Biomedical subjects

P F Piguet

Publications and source records attributed to P F Piguet.

At least 19 recordsLinked to original sources

PubMed1999

Urokinase receptor (uPAR, CD87) is a platelet receptor important for kinetics and TNF-induced endothelial adhesion in mice.

BACKGROUND: Urokinase plasminogen activator receptor (uPAR, CD87) is a widely distributed 55-kD, glycoprotein I-anchored surface receptor. On binding of its ligand uPA, it is known to increase leukocyte adhesion and traffic. Using genetically deficient mice, we explored the role of uPAR in platelet kinetics and TNF-induced platelet consumption. METHODS AND RESULTS: Anti-uPAR antibody stained platelets from normal (+/+) but not from uPAR-/- mice, as seen by fluorescence-activated cell sorter analysis. 51Cr-labeled platelets from uPAR-/- donors survived longer than those from +/+ donors when injected into a +/+ recipient. Intratracheal TNF injection induced thrombocytopenia and a platelet pulmonary localization, pronounced in +/+ but absent in uPAR-/- mice. Aprotinin, a plasmin inhibitor, decreased TNF-induced thrombocytopenia. TNF injection markedly reduced the survival and increased the pulmonary localization of 51Cr-labeled platelets from +/+ but not from uPAR-/- donors, indicating that it is the platelet uPAR that is critical for their response to TNF. As seen by electron microscopy, TNF injection increased the number of platelets and polymorphonuclear neutrophils (PMNs) in the alveolar capillaries of +/+ mice, whereas in uPAR-/- mice, platelet trapping was insignificant and PMN trapping was slightly reduced. Platelets within alveolar capillaries of TNF-injected mice were activated, as judged from their shape, and this was evident in +/+ but not in uPAR-/- mice. CONCLUSIONS: These results demonstrate for the first time the critical role of platelet uPAR for kinetics as well as for activation and endothelium adhesion associated with inflammation.

Animals↗

PubMed1999

Keratinocyte growth factor protects alveolar epithelium and endothelium from oxygen-induced injury in mice.

Keratinocyte growth factor (KGF) has been used successfully to prevent alveolar damage induced by oxygen exposure in rodents. However, this treatment was used intratracheally and before oxygen exposure, which limited its clinical application. In the present study, mice were treated with the recombinant human KGF intravenously before (days -2 and -1) or during (days 0 and +1) oxygen exposure. In both cases, lung damage was attenuated. KGF increased the number of cells incorporating bromodeoxyuridine (BrdU) in the septa and in bronchial epithelium of air-breathing mice but not of oxygen-exposed mice, indicating that the protective effect of KGF is not necessarily associated with proliferation. Oxygen-induced damage of alveolar epithelium and, unexpectedly, of endothelium was prevented by KGF treatment as seen by electron microscopy. We investigated the effect of KGF on different mechanisms known to be involved in oxygen toxicity. The induction of p53, Bax, and Bcl-x mRNAs during hyperoxia was to a large extent prevented by KGF. Surfactant proteins A and B mRNAs were not markedly modified by KGF. The anti-fibrinolytic activity observed in the alveoli during hyperoxia was to a large extent prevented by KGF, most probably by suppressing the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. As PAI-1 -/- mice are more resistant to hyperoxia, KGF might act, at least in part, by decreasing the expression of this protease inhibitor and by restoring the fibrinolytic activity into the lungs.

Animals↗

PubMed1999

TNF-induced enterocyte apoptosis and detachment in mice: induction of caspases and prevention by a caspase inhibitor, ZVAD-fmk.

Injection of mouse recombinant TNF to mice induced apoptosis and detachment of the enterocytes of the tip of the villi, evident after 30 to 90 minutes, which resulted in a shrinkage of the villi. Injection of TNF increased the expression of caspase 1, 2, 3, and 6 as well as of cathepsin D in the mucosal wall, which was maximal 30 minutes after TNF injection. Caspase 1 and 3 were not induced in TNFR1-deficient mice in which TNF does not induce apoptosis and detachment. The administration of a caspase inhibitor (ZVAD-fmk, 300 microg) decreased enterocyte detachment and apoptosis, as well as villus atrophy, whereas a caspase 3 inhibitor (Z-DEVD-cmk) had no effect. The results indicate that the induction of caspases by TNF is the cause of their detachment in the lumen and of the resulting villus atrophy.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Role of TNFR1 and TNFR2 in TNF-induced platelet consumption in mice.

An injection of TNF in mice induced profound thrombocytopenia, due to an increase of platelet consumption, that was evident after 1 h and lasted for 3 days. This process was evident in mice that were genetically deficient in TNFR2 (p75) but not in mice lacking TNFR1 (p55), indicating that the process is mediated by TNFR1-bearing cells. To explore the site of action of TNF, labeled platelets from TNFR1 -/- or +/+ donors were transferred to TNFR1 -/- or +/+ recipients. TNF induced the consumption of platelets from TNFR1 -/- donors when injected into +/+ recipients, while platelets from +/+ donors were not consumed when present in TNFR1 -/- recipients; this finding indicates that TNF acts on the TNFR1 of host cells but does not act on platelets. The expression of TNFRs is consistent with this interpretation, since TNFRs were not detected on platelets by flow cytometry. In megakaryocytes, the expression of TNFR1 was detected by immunohistochemistry. These results indicate that TNF induces platelet consumption by acting not on platelets directly but on the TNFR1 of other cells, presumably increasing the release of factors with agonist activity for platelets.

Animals↗

PubMed1998

TNF-induced enterocyte apoptosis in mice is mediated by the TNF receptor 1 and does not require p53.

Injection of recombinant mouse TNF into mice is known to induce a shrinkage of the duodenal villi, which becomes evident 30-90 min later and is associated with a detachment of enterocytes in the lumen. These cells can be collected by lavage and are all apoptotic, i.e. hypodiploid as seen by flow cytometric analysis. Thus the count of detached cells was used as an evaluation of the TNF-induced cell loss and apoptosis in the mucosa. TNF injection induced a cell loss of similar magnitude in wild-type (+/+) or in mice lacking the TNF receptor (TNFR)2 (p75, TNFR2-/-), while mice lacking the TNFR1 (p55, TNFR1-/-) were completely resistant to this effect. TNF increased the expression of p53 tumor suppressor gene in the enterocytes from the crypts but not from the villi, as seen by Western blots and histochemistry. TNF increased the expression of p53 in both TNFR2-/- and TNFR1-/- mice. Furthermore, enterocyte cell loss was not attenuated in p53-/- mice. The results indicate that TNF, acting on its receptor 1, induces an apoptotic detachment of the enterocytes from the tip of the villi (i.e. the old enterocytes), while in the enterocytes from the crypts (the young enterocytes) TNF increases, via either TNFR1 or TNFR2, the expression of p53, without inducing apoptosis.

Animals↗

PubMed1998

Mortality and platelet depletion occur independently of fibrinogen consumption in murine models of tumour necrosis factor-mediated systemic inflammatory responses.

Tumour necrosis factor (TNF) is known to have procoagulant activity, and platelet depletion is a feature of TNF-mediated systemic inflammatory responses. The aim of this study was to investigate the role of fibrinogen consumption in the development of TNF-mediated systemic inflammatory responses and in the associated depletion of platelets. Three murine models of TNF-mediated systemic inflammatory responses were examined: the systemic toxicity reactions (STR) induced by TNF or lipopolysaccharide (LPS) and severe malaria (SM), a prominently neurological complication of Plasmodium berghei ANKA infection in susceptible mice. There was an acceleration in the consumption of fibrinogen during TNF-STR but not during LPS-STR or SM. However, a concomitant reduction in platelet count was found in all conditions. Mice preliminarily depleted in fibrinogen by treatment with ancrod, an enzyme that specifically degrades fibrinogen, showed no protection against mortality during TNF- or LPS-STR or SM, although they were protected against tissue damage during a modification of the classical local Shwartzman reaction. During TNF- and LPS-STR platelets were even lower in ancrod-treated than control mice and during SM they were not significantly different. This study shows that fibrinogen consumption, although accelerated by the direct injection of TNF, is not necessary for the development of TNF-mediated systemic inflammatory responses in mice, at variance with local pathology, and does not contribute to the associated depletion of platelets.

Ancrod↗

PubMed1998

Stimulation of thrombocytopoiesis decreases platelet beta2 but not beta1 or beta3 integrins.

The expression of CD29, CD61, CD18 and CD11a on platelets was examined by flow cytometry in mice treated with leukaemia inhibitory factor (LIF) or megakaryocyte growth and development factor (PEG-rHuMGDF or mpl-ligand). Treatment for 7-14 d with PEG-rHuMGDF or LIF increased the number of platelets in peripheral blood from 0.9 up to <2.0 x 10(6)/microl. These treatments decreased the expression of CD11a and CD18, whereas that of CD29 or CD61 was not markedly changed. Study after various doses or times of PEG-rHuMGDF administration indicated that a decrease of CD18 expression occurred when platelet counts started to rise. Platelet RNA content was increased in mice treated with PEG-rHuMGDF but double staining indicated that expression of CD18 was not correlated with RNA content. To evaluate integrin expression as a function of time in circulation, platelets were biotinylated in vivo. In normal or PEG-rHuMGDF-treated mice, the expression of CD29 or CD61 did not change, whereas that of CD18 decreased significantly as a function of time in circulation. These findings indicate, firstly, that stimulation of thrombocytopoiesis leads to the release of platelets with a low content of beta2 integrin and, secondly, that this integrin is also selectively lost while in the circulation.

Animals↗

PubMed1998

Role of mast cells and monoamines in the thrombocytopaenia and mortality elicited by tumour necrosis factor in mice.

We explored the thrombocytopaenia elicited by the i.v. injection of mouse recombinant tumour necrosis factor (TNF) in mice. Injection of 10 micrograms of TNF led to a thrombocytopaenia (evident after 0.5 hr) which was caused by decreased platelet survival, as seen by the injection of labelled platelets. TNF-induced thrombocytopaenia was not prevented by heparin, nor by depletion of either fibrinogen or C'. TNF-induced thrombocytopaenia was markedly attenuated in mice treated with reserpine, an agent that depletes monoamines from mast cells and other cells, and in the mast-cell-deficient WWv mice. In vitro, TNF elicited a modest release of monoamine from peritoneal mast cells and from a mast cell line. When mice are injected with 3H-serotonin (3H-5HT) before TNF, TNF injection increased the plasma 3H-5HT content 1 hr later, modifications absent in reserpine pretreated or mast-cell-deficient mice. 3H-5HT content of the small intestine was markedly depleted in TNF-injected mice, suggesting that this organ is the source of the plasma 3H-5HT. Drop in body temperature and mortality induced by TNF were also attenuated in mast-cell-deficient, and in reserpine pretreated mice. These results indicate that TNF can induce a release of monoamines from mast cells, mainly from those of the small intestine, a process that contributes to TNF-induced thrombocytopaenia and mortality.

Adrenergic Uptake Inhibitors↗

PubMed1998

Oxygen toxicity in mouse lung: pathways to cell death.

Mice exposed to 100% O2 die after 3 or 4 d with diffuse alveolar damage and alveolar edema. Extensive cell death is evident by electron microscopy in the alveolar septa, affecting both endothelial and epithelial cells. The damaged cells show features of both apoptosis (condensation and margination of chromatin) and necrosis (disruption of the plasma membrane). The electrophoretic pattern of lung DNA indicates both internucleosomal fragmentation, characteristic of apoptosis, and overall degradation, characteristic of necrosis. Hyperoxia induces a marked increase in RNA or protein levels of p53, bax, bcl-x, and Fas, which are known to be expressed in certain types of apoptosis. However, we did not detect an increased activity of proteases belonging to the apoptosis "executioner" machinery, such as CPP32 (caspase 3), ICE (caspase 1), or cathepsin D. Furthermore, administration of an ICE-like protease inhibitor did not significantly enhance the resistance to oxygen. Additionally, neither p53-deficient mice nor lpr mice (Fas null) manifested an increased resistance to hyperoxia-induced lung damage. These results show that both necrosis and apoptosis contribute to cell death during hyperoxia. Multiple apoptotic pathways seem to be involved in this, and an antiapoptotic strategy does not attenuate alveolar damage.

Amino Acid Chloromethyl Ketones↗

PubMed1997

Resistance of TNF/LT alpha double deficient mice to bleomycin-induced fibrosis.

In order to evaluate the role and mode of action of TNF in bleomycin-induced lung fibrosis, mice deficient for TNF and LT alpha (delta TNF/LT alpha) were examined at 2 months of age and after 3 weekly i.v. injections of bleomycin. The body weight of the delta TNF/LT alpha mice was 88 +/- 11% of that of the wild type littermates. Lung collagen, evaluated by its hydroxyproline content, was also lower (81 +/- 9%) in mutant than in wild type littermates. Bleomycin induced a diffuse alveolitis with focal areas of alveolar remodelling in wild type but not in delta TNF/LT alpha, mice. Lymphoid infiltration was also prominent in wild type, but absent from delta TNF/LT alpha, mice. Bleomycin injections increased collagen deposition, as evaluated by the lung hydroxyproline content, more markedly in wild type, than in delta TNF/LT alpha, mice. Cell trapping in the alveolar capillaries was evaluated by semi-quantitative electron microscopy. Bleomycin markedly increased platelet trapping in the alveolar capillaries of wild type, but not of delta TNF/LT alpha, mice. This study indicates that the expression of TNF/LT alpha genes increases the deposition of collagen in both untreated and inflamed lung and that these genes may act, at least in part, by promoting platelet trapping.

Animals↗

PubMed1997

Platelets play a role in the pathogenesis of the irritant reaction in mice.

The irritant reaction is a model of local inflammation that results from the epicutaneous application of a small molecule with irritating properties such as trinitrochlorobenzene (TNCB). The irritant reaction is mediated by tumor necrosis factor (TNF) and is characterized by skin edema and neutrophil (PMN) infiltration. The aim of this study was to explore the role of platelets in the pathogenesis of the irritant reaction. Mice depleted of platelets by an anti-platelet antibody showed a decrease in edema upon the application of a low dose of TNCB--chosen for causing an irritant reaction that is not complicated by intravascular fibrin deposition and hemorrhage--but no significant change in PMN infiltration. There was platelet trapping in TNCB-treated ears that was maximum between 2 and 6 h after TNCB application. Platelets lined the venular endothelium, which was intact in the absence of hemorrhage, and were not accompanied by fibrin. Mice treated with anti-TNF, anti-CD11a, anti-CD18, or anti-CD54 antibodies showed a decrease in platelet trapping, edema, and PMN infiltration. Platelets contribute to the pathogenesis of the irritant reaction and are necessary for edema to develop but not for PMN infiltration. The role of platelets implicates their early localization in the dermal venules, which depends, at least in part, on TNF and on the adhesion molecules involved in the interaction between CD11a/CD18 and CD54.

Administration, Cutaneous↗

PubMed1996

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.

Amyloid beta-Protein Precursor↗

PubMed1996

Corynebacterium parvum- and Mycobacterium bovis bacillus Calmette-Guerin-induced granuloma formation is inhibited in TNF receptor I (TNF-RI) knockout mice and by treatment with soluble TNF-RI.

The aim of this study was to examine the role of TNF receptor I (TNF-RI) in the pathogenesis of heat-killed Corynebacterium parvum- and live bacillus Calmette-Guerin (BCG)-induced granulomas. Granuloma formation was analyzed in TNF-RI knockout mice and after treatment with soluble TNF-RI (sTNF-RI). TNF-RI knockout mice injected with C. parvum or BCG developed fewer and smaller granulomas than wild-type control mice. Mice treated with sTNF-RI from days 7 to 13 after injection of C. parvum or BCG developed fewer and smaller granulomas than saline-treated control mice. Established granulomas regressed in rats treated with sTNF-RI from days 10 to 13 after injection of C. parvum. In conclusion, TNF signaling via TNF-RI contributes to the pathogenesis of C. parvum- and BCG-induced granulomas. sTNF-RI inhibits the development of granulomas and can cause the regression of established granulomas.

Animals↗

PubMed1996

Heparin attenuates bleomycin but not silica-induced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis.

Pulmonary fibrosis was elicited in mice or rats by the intratracheal instillation of bleomycin or silica. Daily injections of heparin significantly reduced the collagen deposition in bleomycin, but not in silica, injected mice, as evaluated by the lung hydroxyproline content on day 15 after instillation. Heparin also reduced the bleomycin-induced morbidity and mortality. Study of the broncho-alveolar lavage fluid (BAL) detected no significant change in the number of leucocytes or the amount of protein in heparin treated mice. Histologies of bleomycin instilled mice suggested that heparin did reduce the alveolar remodelling but not the alveolitis, evidenced by leucocytic infiltration. As detected by electron microscopy (EM), bleomycin increased the number of leucocytes and platelets within the alveolar capillaries but this was not significant ly reduced by heparin. The phenotype of the interstitial cell involved in these two types of pulmonary fibrosis was investigated by immunohistochemistry and EM. While in bleomycin injected animals the interstitial cells had the phenotype of an actin (alpha-actin in the rat) and lipid containing interstitial cell, with a poorly developed RE, in silica injected animals in contrast, the interstitial cells were without cytoplasmic actin or lipid but with a markedly developed endoplasmic reticulum (ER). Thus bleomycin and silica induced the growth of two different types of interstitial cells, the myofibroblast and the regular fibroblast, which might be a reason why heparin selectively inhibits bleomycin but not silica-induced fibrosis.

Animals↗

PubMed1996

Activated human platelets express beta2 integrin.

The expression of molecules of the beta2 integrin family (CD11a, CD11b , CD11c, and CD18) was explored on 2 human megakaryocytic cell lines and on platelets from different donors by immunofluorescence and flow cytometry using a large panel of mAb. CD11a, CD11b, CD11c and CD18 were detected on the megakaryocytic cell lines DAMI and HEL. A low and variable expression of CD11a, CD11b and CD18 determinants was also detected on resting platelets; this expression was markedly increased when platelets were activated by thrombin. Expression of CD18 was closely correlated to that of CD11a or CD11b when comparing the fluorescence intensity observed in different experiments. In presence of Ca++, platelets did bind to a RAJI cell line which exhibits a high expression level of CD54. This binding was increased when platelets were activated by thrombin and was decreased by an anti CD11a, CD18 and anti CD54 mAb. This study indicates that human platelets express molecules of the beta2 integrin family, when activated, which allows them to bind to CD54 bearing cells.

Animals↗

PubMed1996

Hyperoxia induces platelet activation and lung sequestration: an event dependent on tumor necrosis factor-alpha and CD11a.

Mice were exposed to pure oxygen for various times to explore the pulmonary platelet trapping associated with alveolar damage, its mechanism, and its role in the lesions. Platelet sequestration, evaluated by electron microscopy and by injection of radiolabeled platelets, was detectable after 72 h and reached a maximum after 96 h of exposure (i.e., shortly before death). Circulating platelets (analyzed by Facscan) showed some increase in the expression of CD11a and CD62, but little change in CD31 and CD61. Both platelet activation and lung sequestration were dependent on TNF-alpha, since antibody against TNF-alpha reduced the expression of CD11a on circulating platelets and their sequestration in the lung. Lung platelet sequestration was also decreased by anti-CD11a MoAb. Northern blot analysis of lung mRNA isolated at 96 h of oxygen exposure revealed a 7-fold increase in CD54 (intercellular adhesion molecule-1 [ICAM-1]) and a 2.5-fold increase in TNF-alpha mRNAs respectively. These results demonstrate that the platelet pulmonary trapping induced by hyperoxia is dependent upon TNF-alpha and the CD11a-CD54 adhesion molecules. However, platelet trapping does not appear to play an important pathogenic role in acute oxygen injury, since treatments that decrease trapping (anti-TNF-alpha, anti-CD11a, or antibody-induced thrombocytopenia) did not markedly attenuate the alveolar damage.

Animals↗

PubMed1995

Involvement of granulocytes and the adhesion receptors intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 in tissue inflammation induced by Th2-type helper cells.

We reported recently that subcutaneously injected, anti-CD3 epsilon-pulsed polyclonal Th2 cells mediate interleukin-4-dependent local tissue inflammation. Because a prominent polymorphonuclear infiltrate was observed in the lesions at the time of maximal tissue swelling, we investigated the involvement of polymorphonuclear leukocytes and their adhesion molecules lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in Th2-cell-mediated inflammation. Pretreatment of recipient mice with a depleting monoclonal antibody to neutrophils or with blocking antibodies to LFA-1 or to ICAM-1 completely abrogated tissue swelling in Th2-cell-mediated inflammation. Granulocyte infiltration at 6 h was also inhibited by the antibodies to neutrophils and to ICAM-1, but not by that to LFA-1. Tissue swelling mediated by Th1 cells had different kinetics and was not prevented by administration of anti-neutrophil antibody: maximal edema formation occurred at 24-48 h, when the predominant cellular infiltrate was mononuclear. Because the Th1-cell-induced infiltrate at 6 h also consisted of granulocytes but was not associated with pronounced edema, the mere presence of infiltrating polymorphonuclear leukocytes seems not to be sufficient to induce edema. Because edema but not granulocyte infiltration was inhibited by anti-LFA-1 and because anti-LFA-1 antibodies are known to inhibit several functions of neutrophils, our results suggest that, in inflammation mediated by Th2 cells, granulocytes induce edema through their activation and/or degranulation.

Animals↗

PubMed1995

Administration of anti-TNF-alpha or anti-CD11a antibodies to normal adult mice decreases lung and bone collagen content: evidence for an effect on platelet consumption.

Administration of anti-tumor necrosis factor-alpha (anti-TNF-alpha) or anti-CD11a (LFA-1) antibodies to normal adult mice for about 10 days markedly decreased the hydroxyproline content in lung and femur. Similar observation was made using the recombinant soluble TNF receptor (rsTNFR) as tumor necrosis factor-alpha (TNF-alpha) antagonist. This decrease in collagen content was most likely due to a decrease of collagen synthesis, as evidenced by a decrease of the 3H-proline incorporation in lung and bone and by a decrease of collagen alpha 1 (I) mRNA levels in the lung RNA. The localization of labeled platelets in the lung of normal mice was decreased by anti-TNF-alpha but not by anti-CD11a antibodies. Thrombocytopenia increases the localization of labeled platelets in the lung, and, in this condition, both anti-TNF-alpha and anti-CD11a antibodies decreased pulmonary trapping. TNF-alpha mRNA was detected in the lung of normal adult mice, suggesting that this cytokine is released in the absence of inflammation. These results indicate that in vivo, endogenous TNF-alpha stimulates collagen synthesis, most likely by its influence on platelet trapping, which appears to involve the platelet CD11a since it is decreased by anti-CD11a antibody.

Animals↗