Distinct temporal patterns of macrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury

In the present study the regulation of CXC chemokine expression was evaluated in full-thickness abdominal wounds in mice. During the first 24 h after injury, IL-1alphabeta, KC, macrophage-inflammatory protein (MIP)-2, and monocyte chemoattractant protein-1 were the predominant cytokines and chemokines produced; TNF-alpha was not detected. Chemokine mRNA expression and protein secretion occurred in two temporal stages. The first, which reached a maximum at 6 h, was associated with high levels of IL-1alpha and KC and low levels of MIP-2. This stage could be reproduced by intradermal injection of IL-1alpha or IL-1beta and was partially blocked by injection of neutralizing Ab against IL-1alpha but not IL-1beta. In animals depleted of circulating neutrophils, chemokine expression was reduced by nearly 70% during this stage. In the second stage, which peaked at 24 h after injury, modest but significant levels of IL-1beta were detected in association with low levels of KC and high levels of MIP-2. This pattern of chemokine expression could not be mimicked by injection of IL-1alpha or IL-1beta (even with prolonged exposure), although MIP-2 expression could be partially inhibited by intradermal injection of neutralizing Ab against IL-1beta. Surprisingly, neutrophil depletion before injury resulted in sustained high levels of both KC and MIP-2 expression. These observations demonstrate that these two closely related chemokines are under distinct regulatory controls in vivo that are likely to reflect the temporally ordered participation of different cell types and/or extracellular stimuli and inhibitors.     

Impairment of NF-kappaB activation and modulation of gene expression by calpastatin

To address the involvement of the calpain systemin both basal and silica-induced nuclear factor (NF)-B activation,several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (IB)degradation and NF-B activation were observed in cells stablyoverexpressing calpastatin. In addition, the cells in which calpain wasconstitutively inhibited by the overexpression of calpastatin exhibiteda notable morphological change. Whereas empty vector-transfected cellsdisplayed a morphology indistinguishable from that of parental cells,cells overexpressing calpastatin exhibited a mosaic morphologicalchange with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genescan be evaluated for their expression at the same time, showed thatcalpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen 2,DAZ, and mitochondrial capsule selenoprotein. These results suggestthat, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.

     

Human B cells become highly responsive to macrophage-inflammatory protein-3 alpha/CC chemokine ligand-20 after cellular activation without changes in CCR6 expression or ligand binding

CCR6 is the only known receptor for the chemokine macrophage-inflammatory protein (MIP)-3alpha/CC chemokine ligand (CCL)20. We have shown previously that CCR6 is expressed on peripheral blood B cells, but CCR6 activity on these cells is low in in vitro assays. We report that MIP-3alpha/CCL20-induced calcium flux and chemotaxis can be enhanced significantly on peripheral blood and tonsillar B cells after activation by cross-linking surface Ag receptors. Of particular interest is the fact that the enhanced activity on B cells was not associated with an increase in CCR6 expression as assessed by levels of receptor mRNA, surface staining, or MIP-3alpha/CCL20 binding sites, or by a change in the affinity of the receptor for ligand. These data convincingly demonstrate that responses to a chemokine can be regulated solely by changes in the downstream pathways for signal transduction resulting from Ag receptor activation, and establish CCR6 as an efficacious receptor on human B cells.     

Homocysteine stimulates monocyte chemoattractant protein-1 expression in mesangial cells via NF-kappaB activation

Hyperhomocysteinemia is regarded as an independent risk factor for cardiovascular disorders. Although renal dysfunction or failure is one of the important factors causing hyperhomocysteinemia, the role of homocysteine (Hcy) in the development of glomerulosclerosis is largely unknown. One of the key events in the pathogenesis of glomerulosclerosis is the infiltration of circulating monocytes into affected glomeruli. The objective of the present study was to investigate the effect of Hcy on the expression of monocyte chemoattractant protein-1 (MCP-1) in kidney mesangial cells and the mechanisms involved. Levels of MCP-1 and mRNA were significantly elevated in Hcy-treated rat mesangial cells. This increase was associated with activation of NF-kappaB as a result of increased phosphorylation of the inhibitor protein IkappaBalpha. Monocyte chemotactic activity in these cells was also enhanced. In addition, there was a significant elevation of superoxide anion produced by Hcy-treated cells, which preceded the increased phosphorylation of IkappaBalpha. Addition of superoxide dismutase or NF-kappaB inhibitors to the culture medium abolished Hcy-induced NF-kappaB activation and MCP-1 expression. Taken together, these results indicate that Hcy induced MCP-1 expression in mesangial cells. Such a process was mediated by oxidative stress and NF-kappaB activation. This may further aggravate renal function in patients with hyperhomocysteinemia.     

Temporal control of NF-kappaB activation by ERK differentially regulates interleukin-1beta-induced gene expression

In cultured rat vascular smooth muscle cells, sustained activation of ERK is required for interleukin-1beta to persistently activate NF-kappaB. Without ERK activation, interleukin-1beta induces only acute and transient NF-kappaB activation. The present study examined whether the temporal control of NF-kappaB activation by ERK could differentially regulate the expression of NF-kappaB-dependent genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1), and manganese-containing superoxide dismutase (Mn-SOD). Treatment of vascular smooth muscle cells with interleukin-1beta induced the expression of iNOS, COX-2, VCAM-1, and Mn-SOD in a time-dependent manner, but with different patterns. Either PD98059 or U0126, selective inhibitors of MEK, or overexpression of a dominant negative MEK-1 inhibited interleukin-1beta- induced ERK activation and the expression of iNOS and COX-2 but had essentially no effect on the expression of VCAM-1 and Mn-SOD. The expression of these genes was inhibited when NF-kappaB activation was down-regulated by MG132, a proteasome inhibitor, or by overexpression of an I-kappaBalpha mutant that prevented both the transient and the persistent activation of NF-kappaB. Inhibition of ERK did not affect interleukin-1beta-induced I-kappaBalpha phosphorylation and degradation but attenuated I-kappaBbeta degradation. Thus, although NF-kappaB activation was essential for interleukin-1beta induction of each of the proteins studied, gene expression was differentially regulated by ERK and by the duration of NF-kappaB activation. These results reveal a novel functional role for ERK as an important temporal regulator of NF-kappaB activation and NF-kappaB-dependent gene expression.     

Flavivirus induces interferon-beta gene expression through a pathway involving RIG-I-dependent IRF-3 and PI3K-dependent NF-kappaB activation

In this study, we found that infection with flaviviruses, such as Japanese encephalitis virus (JEV) and dengue virus serotype 2 (DEN-2), leads to interferon-beta (IFN-beta) gene expression in a virus-replication- and de novo protein-synthesis-dependent manner. NF-kappaB activation is essential for IFN-beta induction in JEV- and DEN-2-infected cells. However, these two viruses seem to preferentially target different members of the interferon regulatory factor (IRF) family. The activation of constitutively expressed IRF-3, characterized by slower gel mobility, dimer formation, and nuclear translocation, is more evident in JEV-infected cells. Other members of the IRF family, such as IRF-1 and IRF-7 are also induced by DEN-2, but not by JEV infection. The upstream molecules responsible for IRF-3 and NF-kappaB activation were further studied. Evidently, a cellular RNA helicase, retinoic acid-inducible gene I (RIG-I), and a cellular kinase, phosphatidylinositol-3 kinase (PI3K), are required for flavivirus-induced IRF-3 and NF-kappaB activation, respectively. Therefore, we suggest that JEV and DEN-2 initiate the host innate immune response through a molecular mechanism involving RIG-I/IRF-3 and PI3K/NF-kappaB signaling pathways.     

Blunted activation of NF-kappaB and NF-kappaB-dependent gene expression by geranylgeranylacetone: involvement of unfolded protein response

Geranylgeranylacetone (GGA), an anti-ulcer agent, has anti-inflammatory potential against experimental colitis and ischemia-induced renal inflammation. However, molecular mechanisms involved in its anti-inflammatory effects are largely unknown. We found that, in glomerular mesangial cells, GGA blocked activation of nuclear factor-κB and consequent induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines. It was inversely correlated with induction of unfolded protein response (UPR) evidenced by expression of 78 kDa glucose-regulated protein (GRP78) and suppression of endoplasmic reticulum stress-responsive alkaline phosphatase. Various inducers of UPR including tunicamycin, thapsigargin, A23187, 2-deoxyglucose, dithiothreitol, and AB₅ subtilase cytotoxin reproduced the suppressive effects of GGA. Furthermore, attenuation of UPR by stable transfection with GRP78 diminished the anti-inflammatory effects of GGA. These results disclosed a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of GGA.     

Restoration of NF-kappaB activation by tumor necrosis factor alpha receptor complex-targeted MEKK3 in receptor-interacting protein-deficient cells

Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. However, the mechanism by which RIP mediates TNF-alpha-induced signal transduction is not fully understood. In this study, we reconstituted RIP-deficient Jurkat T cells with a fusion protein composed of full-length MEKK3 and the death domain of RIP (MEKK3-DD). In these cells, MEKK3-DD substitutes for RIP and directly associates with TRADD in TNF receptor complexes following TNF-alpha stimulation. We found that TNF-alpha-induced NF-kappaB activation was fully restored by MEKK3-DD in these cells. In contrast, expression of a fusion protein composed of NEMO, a component of the IkappaB kinase complex, and the death domain of RIP (NEMO-DD) cannot restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells. These results indicate that the role of RIP is to specifically recruit MEKK3 to the TNF-alpha receptor complex, whereas the forced recruitment of NEMO to the TNF-alpha receptor complex is insufficient for TNF-alpha-induced NF-kappaB activation. Although MEKK2 has a high degree of homology with MEKK3, MEKK2-DD, unlike MEKK3-DD, also fails to restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells, indicating that RIP-dependent recruitment of MEKK3 plays a specific role in TNF-alpha signaling.     

Macrophage inflammatory protein-3alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells

Macrophage inflammatory protein (MIP)-3alpha and beta-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3alpha and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3alpha and BD-2 can bind. Real-time PCR showed that MIP-3alpha and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3alpha. MIP-3alpha and BD-2 increased OPN mRNA levels very slightly. MIP-3alpha and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3alpha was lower than that of BD-2. These findings suggest the MIP-3alpha and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.     

Induction of apoptosis and activation of NF-kappaB by CD95 require different signalling thresholds

Mutations in the death domain of the death receptor CD95 (APO-1/Fas) cause lymphoproliferation and autoimmune disease in both lpr(cg) mice and in patients with autoimmune lymphoproliferative syndrome (ALPS) type Ia. By testing lymphocytes from ALPS type Ia patients, comparing heterozygous with homozygous lpr(cg) mice and coexpressing wild-type and mutant CD95 receptors, we demonstrate that induction of apoptosis requires two wild-type alleles of CD95. By contrast, nuclear factor-kappaB (NF-kappaB) can be fully activated in cells expressing both a mutant and a wild-type CD95 allele, suggesting different thresholds to activate the two signalling pathways. This was confirmed by testing lymphocytes from heterozygous lpr mice, which showed reduced sensitivity to CD95-mediated apoptosis but normal activation of NF-kappaB when compared with wild-type mice. Mutations in CD95 may eliminate the tumour-suppressive function of CD95, at the same time allowing induction of survival or proliferative pathways, which could contribute to the increased risk for lymphoma seen in ALPS type Ia patients.