Hepatic PPARs: their role in liver physiology, fibrosis and treatment.

Complex molecular and cellular mechanisms are involved in the pathway of liver fibrosis. Activation and transformation of hepatic stellate cells (HSCs) are considered the two main reasons for the cause and development of liver fibrosis. The peroxisome proliferator-activated receptors (PPARs) belonging to the family of ligand-activated transcription factors play a key role in liver homeostasis, regulating adipogenesis and inhibiting fibrogenesis in HSCs. Normal transcriptional function of PPARs contributes to maintain HSCs in quiescent phase. A reduced expression of PPARs in HSCs greatly induces a progression of liver fibrosis and an increased production of collagen. Here, we discuss role and function of PPARs and we take into consideration molecular factors able to reduce PPARs activity in HSCs. Finally, although further validations are needed, we illustrate novel strategies available from in vitro and animal studies on how some PPARs-agonists have been proved effective as antifibrotic substances in liver disease.

Sudden coronary death, fatal acute myocardial infarction and widespread coronary and myocardial inflammation.

BACKGROUND: T-lymphocyte activation within atherosclerotic plaque, and widespread to the myocardium, has been shown in patients with acute coronary syndromes. OBJECTIVE: To investigate the presence of T-lymphocyte infiltrate at different stages of acute coronary syndromes by studying patients with sudden coronary death, acute myocardial infarction (AMI) and healed infarction, in comparison with patients with myocarditis and patients with non-ischaemic heart failure. METHODS: 72 cases were studied at autopsy: 12 dying of sudden coronary death (group 1), 12 dying <4 weeks (group 2) and 12 dying >4 months after AMI (group 3), 12 with active lymphocytic myocarditis (group 4), 12 with hypertensive heart disease (group 5), and 12 control subjects (group 6). Light microscopy was performed to measure the number of activated T-lymphocytes (CD3+/DR+) in the myocardium and coronary artery wall, and intercellular adhesion molecule-1 (ICAM-1) expression in the myocardium. RESULTS: Activated T-lymphocyte infiltrates and ICAM-1 myocardial expression in both remote and peri-infarction regions and activated T-lymphocytes within the epicardial coronary artery wall of both the infarct- and non-infarct-related arteries were found in groups 1, 2 and 3, whereas myocardial, but not coronary, infiltrates were found in groups 4 (p<0.001 vs groups 1, 2 and 3 for coronary infiltrates). Groups 5 and 6 had no evidence of myocardial or coronary inflammation (p<0.001 vs groups 1, 2 and 3). CONCLUSIONS: The study shows the presence of a lymphocytic infiltrate in both coronary arteries and myocardium and a proinflammatory phenotype shift in the myocardium associated with acute coronary thrombosis in patients dying suddenly, shortly, or even late after coronary thrombosis.

Prostacyclin in sepsis: a systematic review.

According to current literature, infective processes greatly modify both vascular hemodynamics and anti-oxidant properties of affected tissues, causing a change in homeostasis that regulates the correct functioning of all cells responsible for the physiological and metabolic balance of various organs. As a consequence, the response to the infection that has caused the change is also likely to be weaker and, in the case of septic shock, ineffective. In this review, we will take into consideration these mechanisms and then focus on a group of vasodilator drugs (prostacyclin and its analogs) which, though have been used for over 20 years mainly to treat obstructive vascular diseases, have such hemodynamic and anti-inflammatory properties which prevent homeostatic changes. It is obvious that prostacyclin does not definitively have anti-infective characteristics; however, in association with anti-infective drugs (antibiotics, etc.), the effectiveness of the latter appears improved, at least in some circumstances. Similarly, the fact that prostacyclin and its analogs have a cytoprotective effect on the liver and reduce the ischemia-reperfusion damage following liver transplant is not a novelty and evidence that they improve hepatic hemodynamics suggests their use in those pathologies characterized by possible reduced perfusion or ascertained ischemia of the liver.

Right ventricular dilatation after left ventricular acute myocardial infarction is predictive of extremely high peri-infarctual apoptosis at postmortem examination in humans.

BACKGROUND: Cardiac remodelling after acute myocardial infarction (AMI) is characterised by molecular and cellular mechanisms involving both left and right ventricles, and biventricular failure identifies patients with an extremely unfavourable prognosis. AIMS: To assess whether a link exists between increased myocardial apoptotic rates (AR) at sites of recent infarction and patterns of unfavourable cardiac remodelling, such as biventricular enlargement after left ventricular (LV) infarction. METHODS: Twelve patients with recent AMI involving the LV and not the right ventricle (RV) and with permanent infarct related artery occlusion were selected at necropsy. Gross pathological characteristics, such as LV and RV dilatation, and AR at site of infarction were assessed. Potential false positive results (DNA synthesis and RNA splicing) were excluded from the cell count. RESULTS: RV enlargement, defined as a tricuspidal ring greater than 120 mm, was found in five cases and was associated with LV dilatation. These patients showed significantly higher AR than the others. When the subjects were divided into three groups according to progressive cardiac remodelling (absence of cardiac dilatation, isolated LV dilatation, and biventricular enlargement), the last group had significantly higher ARs than the other two groups, showing that myocardiocyte apoptosis is increased in more unfavourable forms of cardiac remodelling. CONCLUSION: Patients with severely unfavourable cardiac remodelling, such as biventricular enlargement, have extremely high myocardiocyte apoptosis at necropsy, even late after LV myocardial infarction, supporting the role of myocardiocyte loss in determining post-infarction adverse remodelling.

High-risk clinical features predict increased post-infarction myocardial apoptosis and the benefits as a result of an open infarct-related artery.

BACKGROUND: Infarct-related artery (IRA) patency after acute myocardial infarction (AMI) is associated with a more favourable clinical course, in particular in patients with high-risk features. As it has been recently reported that IRA patency is associated with a reduced postinfarction apoptotic rate (AR), the aim of our study was to assess whether IRA status late after AMI had a different impact on AR in high- vs. low-risk patients. METHODS AND RESULTS: Co-localization of TUNEL and caspase-3 was used to calculate the AR at the site of infarction at the time of death in 30 subjects. The Norris coronary prognostic index (NI) was calculated (computing age, presence of pulmonary congestion, heart size and history of previous additional AMI) in order to define the patients' individual risk at the time of hospitalization. According to the NI (< or =7 vs. >7), subjects were divided into low and high risk, as NI >7 carries an approximate threefold higher risk of death. The NI was significantly correlated with the AR at the time of death both in infarct and remote areas. Twenty subjects had IRA occlusion at the time of death, and in these patients AR was significantly higher both in infarct and remote areas (P<0.001 and P=0.009 vs. the others, respectively). However the impact of IRA occlusion on AR was significantly different comparing high- vs. low-risk subjects. In particular, AR at the infarct site was 10-fold higher in the high-risk subjects with IRA occlusion (26.1%[20.4-28.7%]) vs. those with open IRA (2.3%[0.6-3.5%]; P=0.002) and was nonsignificantly different in the low-risk subjects vs. those without IRA occlusion (8.2%[2.5-17.5%] vs. 5.4%[1.5-7.9%]; P=0.48). Similarly, in the high-risk subjects, AR in remote areas was significantly greater in cases with occluded vs. open IRA (0.7%[0.4-0.9%] vs. 0.3%[0.3-0.32%]; P=0.009). CONCLUSION: A significantly higher AR is associated with IRA occlusion late post AMI in subjects with high-risk clinical features, and not in low-risk patients. The diverse impact of IRA occlusion on AR in subjects with different risk profiles may explain the greater benefit associated with coronary reperfusion in high-risk subjects. The overall lower AR in low-risk subjects, independently from the IRA status, may be correlated with the better long-term prognosis after AMI in this case.

Interaction between chronically HIV-infected promonocytic cells and human umbilical vein endothelial cells: role of proinflammatory cytokines and chemokines in viral expression modulation.

HIV type 1 expression was significantly up-regulated in chronically infected promonocytic cell line (U1) co-cultured with human umbilical vein endothelial cells (HUVEC). Virus replication, evaluated as supernatant p24 release, was higher when U1 were co-cultured with IL-1beta-activated HUVEC than with unstimulated HUVEC. When non-adherent U1 were removed from co-cultures, the remaining U1 cells adherent to the endothelial monolayer still showed enhanced HIV replication in comparison with an equal number of U1 cultured alone. While addition of adhesion molecule blocking antibodies (anti-intercellular adhesion molecule-1 (ICAM-1), -vascular cell adhesion molecule-1 (VCAM-1), -CD18 and -very late antigen-4 (VLA-4)) strongly inhibited adherence of U1 cells to endothelial monolayers, such treatment resulted in only a partial reduction in p24 release. Furthermore, HIV replication in U1 cells was enhanced on culture in HUVEC-conditioned media. Such data suggest that soluble mediators secreted by endothelial monolayers may modulate HIV-1 expression. Indeed, addition of cytokine and chemokine antagonists to both U1/HUVEC co-cultures and to U1 cultured in HUVEC-conditioned media clearly down-regulated p24 release. Anti-IL-6, anti-tumour necrosis factor-alpha (TNF-alpha) and, particularly, anti-MCP-1 MoAbs reduced p24 release, while anti-IL-8 polyclonal antiserum and IL-1 receptor antagonist (IL-1Ra) had no significant effect. Thus, the interaction between HUVEC and infected monocytic cells up-regulates HIV-1 replication predominantly through production of endothelium-derived soluble factors including MCP-1, TNF-alpha and IL-6. This phenomenon may influence the passage of HIV-1 from latency to productive replication and enhance virus spreading during physiological and/or pathological contact of monocytes with endothelium.

Atherosclerosis and inflammation. Patterns of cytokine regulation in patients with peripheral arterial disease.

Inflammatory phenomena at sites of atherosclerotic plaques are increasingly thought to be major determinants of the progression and clinical outcome of atherosclerotic disease. Therefore, attention is being paid to systemic markers/mediators which may reflect the inflammatory activity in the plaques. This study evaluates the pattern of the main proinflammatory cytokines tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), their soluble receptors/antagonist, and a variety of inflammatory markers, in patients with peripheral arterial disease (PAD). Eight patients with PAD suffering from claudicatio intermittens (CI), eight with critical limb ischemia (CLI) and eight controls (C) were studied. Blood samples were collected at baseline in all groups and. for C and CI, immediately after and 4 h after a 30-min treadmill test. Baseline: no differences in cytokine plasma levels were detected among the three groups. In contrast, soluble receptors of TNF (type I and II) and of IL-6, and IL-1beta receptor antagonist (IL-1ra) were increased in CI and CLI patients, as compared to C. Of note, IL-Ira correlated with the occurrence and stage of the disease in a highly significant proportion of the patients, reaching a predictive value for the disease of P < 0.0001. The opposite trend was observed for the soluble receptor of IL-1beta. Notably, in the patients no alterations could be found in white blood cell counts, expression of CD11c adherence molecule by circulating monocytes or, in vitro. O2- release from zymosan-activated neutrophils. Moreover, plasma levels of platelet activating factor (PAF), of neutrophil elastase and of the acute phase reactants C-reactive protein (CRP) and alpha1-acid glycoprotein were not found to be significantly altered. In contrast, the acute-phase proteins alpha1-antitrypsin (alpha1AT) and haptoglobin (HG) were found to be increased. Effect of treadmill: IL-1beta and TNFalpha remained at baseline levels following exercise, and IL-6 dropped to undetectable levels. Among cytokine antagonists, again the most relevant changes concerned the IL-1ra, which was significantly increased immediately after the treadmill test, both in CI and C, and returned to baseline levels after 4 h. In contrast, soluble TNFalpha, IL-1beta and IL-6 receptors, PAF, and the other markers of leukocyte activation were not found to be altered. Soluble TNFalpha and IL-6 receptors were shown to inhibit the biological effects of their ligands. Similarly, IL-1ra and the acute phase proteins alpha1AT and HG have been reported to exert anti-inflammatory functions. The increased plasma levels of these agents, together with low levels of inflammatory cytokines and other pro-inflammatory mediators such as PAF and alpha1-acid glycoprotein, appear to draw an undescribed picture, so far, of upregulation of a composite systemic anti-inflammatory mechanism in atherosclerotic patients. IL-1ra appears to be a reliable marker of the state of activation of this mechanism. These results may provide a basis for developing new insights into the pathogenesis of the atherosclerotic disease.

Complement-endothelial cell interactions: pathophysiological implications.

The function of the endothelial cells can be modulated by humoral factors present in the circulation and in the extravascular fluid, including proteins of the complement system. This review examines the multiple interactions between the complement system and the endothelial cells and their functional consequences on inflammation, coagulation and regulation of vascular tone. The implications of these interactions in the induction and progression of the vascular lesions occurring in atherosclerosis, ischemia/reperfusion and xenotransplantation and the possible therapeutic approaches in terms of complement regulation are also discussed.

Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors.

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca2+]i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T. M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371-1380). To determine whether stimulation of [Ca2+]i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca2+]i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca2+]i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti-P- and anti-E-selectin mAb, as well as anti-VCAM-1 mAb, induced transient increases in EC [Ca2+]i that were comparable to those induced by 200 microM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca2+]i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca2+]i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca2+]i changes in the 5 h-treated EC, whereas the anti-VLA-4 mAb alone was sufficient to inhibit [Ca2+]i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti-PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca2+]i, i.e. , mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.

The cytolytically inactive terminal complement complex activates endothelial cells to express adhesion molecules and tissue factor procoagulant activity.

The membrane attack complex of complement (C) in sublytic concentrations stimulates endothelial cells (EC) to express adhesion molecules and to release biologically active products. We have examined the ability of a cytolytically inactive form of this complex, which is incapable of inserting into the cell membrane, to upregulate the expression of adhesion molecules and of tissue factor (TF) procoagulant activity. The inactive terminal C complex (iTCC) was prepared by mixing C5b6, C7, C8, and C9 and was purified by fast protein liquid chromatography on a Superose 12 column. Binding of this complex to EC was found to be dose dependent and was inhibited by anti-C9 antibodies, as assessed both by ELISA using an mAb anti-C9 neoantigen and by measuring cell-bound 125I-labeled iTCC. Exposure of EC to iTCC resulted in a dose- and time-dependent expression of endothelial leukocyte adhesion molecule 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 accompanied by increased levels of the corresponding mRNA, but not in the rapid expression of P-selectin. Inactive TCC also induced increased TF activity evaluated by a chromogenic assay that measures the formation of factor Xa. These effects were inhibited by anti-C9 antibodies. The data support the conclusion that iTCC may induce proinflammatory and procoagulant activities on EC.

Leptospira interrogans and Leptospira peptidoglycans induce the release of tumor necrosis factor alpha from human monocytes.

Elevated plasma concentrations of the cytokine tumor necrosis factor alpha (TNF alpha) have been observed in patients affected by leptospirosis. In this study we found that a preparation of peptidoglycan of Leptospira interrogans, serovar copenhageni, was able to induce the release of TNF alpha from peripheral blood mononuclear cells. TNF alpha induction occurred in a dose dependent manner and was not affected by the endotoxin inhibitor polymixin B. This is the first report on induction of TNF alpha release by a peptidoglycan of spirochetes. Our findings are consistent with existing clinical data and provide a potential mechanism for TNF alpha production.

Leptospira icterohemorrhagiae and leptospire peptidolgycans induce endothelial cell adhesiveness for polymorphonuclear leukocytes.

We have examined the effect of the virulent Leptospira interrogans strain Teramo, serotype icterohemorrhagiae, on the adherence of human neutrophilic polymorphonuclear leukocytes (PMN) to cultured human umbilical vein endothelial cells (HEC). Selective pretreatment of HEC with intact or sonicated leptospires caused a dose- and time-dependent increase of HEC-PMN adhesion (13.2% +/- 2.5% adherence to untreated HEC versus 46.3% +/- 5.6% adherence to HEC pretreated for 4 h with 10(8) intact leptospires per ml [mean +/- standard error of six experiments; P < 0.001]). In contrast, selective leptospire pretreatment of PMN or the addition of leptospires during the adherence assay did not alter HEC-PMN adherence. Leptospire induction of endothelial-cell adhesiveness occurred without detectable HEC damage and was prevented by RNA and protein synthesis inhibitors and by monoclonal antibodies to the CD11/CD18 adhesion complex of neutrophils and to the endothelial-leukocyte adhesion molecule 1 (ELAM-1) of endothelial cells. Similar results were obtained with pretreatment of HEC with interleukin-1 or with the lipopolysaccharide (LPS) of the gram-negative bacterium Escherichia coli. The possibility that contamination by the LPS of gram-negative bacteria could be involved in the induction of HEC adhesiveness was ruled out by the observation that the LPS inhibitor polymyxin B, which abolished the proadhesive effect of E. coli LPS, was ineffective in inhibiting leptospire- as well as interleukin-1-induced adherence. Similarly, leptospire LPSs seemed to have no role in the increase of endothelial-cell adhesiveness, since pretreatment of HEC with a leptospire LPS extract (phenol-water method) or with a leptospire total lipid extract failed to induce the proadhesive phenotype for neutrophils. Instead, peptidoglycans extracted from our leptospires actively stimulated the endothelial proadhesive activity for neutrophils (16.5% +/- 2.1% adherence to untreated HEC versus 51.2% +/- 2.9% adherence to HEC pretreated for 4 h with 1 microgram of peptidoglycan per ml; [mean +/- standard error of four experiments; P < 0.001]). This peptidoglycan-induced activity was inhibited by monoclonal antibodies to the CD11/CD18 adhesion complex and to ELAM-1 but not by polymyxin B. We conclude that peptidoglycans from pathogenic leptospires are among the molecules that can directly activate vascular endothelial cells to increase their adhesiveness for neutrophilic granulocytes. These observations may contribute to a better understanding of the mechanisms whereby non-gram-negative bacteria modulate the local and systemic inflammatory reaction.

Biological activity of a peptidoglycan extracted from Leptospira interrogans: in vitro studies.

Peptidoglycan (PG) has been isolated from some species of spirochaetes, including Leptospira interrogans. Although leptospiral PG has been chemically characterized, no study has been carried out on its potential biological activity. Since PG of Treponema and Borrelia is biologically active both in vivo and in vitro, we investigated the capacity of a leptospiral PG preparation to induce relevant biological effects. PG extracted from L. interrogans strain Teramo was mitogenic at 0.1 microgram ml-1 for human peripheral blood mononuclear cells (PBMC) since it increased the PBMC fraction positive for Ki-67, an antigen expressed by human proliferating cells; at 4 micrograms ml-1, PG was able to induce complement consumption and to stimulate leucocyte phagocytosis and the metabolic burst of resting as well as phagocytosing leucocytes. These findings indicate that Leptospira PG may play a role in modulating the immunocompetent cell functions and suggest that PG can contribute to the host response during Leptospira infection.

Effects of endotoxin in mice bearing solid metastasizing tumors and treated with lysozyme hydrochloride.

The effects of the i.v. administration of endotoxin (6.25-50 micrograms/mouse on day 13 after tumor implantation) in mice treated orally with lysozyme hydrochloride (100 mg/kg on days 5-12 from tumor implantation) were examined using Lewis lung carcinoma in the C57Bl mouse and MCa mammary carcinoma of CBA mice. On primary tumor growth, endotoxin alone causes a dose-dependent and statistically significant reduction with a nadir on day +2 from endotoxin treatment. Combined with lysozyme, endotoxin causes an effect independent of the dose used, corresponding to the effect caused by endotoxin alone at the dose of 25 micrograms/mouse. No tumor regression was recorded in any of the treated groups. Endotoxin is virtually devoid of effects at the metastatic level. In the same conditions, lysozyme causes a reduction of primary tumor growth and a more pronounced inhibition of lung metastasis formation as expected from its already reported effects. The antitumor activity of endotoxin, unlike lysozyme, can be ascribed to tumor hemorrhagic necrosis due to tumor necrosis factor (TNF) production, as determined in tumor homogenates. Endotoxin does not increase the antitumor effects in mice treated with lysozyme, as expected from the data obtained with the more immunogenic SA1 sarcoma, although lysozyme increased the mitogenic response to ConA of ex vivo isolated splenocytes, in vitro cultured in the presence of IL-2.

Mechanisms of eosinophil adherence to cultured vascular endothelial cells. Eosinophils bind to the cytokine-induced ligand vascular cell adhesion molecule-1 via the very late activation antigen-4 integrin receptor.

We have examined the mechanisms involved in the adherence of normal peripheral blood eosinophils to cultured human umbilical vein endothelial cells (HEC) under three conditions: (a) adherence in the absence of treatment of HEC or eosinophils with activating agents (basal adherence); (b) adherence induced by stimulation of eosinophils with phorbol ester (eosinophil-dependent adherence); and (c) adherence induced by pretreatment of HEC with LPS, tumor necrosis factor (TNF), or IL-1 (endothelial-dependent adherence). A mechanism was identified that was equally active in basal, eosinophil-dependent, and endothelial-dependent adherence. This mechanism was optimally active in the presence of both Ca++ and Mg++, and reduced in the presence of Ca++ only or Mg++ only. Furthermore, like the other mechanisms of eosinophil adherence, it was active at 37 degrees C but not at 4 degrees C. A second mechanism of adherence was involved in eosinophil- and in endothelial-dependent adherence. This mechanism was dependent on the CD11/CD18 adhesion complex of eosinophils (i.e., inhibited by anti-CD18 MAb) and it was active in the presence of Ca++ and Mg++ or Mg++ only, but not Ca++ only. The third mechanism of adherence was specific for endothelial-dependent adherence. It involved the endothelial ligand vascular cell adhesion molecule-1 (VCAM-1) and the eosinophil receptor very late activation antigen-4 (VLA-4, CD49d/CD29, i.e., inhibited by anti-VCAM-1 MAb or anti-VLA-4 MAb). This mechanism was active in the presence of Ca++ and Mg++ but not of Ca++ only or Mg++ only, and was not up- or downregulated when eosinophils were stimulated with phorbol ester. In contrast, the endothelial leukocyte adhesion molecule-1 (ELAM-1), that binds neutrophils and monocytes, was not involved in eosinophil adherence to LPS-, TNF-, or IL-1-stimulated HEC (i.e., not inhibited by anti-ELAM-1 MAb). We conclude that eosinophils, like monocytes and lymphocytes, bind to the cytokine-induced endothelial ligand VCAM-1 via the integrin receptor VLA-4.

Identification and partial characterization of a cytolytic toxin produced by Gardnerella vaginalis.

Generation and release into the culture medium of a cytolytic toxin by Gardnerella vaginalis has been demonstrated. Addition of starch and of the nonionic detergent Tween 80 to the culture medium was essential to recover cytolytic activity. A protein with an apparent molecular mass of 61 to 63 kDa was purified from the culture supernatants showing lytic activity towards erythrocytes and nucleated cells, such as human endothelial cells and human neutrophils. The protein had marked selectivity for human erythrocytes, while erythrocytes from other species were not lysed or were lysed at much higher concentrations of the protein than those needed for human erythrocytes. The cytolytic activity was remarkably unstable in polar media, but was stabilized by nonionic detergents, by binding, or by insertion into the target cell membrane, suggesting its amphiphilic nature.

Multiple receptors on human monocytes are involved in adhesion to cultured human endothelial cells.

Monocytes exhibit significant basal (unstimulated) adherence to human umbilical vein endothelium (HUVE), which is only partially inhibited by an anti-CD18 monoclonal antibody (mAb) (60.3). We examined factors modulating the residual, CD18-independent monocyte binding to HUVE by pretreating monocytes with mAb 60.3 to eliminate CD18-dependent binding. Basal adherence was reduced from 32% +/- 2% to 14% +/- 2% with mAb 60.3 (means +/- SE of eight experiments; P less than 0.01). mAb 60.3-treated monocytes were incubated with tumor necrosis factor-gamma (TNF-alpha), interleukin-1 (IL-1), lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalamine (FMLP), or phorbol myristate acetate (PMA). Only PMA affected CD18-independent binding. Pretreatment with PMA alone reduced adherence to 21% +/- 2% (mean +/- SE of eight experiments; P less than 0.01). In conjunction with mAb 60.3, PMA virtually eliminated monocyte adherence to HUVE (7% +/- 1%, mean +/- SE of eight experiments; P less than 0.01). We also examined CD18-independent monocyte binding to endothelial-leukocyte adhesion molecules (E-LAMs) induced by pretreatment of HUVE with LPS. Monoclonal antibody 60.3-treated monocytes increased adherence from 14% +/- 2% with unstimulated HUVE to 37% +/- 2% with LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). Monocytes pretreated with both mAb 60.3 and PMA increased adherence from 5% +/- 1% with the unstimulated HUVE to 18% +/- 1% with the LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). This result implies the presence of a CD18-independent and PMA-insensitive receptor on human monocytes for an E-LAM induced by LPS. In summary, we have identified two CD18-independent mechanisms of monocyte adherence to HUVE; a PMA-sensitive mechanism mediating basal adherence and a PMA-insensitive mechanism involved in binding to E-LAMs.

Tumor necrosis factor-alpha exhibits greater proinflammatory activity than lymphotoxin in vitro.

Tumor necrosis factor-alpha/cachectin (TNF-alpha) and lymphotoxin (LT, TNF-beta) are primarily products of monocytes and lymphocytes, respectively. The proteins are 51% homologous in their primary structure, cause necrosis of Meth A sarcoma in vivo, are toxic to selected tumor cells in vitro, and bind to the same receptor on cells in vitro. However, some recent studies have indicated both qualitative and quantitative differences between recombinant human (rh) LT and rhTNF with respect to inducing human umbilical vein endothelial cell (HEC) adhesiveness for neutrophils and release of hematopoietic growth factor and interleukin-1 (IL-1) from HEC. The rhLT used in these studies was expressed in bacteria and was consequently not glycosylated, whereas natural LT is glycosylated. Therefore, we have compared various preparations of glycosylated and nonglycosylated rhLT with two preparations of rhTNF with respect to their proinflammatory characteristics. We now report that the same LT cDNA, when expressed in mammalian cell line and appropriately glycosylated, is also markedly less potent than rhTNF on a molar basis in inducing endothelial adhesiveness for neutrophils and in directly activating neutrophil adherence to albumin-coated plastic. All recombinant proteins, however, were equipotent on a molar basis in producing cytotoxicity in L929 cells. We conclude that differences in the primary structure of rhTNF and rhLT, rather than alterations induced by prokaryote protein processing, account for the disparate proinflammatory activity in vitro.

Phorbol ester causes down-regulation of CD11/CD18-independent neutrophil adherence to endothelium.

Neutrophils adhere to interleukin-1 (IL-1)-, tumour necrosis factor (TNF)- or lipopolysaccharide (LPS)-pretreated human umbilical vein endothelial cells (HEC) by CD11/CD18-dependent and independent mechanisms. We investigated CD11/CD18-independent neutrophil adherence to LPS-pretreated HEC by: (i) pretreating neutrophils with the anti-CD18 monoclonal antibody mAb 60.3; (ii) performing assays in the absence of Mg2; or (iii) using neutrophils isolated from a patient with leucocyte adhesion deficiency (CD11/CD18-deficiency). Under each of these conditions, CD11/CD18-independent neutrophil adherence to LPS-pretreated HEC was significantly greater than adherence to untreated HEC (15-18% versus 3-7%). In each case, however, stimulation of neutrophils with phorbol ester (PMA) abolished CD11/CD18-independent adherence to LPS-pretreated HEC (less than 5% adherence). Stimulation of neutrophils with bacterial chemotactic peptide (FMLP) or calcium ionophore (A23187) likewise reduced CD18-independent adherence to LPS-pretreated HEC. PMA also inhibited CD11/CD18-independent neutrophil adherence to HEC pretreated with IL-1 or TNF (80-90% inhibition). In contrast, PMA markedly enhanced CD11/CD18-dependent adherence to untreated or LPS-treated HEC. We conclude that stimulation of neutrophils with phorbol ester or other direct agonists down-regulates the CD11/CD18-independent mechanism of neutrophil adherence to IL-1, TNF- or LPS-pretreated HEC.