Korbinian Brand

Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

A Critical Role of Platelet Adhesion in the Initiation of Atherosclerotic Lesion Formation

The contribution of platelets to the process of atherosclerosis remains unclear. Here, we show in vivo that platelets adhere to the vascular endothelium of the carotid artery in ApoE − / − mice before the development of manifest atherosclerotic lesions. Platelet–endothelial cell interaction involved both platelet glycoprotein (GP)Ibα and GPIIb-IIIa. Platelet adhesion to the endothelium coincides with inflammatory gene expression and preceded atherosclerotic plaque invasion by leukocytes. Prolonged blockade of platelet adhesion in ApoE − / − mice profoundly reduced leukocyte accumulation in the arterial intima and attenuated atherosclerotic lesion formation in the carotid artery bifurcation, the aortic sinus, and the coronary arteries. These findings establish the platelet as a major player in initiation of the atherogenetic process.

Nuclear factor κB is activated in macrophages and epithelial cells of inflamed intestinal mucosa

BACKGROUND & AIMS Transcription factors of the nuclear factor kappaB (NF-kappaB) family play an important role in the regulation of genes involved in inflammation. In inflammatory bowel diseases, proinflammatory cytokines known to be regulated by NF-kappaB are involved. The aim of this study was to investigate the role of NF-kappaB activation during mucosal inflammation in situ. METHODS A monoclonal antibody, alpha-p65mAb, was applied for immunofluorescence and immunohistochemical analysis that recognizes activated NF-kappaB. Electrophoretic mobility shift assay was used to directly demonstrate the presence of active DNA-binding NF-kappaB. RESULTS Using the alpha-p65mAb antibody, activated NF-kappaB could be found in biopsy specimens from inflamed mucosa but was almost absent in uninflamed mucosa. The number of cells showing NF-kappaB activation correlated with the degree of mucosal inflammation but was not significantly different between inflamed mucosa from patients with Crohns disease, ulcerative colitis, and nonspecific colitis or diverticulitis. NF-kappaB activation was localized in macrophages and in epithelial cells as identified by double-labeling techniques. Electrophoretic mobility shift assay with isolated lamina propria mononuclear cells and epithelial cells confirmed these results. CONCLUSIONS This study shows for the first time the activation of NF-kappaB during human mucosal inflammation in situ. In addition to macrophages, epithelial cells contained activated NF-kappaB, indicating an involvement in the inflammatory process.

Induction of Cytokine Expression in Leukocytes by Binding of Thrombin-Stimulated Platelets

BACKGROUND Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI. METHODS AND RESULTS We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean +/- SE of fluorescence [channels] before PTCA: 77 +/- 16 versus 35 +/- 9; P = .003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL),1 beta, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1 beta and IL-8 concentration of the cell-free supernatant had increased by 268 +/- 36% and 210 +/- 7%, respectively, and cellular MCP-1 content had increased by 170 +/- 8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-kappa B activation. Inhibition of binding by anti-P selectin antibodies reduced the effect of activated platelets on cytokine production. CONCLUSIONS In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1 beta, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.

Activated Platelets Induce Monocyte Chemotactic Protein-1 Secretion and Surface Expression of Intercellular Adhesion Molecule-1 on Endothelial Cells

BACKGROUND Platelet/endothelium interaction plays an important role in the pathophysiology of inflammation and atherosclerosis. The role of platelets for monocyte chemotactic protein-1 (MCP-1) secretion and surface expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells has been assessed. METHODS AND RESULTS Monolayers of human umbilical vein endothelial cells were incubated with nonstimulated or ADP-activated platelets for 6 hours, and secretion of MCP-1 and surface expression of ICAM-1 were determined by ELISA and flow cytometry, respectively. In the presence of ADP-activated platelets, both MCP-1 secretion and ICAM-1 surface expression were significantly increased compared with nonstimulated platelets (P<0.02). Activation of the transcription factor nuclear factor-kappaB (NF-kappaB) determined by electrophoretic mobility shift assay and kappaB-dependent transcriptional activity was enhanced in the presence of activated platelets. In addition, ADP-activated platelets induced MCP-1 and ICAM-1 promoter-dependent transcription. Liposomal transfection of a double-stranded kappaB phosphorothioate oligonucleotide, but not of the mutated form, inhibited MCP-1 secretion and surface expression of ICAM-1 on activated endothelium (P<0.05). CONCLUSIONS The present study indicates that activated platelets modulate chemotactic (MCP-1) and adhesive (ICAM-1) properties of endothelial cells via an NF-kappaB-dependent mechanism. Platelet-induced activation of the NF-kappaB system might contribute to early inflammatory events in atherogenesis.

Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism. Implications for atherogenesis

Platelets and alterations of chemotactic and adhesive properties of endothelium play an important role in the pathophysiology of atherosclerosis. We investigated the effect of platelets on secretion of monocyte chemotactic protein-1 (MCP-1) and on surface expression of intercellular adhesion molecule-1 (ICAM-1) of cultured endothelium. Pretreatment of cultured monolayers of endothelial cells with alpha-thrombin-activated platelets significantly enhanced secretion of MCP-1 and ICAM-1 surface expression (P<0.01) that could be inhibited by interleukin-1 (IL-1) antagonists by approximately 40%. Activation of transcription factor nuclear factor-kappaB (NF-kappaB) which regulates transcription of early inflammatory response genes such as MCP-1, was significantly increased in endothelial cells treated with activated platelets via an IL-1 mediated mechanism as determined by electrophoretic mobility shift assay (EMSA) and kappaB-dependent transcriptional activity. In trans-well experiments, alpha-thrombin-activated platelets enhanced IL-1-dependent surface expression of vitronectin receptor (alpha(v)beta(3)) on the luminal aspect of endothelial monolayers and promoted alpha(v)beta(3)-mediated platelet/endothelium adhesion that could be inhibited by the antiadhesive peptides GRGDSP and c(RGDfV). We conclude that activated platelets induce significant changes in chemotactic (secretion of MCP-1) and adhesive (surface expression of ICAM-1 and alpha(v)beta(3)) properties of cultured endothelium. These findings imply a potential pathophysiological mechanism of platelets in an early stage of atherogenesis.

Predominant Role of Toll-Like Receptor 2 Versus 4 in Chlamydia pneumoniae-Induced Activation of Dendritic Cells

Chlamydia pneumoniae is an obligate intracellular human pathogen causing diseases such as pneumonia, bronchitis, and pharyngitis. Because of its intracellular replication, cell-mediated immune responses are needed to mediate successful defenses of the host. Because dendritic cells play a central role in linking innate immunity and Ag-specific cell-mediated immune responses we asked whether dendritic cells are activated upon contact with C. pneumoniae and whether known Toll like receptors (TLR) are involved in this process. Here we show that C. pneumoniae was taken up by bone marrow-derived murine dendritic cells. Ingested C. pneumoniae appeared to be unable to develop mature inclusion inside dendritic cells. Furthermore, upon contact with C. pneumoniae dendritic cells were potently stimulated because NF-κB was activated and translocated to the nucleus, cytokines like IL-12p40 and TNF-α were secreted, and expression of MHC class II molecules, CD40, CD80, and CD86 was up-regulated. Importantly, secretion of cytokines as well as translocation of NF-κB were dependent on the presence of TLR2 and independent from TLR4 with the exception of IL-12p40 secretion, which was attenuated in the absence of either a functional TLR2 or 4. In conclusion, we show here that recognition of the Gram-negative bacterium C. pneumoniae depends largely on TLR2 and only to a minor extent on TLR4.

Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity.

T lymphocytes, macrophages, and oxidized low-density lipoprotein (Ox-LDL) are collocalized in early atherosclerotic lesions. Using a low-endotoxin in vitro system, we observed that Ox-LDL but not native LDL induced the production, by both freshly adherent human peripheral blood monocytes and human monocytic THP-1 cells, of the alpha chemokine interleukin (IL)-8, a potent chemoattractant for T lymphocytes. Marked IL-8 induction by Ox-LDL did not require IL-1 beta generation in THP-1 cells. Ox-LDL-induced chemokine production was selective, as Ox-LDL did not stimulate the production by THP-1 cells of the T-lymphocyte chemotactic beta chemokine macrophage inflammatory protein (MIP)-1 alpha. IL-8 induction increased in proportion to the extent of oxidation of LDL as measured by the content of lipid oxidation end products. To identify potentially active components of Ox-LDL, we tested malondialdehyde, an arachidonate-derived lipid oxidation product, and 9-hydroxyoctadecadienoic acid, an oxidation product of linoleate, the major polyunsaturated fatty acid in LDL, and observed that they induced IL-8 generation in the absence of Ox-LDL. Furthermore, when most free lipid oxidation products were removed from Ox-LDL by dialysis, some IL-8-inducing activity was released into the dialysate. However, the major IL-8-inducing activity was not dialyzable. To address the nature of the LDL particle modification required to induce IL-8, acetylated or malondialdehyde-treated native LDL particles were monitored for activity. Neither procedure rendered LDL capable of inducing IL-8. However, phospholipase A2-treated LDL induced THP-1 cell expression of IL-8.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Hydroxynonenal Prevents NF-κB Activation and Tumor Necrosis Factor Expression by Inhibiting IκB Phosphorylation and Subsequent Proteolysis

Extensively oxidized low density lipoprotein (ox-LDL), a modulator of atherogenesis, down-regulates the lipopolysaccharide (LPS)-induced activation of transcription factor NF-κB. We investigated whether 4-hydroxynonenal (HNE), a prominent aldehyde component of ox-LDL, represents one of the inhibitory substances. NF-κB activation by stimuli such as LPS, interleukin (IL)-1β, and phorbol ester, but not tumor necrosis factor (TNF), was reversibly inhibited by HNE in a dose-dependent manner in human monocytic cells, whereas AP-1 binding was unaffected. Using similar HNE concentrations, LPS-induced κB- and TNF or IL-8 promoter-dependent transcription was prevented. Furthermore, pretreatment with HNE suppressed TNF production but not lactate dehydrogenase levels. Under these conditions the binding of LPS to monocytic cells was not significantly affected. However, induced proteolysis of the inhibitory proteins IκB-α, IκB-β, and, at a later time point, IκB-ε was prevented. This is not due to inhibition of the proteasome, the major proteolytic activities of which remain unaffected, but rather to a specific prevention of the activation-dependent phosphorylation of IκB-α. This is the first report which demonstrates that HNE specifically inhibits the NF-κB/Rel system. Down-modulation of NF-κB-regulated gene expression may contribute at certain stages of atherosclerosis to low levels of chronic inflammation and may also be involved in other inflammatory/degenerative diseases.

Involvement of NF-κB signalling in skin physiology and disease

Transcription factors of the nuclear factor-κB (NF-κB)/Rel family play a crucial role in gene regulation during a variety of different cellular processes. This review focuses on the increasing knowledge of the role of NF-κB in skin physiology and pathology. Several studies demonstrate that NF-κB, or components of the system such as IκB kinase (IKK)-α, seem to be involved in epidermal development and differentiation. Furthermore, a dysregulation of NF-κB is suggested to play an important role in skin pathology, including proliferative disorders, e.g. psoriasis, inflammatory processes such as incontinentia pigmenti (IP), sunburn, Lyme disease, allergic contact dermatitis and autoimmune diseases, as well as also in skin carcinogenesis. However, although the knowledge concerning the role of NF-κB in the homeostasis of the skin is steadily increasing, many more questions need to be answered.