Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development.

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'-region, named sopB4 and sopB5, were each fused to the gvpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

The inhibitory action of quercetin on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells.

The effect of quercetin on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied. Quercetin pretreatment significantly inhibited NO production in an LPS-stimulated RAW 264.7 murine macrophage cell line. Post-treatment with quercetin partially inhibited NO production. The inhibitory action of quercetin was due to neither the cytotoxic action nor altered LPS binding. The expression of inducible-type NO synthase (iNOS) was markedly down-regulated by quercetin. Quercetin suppressed the release of free nuclear factor (NF)-kappaB by preventing degradation of IkappaB-alpha and IkappaB-beta. Moreover, quercetin blocked the phosphorylation of extracellular signal regulated kinase 1/2 (Erk 1/2), p38, and c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and, further, the activity of tyrosine kinases in LPS-stimulated RAW cells. Quercetin also inhibited interferon (IFN)-gamma-induced NO production. Taken together, these results indicate that the inhibitory action of quercetin on NO production in LPS- and/or IFN-gamma-stimulated macrophages might be due to abrogation of iNOS protein induction by impairment of a series of intracellular signal pathways.

The expression of membrane-bound CD14 renders mouse B-1 cells susceptible to LPS.

The surface expression of CD14 on mouse B-1 cells and its role on their response to lipopolysaccharide (LPS) were studied by using the murine TH2.52 B-1 cell line and peritoneal B-1 cells. TH2.52 cells with the B-1 phenotype were found to express membrane-bound CD14. Furthermore, CD14 was expressed on physiological peritoneal CD5+ B-1 cells. The stimulation of CD14-expressing TH2.52 cells with a low concentration of LPS resulted in the activation of nuclear factor (NF)-B and a mitogen-activated protein kinase (MAPK). The LPS-induced NF-B and MAPK activation was markedly inhibited by anti-CD14 antibody. These results suggest that B-1 cells may respond to LPS via membrane-bound CD14.

Mouse B1 cell line responds to lipopolysaccharide via membrane-bound CD14.

The role of membrane-bound CD14 in the response of mouse B1 cell lines to lipopolysaccharide (LPS) was studied. The surface profile of mouse TH2.52 B cells was positive for CD5, IgM, B220, CD11b and F4/80, suggesting that TH2.52 cells carried the typical phenotype of B1 cells. Furthermore, TH2.52 B1 cells were found to express membrane-bound CD14, which plays a critical role in LPS recognition. TH2.52 B1 cells responded to a very low concentration of LPS and exhibited: (i) augmentation of DNA synthesis; (ii) activation of nuclear factor (NF)-kappaB; and (iii) phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2). They were markedly inhibited by anti-CD14 antibody. Therefore, the expression of membrane-bound CD14 was suggested to provide high sensitivity to LPS for TH2.52 B1 cells.

Inhibition of p38 mitogen-activated protein kinase augments lipopolysaccharide-induced cell proliferation in CD14-expressing Chinese hamster ovary cells.

CD14-expressing Chinese hamster ovary (CD14-CHO) cells, established by transfection of human CD14 DNA, acquired high responsiveness to lipopolysaccharide (LPS) through membrane-bound CD14 expression. LPS induced DNA synthesis and activated a series of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase, in CD14-CHO cells but not in mock-transfected CHO cells. Anti-CD14 antibody completely abrogated both LPS-induced DNA synthesis and LPS-induced phosphorylation of those MAP kinases, suggesting a critical role of membrane-bound CD14 in LPS signaling. A p38 MAP kinase inhibitor, SB203580, markedly augmented LPS-induced DNA synthesis in CD14-CHO cells, whereas an Erk1/2 inhibitor, PD98059, had no affect. On the other hand, SB203580 exhibited no effect on epidermal growth factor-induced DNA synthesis in CD14-CHO cells, although PD98059 inhibited it significantly. The activation and inactivation of p38 MAP kinase with dominant negative and dominant positive mutants also suggested the participation of p38 MAP kinase in LPS-induced DNA synthesis. It was therefore suggested that the activation of p38 MAP kinase can negatively regulate LPS-induced cell proliferation in CD14-CHO cells.

Inhibition of caspase 3 abrogates lipopolysaccharide-induced nitric oxide production by preventing activation of NF-kappaB and c-Jun NH2-terminal kinase/stress-activated protein kinase in RAW 264.7 murine macrophage cells.

The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-kappaB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-kappaB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-kappaB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.

Production of experimental autoimmune sialadenitis in mice immunized with homologous salivary gland extract and Klebsiella O3 lipopolysaccharide.

An experimental murine model for autoimmune sialadenitis was produced by repeated immunization of homologous salivary gland extract together with Klebsiella O3 lipopolysaccharides as an immunological adjuvant. The cell infiltration was observed in the salivary glands of mice immunized more than twice. Inflammatory cells consisting mainly of CD4+ T cells and CD8+ T cells accumulated at the perivascular regions. There was hyperplasia and enlargement of ductal epithelial cells in the secretory acinar units in salivary glands of repeatedly immunized mice. The repeated immunization developed delayed-type hypersensitivity and autoantibody production to the homologous salivary gland extract. The immunohistochemical analysis showed positive staining on the cuboidal cells in the intercalated ducts, and the columnar pseudostratified cells in the striated ducts. Organ-specific antigens with molecular weights ranging from 20 to 90 kDa were recognized by the sera from immunized mice. Therefore, it was suggested that the sialadenitis was produced by the autoimmune mechanism and might be a new experimental model for characterization of the pathogenesis of autoimmune sialadenitis.

The inhibitory action of sodium arsenite on lipopolysaccharide-induced nitric oxide production in RAW 267.4 macrophage cells: a role of Raf-1 in lipopolysaccharide signaling.

The effect of sodium arsenite (SA) on LPS-induced NO production in RAW 267.4 murine macrophage cells was studied. SA pretreatment of LPS-stimulated RAW cells resulted in a striking reduction in NO production. No significant difference in LPS binding was observed between RAW cells pretreated with SA and control untreated RAW cells, suggesting that SA might impair the intracellular signal pathway for NO production. SA inhibited LPS-induced NF-kappaB activation by preventing loss of IkappaB-alpha and -beta. Furthermore, SA blocked phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), but not phosphorylation of p38 and c-Jun N-terminal kinase. SA treatment resulted in the disappearance of Raf-1, suggesting that it might cause the inhibition of the Erk1/2 mitogen-activated protein (MAP) kinase pathway. The SA-mediated loss of Raf-1 also abolished LPS-induced NF-kappaB activation as well as the Erk1/2 pathway. The dominant negative mutant of MAP kinase kinase 1 inhibited both NO production and NF-kappaB activation in LPS-stimulated RAW cells. Taken together, these results indicate that the inhibitory action of SA on NO production in LPS-stimulated macrophages might be due to abrogation of inducible NO synthase induction, and it might be closely related to inactivation of the NF-kappaB and Erk1/2 MAP kinase pathways through loss of Raf-1.

The inhibitory action of butyrate on lipopolysaccharide-induced nitric oxide production in RAW 264.7 murine macrophage cells.

The effect of butyrate, a natural bacterial product of colonic bacterial flora, on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 murine macrophage cells was studied. Butyrate significantly reduced NO production in LPS-stimulated RAW cells. The inhibition was abolished by the removal of butyrate. Butyrate also inhibited the expression of inducible type NO synthase (iNOS) in LPS-stimulated RAW cells. Furthermore, butyrate prevented the activation of nuclear factor (NF)-kappaB through the stabilization of IkappaB-alpha and IkappaB-beta. Butyrate did not affect the phosphorylation of mitogen-activated protein (MAP) kinases by LPS. It was, therefore, suggested that butyrate down-regulated LPS-induced NO production in RAW cells through preventing the expression of iNOS, and that it was due to the inhibitory action of butyrate on the activation of NF-kappaB.

Augmentation of nitric oxide production by gamma interferon in a mouse vascular endothelial cell line and its modulation by tumor necrosis factor alpha and lipopolysaccharide.

The effect of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) on nitric oxide (NO) production in the mouse vascular aortic endothelial cell line END-D was examined. LPS, TNF-alpha, and a low concentration of IFN-gamma inhibited NO production in END-D cells, while a high concentration of IFN-gamma definitely enhanced it. The NO production induced by a high concentration of IFN-gamma was further augmented by using IFN-gamma in combination with LPS or TNF-alpha. In sequential incubations of LPS and IFN-gamma, the enhancement of NO production required prior treatment with IFN-gamma. Stimulation of END-D cells with a high concentration of IFN-gamma led to the expression of inducible NO synthase (iNOS). The augmentation of NO production by IFN-gamma alone or in combination with LPS or TNF-alpha was completely blocked by several inhibitors of iNOS. It was strongly suggested that a high concentration of IFN-gamma itself enhanced NO production in END-D cells through inducing the expression of iNOS. LPS and TNF-alpha exclusively modulated the activity of iNOS once its expression was triggered by IFN-gamma. On the other hand, a low concentration of IFN-gamma, LPS, and TNF-alpha reduced NO production through down-regulating constitutive NOS (cNOS). The differential regulation of cNOS- and iNOS-mediated NO production by IFN-gamma, TNF-alpha, and LPS is discussed.

Big mitogen-activated kinase regulates multiple members of the MEF2 protein family.

Big mitogen-activated protein (MAP) kinase (BMK1), a member of the mammalian MAP kinase family, is activated by growth factors. The activation of BMK1 is required for growth factor-induced cell proliferation and cell cycle progression. We have previously shown that BMK1 regulates c-jun gene expression through direct phosphorylation and activation of transcription factor MEF2C. MEF2C belongs to the myocyte enhancer factor 2 (MEF2) protein family, a four-membered family of transcription factors denoted MEF2A, -2B, -2C, and -2D. Here, we demonstrate that, in addition to MEF2C, BMK1 phosphorylates and activates MEF2A and MEF2D but not MEF2B. The blocking of BMK1 signaling inhibits the epidermal growth factor-dependent activation of these three MEF2 transcription factors. The sites phosphorylated by activated BMK1 were mapped to Ser-355, Thr-312, and Thr-319 of MEF2A and Ser-179 of MEF2D both in vitro and in vivo. Site-directed mutagenesis reveals that the phosphorylation of these sites in MEF2A and MEF2D are necessary for the induction of MEF2A and 2D transactivating activity by either BMK1 or by epidermal growth factor. Taken together, these data demonstrate that, upon growth factor induction, BMK1 directly phosphorylates and activates three members of the MEF2 family of transcription factors thereby inducing MEF2-dependent gene expression.

Exacerbation of vascular endothelial injury in the generalized Shwartzman reaction by the administration of anti-E-selectin antibody.

Previously, we reported that the consecutive administration of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) induced systemic injury of vascular endothelial cells. The aim of this study was to investigate the participation of vascular adhesion molecules in the vascular endothelial injury of GSR. The administration of anti-E-selectin antibody in GSR-induced mice resulted in massive apoptosis of vascular endothelial cells and congestion in blood vessels. Further, marked hemorrhage was found in the pulmonary alveoli of those mice. GSR, especially lung injury, was definitely exacerbated by the administration of anti-E-selectin antibody. On the other hand, the administration of anti-VCAM-1 antibody did not induce such injury of vascular endothelial cells. The possible role of E-selectin in the exacerbation of vascular endothelial injury in GSR is discussed.

Cytoskeletal alterations in lipopolysaccharide-induced bovine vascular endothelial cell injury and its prevention by sodium arsenite.

Morphological changes, especially cytoskeletal alterations, in lipopolysaccharide (LPS)-induced vascular endothelial cell injury were studied by using LPS-susceptible bovine aortic endothelial cells (BAEC). BAEC in cultures with LPS showed cell rounding, shrinking, and intercellular gap formation. In those cells, LPS caused the disorganization of actin, tubulin, and vimentin. LPS also induced a reduction in the F-actin pool and an elevation in the G-actin pool. Cytoskeletal disorganization affected transendothelial permeability across the endothelial monolayer. Pretreatment of BAEC with sodium arsenite (SA) prevented alterations in LPS-induced BAEC injury. However, posttreatment with SA had no protective effect on them. SA upregulated the expression of heat shock protein in the presence of LPS. The role of SA in prevention of LPS-induced BAEC injury is discussed.

Extracellular matrix components prevent lipopolysaccharide-induced bovine arterial endothelial cell injury by inhibiting p38 mitogen-activated protein kinase.

The effect of extracellular matrix components on lipopolysaccharide-induced vascular endothelial cell injury was studied by using lipopolysaccharide-susceptible bovine aortic endothelial cells. For evaluation of lipopolysaccharide-induced injury, we estimated DNA synthesis and cell detachment of bovine aortic endothelial cells in cultures using extracellular matrix components-coated plastic dishes. Among extracellular matrix components, matrigel almost completely inhibited the reduction in DNA synthesis and the enhancement in cell detachment of bovine aortic endothelial cells in cultures with lipopolysaccharide. The lipopolysaccharide-induced injury was also inhibited by coating with type IV collagen, gelatin, fibronectin, laminin, vitronectin, and heparin sulphate proteoglycan. Extracellular matrix components capable of preventing lipopolysaccharide-induced bovine aortic endothelial cells injury coincidentally inhibited the phosphorylation of p38 mitogen-activated protein kinase in lipopolysaccharide-treated bovine aortic endothelial cells. SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, also prevented the reduction in DNA synthesis and the enhancement in cell detachment of bovine aortic endothelial cells in cultures with lipopolysaccharide. It was therefore suggested that extracellular matrix components might protect bovine aortic endothelial cells from lipopolysaccharide-induced injury through inhibiting the activation of p38 mitogen-activated protein kinase.

Bacterial lipopolysaccharide induces cytoskeletal rearrangement in small intestinal lamina propria fibroblasts: actin assembly is essential for lipopolysaccharide signaling.

Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on the dynamics and organization of the cytoskeletal proteins, actin, vimentin, tubulin and vinculin in human small intestinal lamina propria fibroblasts (HSILPF). A noticeable change in the actin architecture was observed after 30 min incubation with LPS with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 2 h. Reorganization of the vimentin network into vimentin bundling was conspicuous at 2 h. With further increase in the time period of LPS exposure, diffused staining of vimentin along with vimentin bundling was observed. Vinculin plaques distributed in the cell body and cell periphery in the control cells rearrange to cell periphery in LPS-treated cells by 30 min of LPS exposure. However, there was no change in the tubulin architecture in HSILPF in response to LPS. LPS increased the F-actin pool in HSILPF in a concentration-dependent manner with no difference in the level of G-actin. A time-dependent study depicted an increase in the G-actin pool at 10 and 20 min of LPS exposure followed by a decrease at further time intervals. The F-actin pool in LPS-treated cells was lower than the control levels at 10 and 20 min of LPS exposure followed by a sharp increase until 120 min and finally returning to the basal level at 140 and 160 min. Further (35)S-methionine incorporation studies suggested a new pool of actin synthesis, whereas the synthesis of other cytoskeletal filaments was not altered. Cytochalasin B, an actin-disrupting agent, severely affected the LPS induced increased percentage of 'S' phase cells and IL-6 synthesis in HSILPF. We conclude that dynamic and orchestrated organization of the cytoskeletal filaments and actin assembly in response to LPS may be a prime requirement for the LPS induced increase in percentage of 'S' phase cells and IL-6 synthesis

Production of tissue factor in CD14-expressing human umbilical vein endothelial cells by lipopolysaccharide.

Production of tissue factor (TF) in response to lipopolysaccharide (LPS) was examined in human umbilical vein endothelial cells (HUVECs) transfected with human CD14 DNA. The expression of CD14 on HUVECs dramatically enhanced the production of TF at a low concentration of LPS in the absence of fetal calf serum (FCS). On the other hand, mock-transfected HUVECs did not respond to even a high concentration of LPS. TF production in CD14-expressing HUVECs was significantly inhibited by anti-CD14 monoclonal antibody. Addition of FCS to the culture of CD14-expressing HUVECs markedly augmented the LPS-induced TF production, whereas only a marginal effect was observed in mock-transfected HUVECs. The findings suggested that the integration of membrane CD14 rendered HUVECs highly sensitive to LPS in the production of TF irrespective of the presence of FCS.

Modulation of barrier function of small intestinal epithelial cells by lamina propria fibroblasts in response to lipopolysaccharide: possible role in TNFalpha in inducing barrier dysfunction.

Recent evidence suggests an interaction between immune, enteric neural and fibroblasts in the regulation of intestinal function. Earlier, we have reported that lipopolysaccharide (LPS) induced cell proliferation, collagen synthesis and production of proinflammatory mediators in lamina propria fibroblasts. In this report, we investigated the change in transepithelial resistance (TER) as a marker of epithelial barrier function by lipopolysaccharide (LPS) and its modulation by human small intestinal lamina propria fibroblasts (HSILPF). Epithelial cells incubated with LPS alone did not show any change in the TER at any concentration or prolonged exposure. However, co-cultivation of epithelial cells with lamina propria fibroblasts which had been exposed to LPS resulted in a rapid decrease in TER by 2 hr. The decrease in the TER was continued till 8 hr followed by returning to the basal level by 24 hr. The supernatant of LPS-treated HSILPF was less effective in causing a fall in the TER than HSILPF itself. The fall in TER was accompanied by loosening of tight junctions as depicted by increased penetration of horse radish peroxidase (HRP) across the epithelial cells from the apical to the basal side. Increased incorporation of 3[H]thymidine (tritiated thymidine) in epithelial cells was observed at 48 hr in the presence of LPS-treated HSILPF. The decrease in TER during the early time period in epithelial cells was abrogated to 70% by incubating the LPS-treated HSILPF and the conditioned medium of LPS-treated HSILPF with anti-TNFalpha antibody, and not with antibody to other cytokines like IL1alpha, IL1beta, IL6 and IL8. Overall, these results suggest that TNFalpha produced by HSILPF in response to LPS as a soluble form cause a decrease in the TER and loosening of tight junctions, and such early changes in the epithelial barrier may contribute to local inflammation in the gut.

Expression of Fas and Fas ligand on mouse renal tubular epithelial cells in the generalized shwartzman reaction and its relationship to apoptosis.

Previously we reported that the consecutive injection of lipopolysaccharide (LPS) into LPS-sensitized mice for the generalized Shwartzman reaction (GSR) appeared to induce the injury of renal tubular epithelial cells via apoptosis. The aim of this study was to characterize the mechanism of renal tubular epithelial cell injury in GSR. The expression of Fas and Fas ligand was immunohistochemically detected on renal tubular epithelial cells from GSR-induced mice, although neither Fas nor Fas ligand was found in cells from untreated control mice or in cells from mice receiving a single injection of LPS. GSR-induced renal tubular epithelial cell injury was produced in neither Fas-negative MRL-lpr/lpr mice nor Fas ligand-negative MRL-gld/gld mice. The administration of anti-gamma interferon antibody together with a preparative injection of LPS prevented the expression of Fas and Fas ligand and the apoptosis of renal tubular epithelial cells. A provocative injection of tumor necrosis factor alpha into LPS-sensitized mice augmented Fas and Fas ligand expression and the apoptosis of renal tubular epithelial cells. The administration of tumor necrosis factor alpha to interleukin-12-sensitized mice resulted in Fas and Fas ligand expression and the apoptosis. Sensitization with interleukin-12 together with anti-gamma interferon antibody did not cause the apoptosis of renal tubular epithelial cells. It was suggested that the Fas/Fas ligand system probably plays a critical role in the development of renal tubular epithelial cell injury through apoptotic cell death.

Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules.

Fibroblasts are important effector cells having a potential role in augmenting the inflammatory responses in various diseases. In infantile diarrhea caused by enteropathogenic Escherichia coli (EPEC), the mechanism of inflammatory reactions at the mucosal site remains unknown. Although the potential involvement of fibroblasts in the pathogenesis of cryptococcus-induced diarrhea in pigs has been suggested, the precise role of lamina propria fibroblasts in the cellular pathogenesis of intestinal infection and inflammation caused by EPEC requires elucidation. Earlier we reported the lipopolysaccharide (LPS)-induced cell proliferation, and collagen synthesis and downregulation of nitric oxide in lamina propria fibroblasts. In this report, we present the profile of cytokines and adhesion molecules in the cultured and characterized human small intestinal lamina propria fibroblasts in relation to neutrophil migration and adhesion in response to lipopolysaccharide (LPS) extracted from EPEC 055:B5. Upon interaction with LPS (1-10 micrograms/ml), lamina propria fibroblasts produced a high level of proinflammatory mediators, interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and cell adhesion molecules (CAM) such as intercellular cell adhesion molecule (ICAM), A-CAM, N-CAM and vitronectin in a time-dependent manner. LPS induced cell-associated IL-1alpha and IL-1beta, and IL-6, IL-8 and TNF-alpha as soluble form in the supernatant. Apart from ICAM, vitronectin, A-CAM, and N-CAM proteins were strongly induced in lamina propria fibroblasts by LPS. Adhesion of PBMC to LPS-treated lamina propria fibroblasts was ICAM-dependent. LPS-induced ICAM expression in lamina propria fibroblasts was modulated by whole blood, PBMC and neutrophils. Conditioned medium of LPS-treated lamina propria fibroblasts remarkably enhanced the neutrophil migration. The migration of neutrophils was inhibited by anti-IL-8 antibody. Co-culture of fibroblasts with neutrophils using polycarbonate membrane filters exhibited time-dependent migration of neutrophils. These findings indicate that the coordinate production of proinflammatory cytokines and adhesion molecules in lamina propria fibroblasts which do not classically belong to the immune system can influence the local inflammatory reactions at the intestinal mucosal site during bacterial infections and can influence the immune cell population residing in the lamina propria.

Role of nitric oxide in lipopolysaccharide-induced hepatic injury in D-galactosamine-sensitized mice as an experimental endotoxic shock model.

The role of nitric oxide (NO) in lipopolysaccharide (LPS)-induced hepatic injury was studied in D-galactosamine (D-GalN)-sensitized mice. The inducible isoform of NO synthase (iNOS) was immunohistochemically detected on hepatocytes around blood vessels in livers of mice injected with D-GalN and LPS not on hepatocytes in mice injected with D-GalN or LPS alone, although mRNA for iNOS was found in those mice. Nitrotyrosine (NT) was also found in livers of mice injected with D-GalN and LPS. The localization of NT was consistent with that of iNOS, and the time courses of NT and iNOS expression were almost the same. Expression of iNOS and NT was detected exclusively in the hepatic lesions of mice injected with D-GalN and LPS. Anti-tumor necrosis factor alpha neutralizing antibody inhibited iNOS and NT expression and hepatic injury. The results suggested that NO from iNOS may play a role in LPS-induced hepatic injury on D-GalN-sensitized mice as an experimental endotoxic shock model.