Vito L. Burgio

Endothelial activation by angiotensin II through NFκB and p38 pathways: Involvement of NFκB-inducible kinase (NIK), free oxygen radicals, and selective inhibition by aspirin†

Angiotensin‐II (AII), the dominant effector of the renin–angiotensin system, is involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Upregulation of the adhesion molecules VCAM‐1, ICAM‐1, and E‐selectin in endothelial cells by inflammatory cytokines through nuclear factor kappa B (NFκB) activation is implicated in formation and progression of atherosclerotic plaque. Here we show that AII induces NFκB‐dependent transcription in primary endothelial cell lines, leading to the upregulation of ICAM‐1 and VCAM‐1 expression. NFκB activation by AII is mediated by the NFκB‐inducing kinase (NIK), a common mediator of NFκB activation by inflammatory cytokines, such as TNF‐α. However, NFκB stimulation by AII differs from that of TNF‐α since a TNF‐receptor associated factor 2 (TRAF‐2) dominant negative mutant does not prevent AII‐mediated NFκB activation. In analogy with TNF‐α‐dependent activation of NFκB, treatment with either the anti‐oxidant N‐acetyl cysteine (NAC) or the cyclooxygenase (COX) inhibitor acetyl salicylic acid (aspirin), but not indometacin, prevents the induction of NFκB‐dependent transcription by AII. Thus, production of reactive oxygen species, aspirin (asp)‐sensitive enzymes of the arachidonate metabolism, and NIK are common transducers of AII‐ and TNF‐dependent pathways to NFκB. AII also activates the inflammatory p38 kinase in endothelial cells, an effect inhibited by exposure to either NAC or asp. Pharmacological interference of the p38 pathway, with the inhibitor SB 202190, prevented AII‐mediated activation of the NFκB target V‐CAM, without affecting degradation of IκBα. These results support a pro‐inflammatory effect of the vasoactive peptide AII in endothelial cells, through at least two pathways—NFκB and p38—both of which are sensitive to asp and antioxidants.

Peripheral monocyte and naive T-cell recruitment and activation in Crohn's disease

BACKGROUND & AIMS Transmural perivascular mononuclear cell infiltrates are a feature of Crohns disease. The aim of this study was a molecular characterization of the mechanisms leading to the formation of these infiltrates. METHODS Endothelial cell and leukocyte expression of the adhesion molecules directing leukocyte transendothelial migration were studied in situ by immunohistochemical analysis of 10 samples from patients with Crohns disease and 10 samples from normal controls. Double-staining methods were used to characterize the cells forming the infiltrates. RESULTS CD11a+ and L-selectin-positive mononuclear cells seemed to be the major component of perivascular infiltrates. The vast majority of these cells were CD68+, CD31+ monocytes/macrophages surrounded by CD3+, L-selectin-positive, CD31+, CD45RA+, and/or CD45RO+ T lymphocytes. T lymphocytes within the vessels expressed both CD45RA and CD45RO markers. Endothelial cells were intercellular adhesion molecule 1 positive and mostly CD34+. Strong adhesion between L-selectin-positive and CD11a+ intravascular mononuclear cells and CD34+ and intercellular adhesion molecule 1-positive endothelial cells were observed. CONCLUSIONS Data indicate that peripheral mononuclear cells are actively recruited in the submucosa of Crohns disease tissue; endothelial cells express adhesion molecules highly permissive for transendothelial migration of monocytes and both naive and memory T cells contributing to infiltrates generation; and close membrane contact between migrated macrophages and naive T cells leads to the T-cell transition from naive to memory phenotype within Crohns disease.

Reactive oxygen intermediates mediate angiotensin II-induced c-Jun.c-Fos heterodimer DNA binding activity and proliferative hypertrophic responses in myogenic cells

Angiotensin II (Ang-II) receptor engagement activates many immediate early response genes in both vascular smooth muscle cells and cardiomyocytes whether a hyperplastic or hypertrophic response is taking place. Although the signaling pathways stimulated by Ang-II in different cell lines have been widely characterized, the correlation between the generation of different second messengers and specific physiological responses remains relatively unexplored. In this study, we report how in both C2C12 quiescent myoblasts and differentiated myotubes Ang-II significantly stimulates AP1-driven transcription and c-Jun•c-Fos heterodimer DNA binding activity. Using a set of different protein kinase inhibitors, we could demonstrate that Ang-II-induced increase in AP1 binding is not mediated by the cAMP-dependent pathway and that both protein kinase C and tyrosine kinases are involved. The observation that in quiescent myoblasts Ang-II increase of AP1 binding and induction of DNA synthesis and, in differentiated myotubes, Ang-II stimulation of protein synthesis are abolished by the cysteine-derivative and glutathione precursor N-acetyl-L-cysteine strongly suggests a role for reactive oxygen intermediates in the intracellular transduction of Ang-II signals for immediate early gene induction, cell proliferation, and hypertrophic responses.

Nuclear Factor kB-independent Cytoprotective Pathways Originating at Tumor Necrosis Factor Receptor-associated Factor 2

Most normal and neoplastic cell types are resistant to tumor necrosis factor (TNF) cytotoxicity unless cotreated with protein or RNA synthesis inhibitors, such as cycloheximide and actinomycin D. Cellular resistance to TNF requires TNF receptor-associated factor 2 (TRAF2), which has been hypothesized to act mainly by mediating activation of the transcription factors nuclear factor kB (NFkB) and activator protein 1 (AP1). NFkB was proposed to switch on transcription of yet unidentified anti-apoptotic genes. To test the possible existence of NFkB-independent cytoprotective pathways, we systematically compared selective trans-dominant inhibitors of the NFkB pathway with inhibitors of TRAF2 signaling for their effect on TNF cytotoxicity. Blockade of TRAF2 function(s) by signaling-deficient oligomerization partners or by molecules affecting TRAF2 recruitment to the TNF receptor 1 complex completely abrogated the cytoprotective response. Conversely, sensitization to TNF cytotoxicity induced by a selective NFkB blockade affected only a fraction of TNF-treated cells in an apparently stochastic manner. No cytoprotective role for c-Jun amino-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), which are activated by TRAF2 and contribute to stimulation of activator protein 1 activity, could be demonstrated in the cellular systems tested. Although required for cytoprotection, TRAF2 is not sufficient to protect cells from TNF + cycloheximide cytotoxicity when overexpressed in transfected cells, thus indicating an essential role of additional TNF receptor 1 complex components in the cytoprotective response. Our results indicate that TNF-induced cytoprotection is a complex function requiring the integration of multiple signal transduction pathways.

Anaplastic large cell lymphoma (CD30+/Ki-1+): results of a prospective clinico-pathological study of 69 cases

Summary. Sixty‐nine anaplastic large cell lymphomas (ALCLs) were selected from an Italian comparative trial on MACOP‐B and F‐MACHOP. As no significant difference in effectiveness of the protocols emerged, they were considered homogenously treated. The ALCLs were divided into two groups according to previously defined criteria: 41 were common type (ALCLs‐CT) and 28 Hodgkin‐related (ALCLs‐HR). T‐cell phenotype was most common (58%), while B‐cell, null and hybrid forms accounted for 27%, 13% and 2%. Clinically, ALCLs CT and HR differed as to mean age (27 v 34·3 years) and presentation; all ALCLs‐HR showed mediastinal involvement, with bulky disease in 57%, and more frequent occurrence in stage II. In contrast, ALCLs‐CT showed mediastinal masses in 58·5%, infrequently revealed bulky disease (24%), and were not specifically associated to stage. Among the ALCLs‐CT, 68·4% achieved complete remission (CR), 24·4% partial remission (PR), one (2·4%) was resistant to therapy, and two (4·8%) had fatal drug toxicity. Of the ALCLs‐HR, 67·8% reached CR, 14·3% PR, and 17·9% did not respond. In CR, ALCLs‐CT showed a greater tendency to relapse (32·1%v 14·2%). At present, 65·8% of ALCLs‐CT and 67·8% of ALCLs‐HR are alive with overall survival/disease‐free survival averages of 31/27 and 29/24 months respectively. Our data emphasize that, independently of subtype, ALCLs benefit from the application of third‐generation protocols for high‐grade non‐Hodgkins lymphomas.

Effect Of Human Natural Killer and γδ T Cells on the Growth of Human Autologous Melanoma Xenografts in SCID Mice

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, γδ T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and γδ cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vδ1 or Vδ2 γ/δ T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vδ1 γ/δ T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vδ1 γδ T lymphocytes could be detected at the s.c. tumor site. In contrast, Vδ2 γδ T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period. These data support the possibility to exploit NK cells and Vδ1 γδ T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors.

The Human Marginal Zone B Cell

Abstract: This study describes the features of the marginal zone (MZ) B cells of human tonsils and spleens and compares them with those of the follicular mantle (FM) B cells from the same tissues. The two B cell subpopulations displayed marked differences in phenotype, in response capacity to T cell‐independent antigens and polyclonal B cell activators, and in presentation of antigens to T cells. FM B cells expressed surface CD5, and hence should be considered as B1 cells by current nomenclature. Fractionation of MZ B cells according to the presence or absence of surface IgD revealed the presence of two subsets. These subsets were characterized by different properties, including the presence of Ig VH gene mutations and the response capacity to TI‐2 antigens, this latter property being associated with IgD‐positive cells. Comparison of the data with those reported for mice revealed that human MZ B cells had strong analogies with both the murine MZ and B1 cells. In contrast, human B1 cells (that is, CD5‐positive FM cells) were considerably different, an observation that should prompt further studies. Indeed, B cells with characteristics analogous to those of murine B1 cells were detected in small but definite proportions in the peripheral blood and tonsils. If the current distinction into B1 and B2 cells has to be maintained also for humans, it is likely that only these CD5‐positive cells rather than the FM B cells should be called B1 cells.

Characterization of a novel human surface molecule selectively expressed by mature thymocytes, activated T cells and subsets of T cell lymphomas.

We have previously characterized mouse H4 (mH4), a surface glycoprotein recognized by the C398.4A monoclonal antibody. We now show that C398.4A also binds its human putative homolog (hpH4). Both hpH4 and mH4 (1) are selectively expressed by activated T cells and mature thymocytes, (2) are disulfide‐linked dimers of two chains (29/37 kDa in humans, 25/29 kDa in mice), whose N‐deglycosylation produces a single band at 20 – 21 kDa, and (3) display a low association with CD4 and the TCR. The expression pattern of hpH4 and its biochemical features showed that it is different from other known activation molecules, and this was confirmed when analysis of the tryptic digest of the hpH4 29‐kDa band by peptide mass searching using matrix‐assisted laser desorption ionization mass spectrometry did not reveal any significant homology with other molecules. In normal lymphoid tissue, hpH4 is expressed by T cells located at the periphery of lymph node germinal centers and paracortical areas. In T cell neoplasia, expression of hpH4 clusters with a subset of peripheral T cell lymphomas with a large‐cell component, and with cases of angioimmunoblastic T cell lymphomas. Overall, these data provide evidence for a novel T cell activation molecule that could help in the phenotypic categorization of T cell malignancies.

The biological relevance of polykaryons in the immune response

Peripheral blood monocyte-derived multinucleated giant cells are a well-known feature of chronic inflammatory conditions. Similarly, virus-induced syncytia derived from CD4+ cells are considered to be typical of human immunodeficiency virus infection under culture conditions. Here, Stefano Fais and colleagues summarize recent experimental results comparing the mechanisms underlying the formation and fate of these two different polykaryons, discussing their putative role in the immune response.

Reactive Oxygen Intermediates (ROIs) Are Involved in the Intracellular Transduction of Angiotensin II Signal in C2C12 Cells

Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.