IL-R and TNF-R ultimately converge on I-κB kinase (IKK) complex phosphorylation and the subsequent decrease in cytoplasmic I-κB levels. The IKK complex exists in the cytoplasm as a large complex of over 700kDa, and is composed of a trimer of IKKα, IKKβ, and IKKγ/NF-κB essential modulator (NEMO). When activated, IKKα phosphorylates serines 32 and 36 of I-κBα, and IKKβ phosphorylates serines 19 and 23 of I-κBβ. NEMO can be activated by both phosphorylation and ubiquitination. Upon IKK complex activation and subsequent I-κB phosphorylation, I-κB is poly-ubiquitinated and targeted for proteasome-mediated degradation 28, 29). The NF-κB dimer is then liberated from the inhibitory complex and translocates into the nucleus, where it binds to its cognate target sequence (GGGACTTTCC) and transcriptionally induces inflammatory cytokines and apoptosis inhibiting factors via the canonical pathway. Signaling through TLR and IL-R also uses the canonical pathway and results in nuclear translocation of the RelA and p50 dimer.
Recent reports suggest that a non-canonical NF-κB pathway exists 27). In this pathway, IKKα free of NEMO or IKKβ requires phosphorylation by NF-κB inducing kinase (NIK) to become active. When the RelB-p100 dimer gets phosphorylated by IKKα, p100 undergoes limited processing and generates a RelB-p52 dimer. This dimer then undergoes nuclear translocation and activates transcription of NF-κB target genes. Recent studies have shown that the NF-κB dimer is trapped in the nucleus by I-κBζ 29), and that IKKα can localize to both the cytoplasm and nucleus 30). Nuclear I-κBζ gets phosphorylated by nuclear IKKα, which likely leads to I-κBζ degradation and NF-κB activation 29 30). Some studies suggest the possibility that free cytoplasmic RelA can transport IKK into the nucleus by forming a complex. Nuclear IKK can then phosphorylate histone H3 associated with chromatin and increase the transcription of low molecular weight G proteins and immediate early genes such as Fos and Jun 31). Given the lack of a full understanding of the non-canonical pathway, various aspects will need to be elucidated in future research. Nonetheless, while activation of this pathway is observed in leukotriene receptor signaling in immunocompetent cells, its activation in cells of major organs and vascular endothelial cells has been difficult to detect. Furthermore, since the main NF-κB complex in cells of major organs and vascular endothelial cells in sepsis is RelA/p50, it is highly likely that inflammatory signaling during the early stages of sepsis is mediated by the canonical pathway.
Table 1 lists representative proteins that are overproduced as a result of NF-κB activation in SIRS, shock, and disseminated intravascular coagulation. Inflammatory cytokines such as TNF-α，IL-1，IL-6; inducible nitrogen oxide synthase (iNOS) that produces high levels of nitric oxide (NO); inducible cyclooxygenase-2 (COX2) that leads to production of prostanoids such as prostacyclins; granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF), which stimulate the production of immature granulocytes; chemokines and adhesion molecules that function in leukocyte migration and infiltration; and molecules that activate coagulation all depend on NF-κB activation. In order to understand systemic inflammation, it is key to note that NF-κB activation underlies the generation of inflammatory cytokines and inflammatory molecules.
On the other side, glucocorticoids (e.g., methylprednisolone), which were conventionally used to reduce inflammation, exert their NF-κB inhibiting effect via glucocorticoid receptor α (GRα). While inflammatory infiltration cells (e.g., monocytes, neutrophils, and lymphocytes) slightly express GRα, its expression on alert cells of major organs and vascular endothelial cells has not been reported 32). Because alert cells are known to express scavenger receptors such as CLR and LOX-1 14) on their surface in response to NF-κB activation, the success rate of gene therapy by liposome encapsulation markedly increases. While glucocorticoids mainly target cells involved in cell-mediated immunity, such as non-matured leukocytes that abundantly express GRα, NF-κB decoy oligonucleic acids aim to reduce the induction of inflammation by alert cells, phagocytes and fat cells. From this perspective, studies have shown that while NF-κB decoys effectively reduce inflammation in major organs in sepsis 33-35), they also progress to apoptosis. This suggests that NF-κB may function to inhibit apoptosis during inflammation.
4. NF-κB activation and apoptosis
Alert cell apoptosis during inflammatory stage is prevented by NF-κB-mediated induction of anti-apoptotic factors such as FLICE-inhibitory protein (FLIP), inhibitors of apoptosis (IAPs), and BclX 36). As sepsis progresses, apoptotic vascular endothelial cells are detected in serum 37,38).
In sepsis, the main factor leading to apoptosis in major organs and vascular endothelial cells is the overexpression of DR family members or their downstream adapter proteins 39). As established inducers of apoptosis, TNF-R1, CD95 (Fas), DR4 (TRAIL receptor 1), and DR5 (TRAIL receptor 2) are not only expressed in immunocompetent cells, but also in major organs and vascular endothelial cells25, 26). These DR family members form a complex with FADD, procaspase-8, procaspase-10, and c-FLIP to generate the death-inducing signal complex (DISC) 30) (Table 4). Although c-FLIP, which inhibits procaspase-8 processing, increases in response to NF-κB activation, it decreases as sepsis progresses due to the associated reduction in NF-κB activity 40). The transcriptional induction of DR family and FADD in lungs and vascular endothelial cells in sepsis has been confirmed by RT-PCR and Western blot analysis25, 26). The activation of caspase 3 following DISC activation leads to apoptosis 23).
Sepsis can be modeled in male BALB-C mice by ligating the cecum at 5 mm from the distal end, followed by cecal puncture with a 23G needle in a method called cecal ligation and puncture (CLP) 23, 25, 26, 35). These CLP mice die within 2 days of the procedure, and show two peaks of NF-κB activation at around 10 and 15 hours, which is followed by a progressive decrease in activity. A layer of vascular endothelial cells is found on the surface of an approximately five-layered smooth muscle layer of the mouse aorta, which does not exhibit swelling or shedding under normal conditions. Yet, as sepsis progresses, vascular endothelial cells swell and shed, and swollen cells exhibit positive TUNEL staining (Figure 5) 23,). Systemic apoptosis is also observed in the lungs, cardiac atrium, renal tubules, intestines, spleen, and lymph nodes41). In addition to immunocompetent cells, alert cells in major organs are also affected by apoptosis. Caspase inhibitors, FADD inhibitors, and gene therapy using siRNAs targeting these factors that aim to reduce apoptosis following inflammation hold promise as potential therapies23, 25, 26).
to be continued
Prof. Naoyuki Matsuda M.D., Ph.D.
Department of Emergency & Critical Care Medicine
Nagoya University Graduate School of Medicine, Nagoya, Japan
E-mail: nmatsuda@med.nagoya-u.ac.jp
3. NF-κB activation
NF-κB is a collective term for the Rel/NF-κB family, which contains a domain homologous to the proto-oncogene c-rel. The Rel/NF-κB family comprises RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52) 27). These 5 subunits can homo or heterodimerize, and when inactive, they are prevented from binding to DNA through interactions with inhibitory κB (I-κB) proteins (i.e., I-κBα，I-κBβ，I-κBε，I-κBγ，I-κBζ，Bcl-3，p105，p100). NF-κB activation occurs through canonical and non-canonical pathways.
TLR signaling in response to infection or inflammation amplifying signals of IL-R and TNF-R ultimately converge on I-κB kinase (IKK) complex phosphorylation and the subsequent decrease in cytoplasmic I-κB levels. The IKK complex exists in the cytoplasm as a large complex of over 700kDa, and is composed of a trimer of IKKα, IKKβ, and IKKγ/NF-κB essential modulator (NEMO). When activated, IKKα phosphorylates serines 32 and 36 of I-κBα, and IKKβ phosphorylates serines 19 and 23 of I-κBβ. NEMO can be activated by both phosphorylation and ubiquitination. Upon IKK complex activation and subsequent I-κB phosphorylation, I-κB is poly-ubiquitinated and targeted for proteasome-mediated degradation 28, 29). The NF-κB dimer is then liberated from the inhibitory complex and translocates into the nucleus, where it binds to its cognate target sequence (GGGACTTTCC) and transcriptionally induces inflammatory cytokines and apoptosis inhibiting factors via the canonical pathway. Signaling through TLR and IL-R also uses the canonical pathway and results in nuclear translocation of the RelA and p50 dimer.
Recent reports suggest that a non-canonical NF-κB pathway exists 27). In this pathway, IKKα free of NEMO or IKKβ requires phosphorylation by NF-κB inducing kinase (NIK) to become active. When the RelB-p100 dimer gets phosphorylated by IKKα, p100 undergoes limited processing and generates a RelB-p52 dimer. This dimer then undergoes nuclear translocation and activates transcription of NF-κB target genes. Recent studies have shown that the NF-κB dimer is trapped in the nucleus by I-κBζ 29), and that IKKα can localize to both the cytoplasm and nucleus 30). Nuclear I-κBζ gets phosphorylated by nuclear IKKα, which likely leads to I-κBζ degradation and NF-κB activation 29 30). Some studies suggest the possibility that free cytoplasmic RelA can transport IKK into the nucleus by forming a complex. Nuclear IKK can then phosphorylate histone H3 associated with chromatin and increase the transcription of low molecular weight G proteins and immediate early genes such as Fos and Jun 31). Given the lack of a full understanding of the non-canonical pathway, various aspects will need to be elucidated in future research. Nonetheless, while activation of this pathway is observed in leukotriene receptor signaling in immunocompetent cells, its activation in cells of major organs and vascular endothelial cells has been difficult to detect. Furthermore, since the main NF-κB complex in cells of major organs and vascular endothelial cells in sepsis is RelA/p50, it is highly likely that inflammatory signaling during the early stages of sepsis is mediated by the canonical pathway.
Table 1 lists representative proteins that are overproduced as a result of NF-κB activation in SIRS, shock, and disseminated intravascular coagulation. Inflammatory cytokines such as TNF-α，IL-1，IL-6; inducible nitrogen oxide synthase (iNOS) that produces high levels of nitric oxide (NO); inducible cyclooxygenase-2 (COX2) that leads to production of prostanoids such as prostacyclins; granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF), which stimulate the production of immature granulocytes; chemokines and adhesion molecules that function in leukocyte migration and infiltration; and molecules that activate coagulation all depend on NF-κB activation. In order to understand systemic inflammation, it is key to note that NF-κB activation underlies the generation of inflammatory cytokines and inflammatory molecules.
On the other side, glucocorticoids (e.g., methylprednisolone), which were conventionally used to reduce inflammation, exert their NF-κB inhibiting effect via glucocorticoid receptor α (GRα). While inflammatory infiltration cells (e.g., monocytes, neutrophils, and lymphocytes) slightly express GRα, its expression on alert cells of major organs and vascular endothelial cells has not been reported 32). Because alert cells are known to express scavenger receptors such as CLR and LOX-1 14) on their surface in response to NF-κB activation, the success rate of gene therapy by liposome encapsulation markedly increases. While glucocorticoids mainly target cells involved in cell-mediated immunity, such as non-matured leukocytes that abundantly express GRα, NF-κB decoy oligonucleic acids aim to reduce the induction of inflammation by alert cells, phagocytes and fat cells. From this perspective, studies have shown that while NF-κB decoys effectively reduce inflammation in major organs in sepsis 33-35), they also progress to apoptosis. This suggests that NF-κB may function to inhibit apoptosis during inflammation.
4. NF-κB activation and apoptosis
Alert cell apoptosis during inflammatory stage is prevented by NF-κB-mediated induction of anti-apoptotic factors such as FLICE-inhibitory protein (FLIP), inhibitors of apoptosis (IAPs), and BclX 36). As sepsis progresses, apoptotic vascular endothelial cells are detected in serum 37,38).
In sepsis, the main factor leading to apoptosis in major organs and vascular endothelial cells is the overexpression of DR family members or their downstream adapter proteins 39). As established inducers of apoptosis, TNF-R1, CD95 (Fas), DR4 (TRAIL receptor 1), and DR5 (TRAIL receptor 2) are not only expressed in immunocompetent cells, but also in major organs and vascular endothelial cells25, 26). These DR family members form a complex with FADD, procaspase-8, procaspase-10, and c-FLIP to generate the death-inducing signal complex (DISC) 30) (Table 4). Although c-FLIP, which inhibits procaspase-8 processing, increases in response to NF-κB activation, it decreases as sepsis progresses due to the associated reduction in NF-κB activity 40). The transcriptional induction of DR family and FADD in lungs and vascular endothelial cells in sepsis has been confirmed by RT-PCR and Western blot analysis25, 26). The activation of caspase 3 following DISC activation leads to apoptosis 23).
Sepsis can be modeled in male BALB-C mice by ligating the cecum at 5 mm from the distal end, followed by cecal puncture with a 23G needle in a method called cecal ligation and puncture (CLP) 23, 25, 26, 35). These CLP mice die within 2 days of the procedure, and show two peaks of NF-κB activation at around 10 and 15 hours, which is followed by a progressive decrease in activity. A layer of vascular endothelial cells is found on the surface of an approximately five-layered smooth muscle layer of the mouse aorta, which does not exhibit swelling or shedding under normal conditions. Yet, as sepsis progresses, vascular endothelial cells swell and shed, and swollen cells exhibit positive TUNEL staining (Figure 5) 23,). Systemic apoptosis is also observed in the lungs, cardiac atrium, renal tubules, intestines, spleen, and lymph nodes41). In addition to immunocompetent cells, alert cells in major organs are also affected by apoptosis. Caspase inhibitors, FADD inhibitors, and gene therapy using siRNAs targeting these factors that aim to reduce apoptosis following inflammation hold promise as potential therapies23, 25, 26).
to be continued
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