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Pulmonary epithelial cells are continuously exposed to microbial challenges as a result of breathing. It is recognized that immune myeloid cells express Toll-like receptors (TLRs), which play a major role in detecting microbes and initiating innate immune responses. In contrast, little is known concerning the expression of TLR in pulmonary epithelial cells per se, their distribution within the cell, their function, and the signaling pathways involved. In this work, we demonstrated by reverse transcription-PCR and/or immunoblot that TLR4 and the accessory molecule MD-2 are constitutively expressed in distinct human alveolar and bronchial epithelial cells. We further characterized by flow cytometry, biotinylation/precipitation, and confocal microscopy the intracellular localization of TLR4 in these cells. Despite this intracellular compartmentalization of TLR4, pulmonary epithelial cells were responsive to the TLR4 activator lipopolysaccharide (LPS), a potent Gram-negative bacteria-associated molecular pattern. Using respiratory epithelial cells isolated from TLR4 knock-out and wild type mice, we demonstrated that TLR4 is the actual activating receptor for LPS in these cells. Furthermore we showed that this cell response to LPS involves a signaling complex including the kinases interleukin-1 receptor-associated kinase (IRAK), p38, Jnk, and ERK1/2. Moreover, using vectors expressing dominant-negative forms of MyD88 and TRAF6, we established that LPS-induced activation of respiratory epithelial cells is largely dependent on TLR4 signaling intermediates. Altogether these data demonstrate that TLR4 is a key element in the response of pulmonary epithelial cells to molecules derived from Gram-negative bacteria. The intracellular localization of TLR4 in lung epithelia is expected to play an important role in the prevention of the development of chronic inflammatory disease.

Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-kappaB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.

We previously showed that surfactant inhibits the synthesis of type IIA secretory phospholipase A2 (sPLA2-IIA) by alveolar macrophages. These cells have been identified as the main source of this enzyme in an animal model of acute lung injury. The aim of the present study was to identify the surfactant components involved in the inhibition of sPLA2-IIA expression in alveolar macrophages and the signaling pathways that mediate this inhibition. Our results show that various surfactant preparations can inhibit sPLA2-IIA expression in endotoxin-stimulated alveolar macrophages. Both the surfactant protein (SP)-A and the surfactant phospholipid fraction inhibit this expression. The surfactant phospholipid dioleylphosphatidylglycerol (DOPG) abolishes sPLA2-IIA expression, whereas dipalmitoylphosphatidylcholine does not. Chromatographic analysis and confocal microscopy revealed that phosphatidylglycerol was rapidly incorporated and metabolized by alveolar macrophages and that its metabolites accumulate in the cytosol. Nuclear factor-kappaB (NF-kappaB) modulates sPLA2-IIA expression in endotoxin-activated alveolar macrophages, and surfactant preparations, surfactant phospholipid fraction, SP-A, and DOPG indeed suppressed NF-kappaB activation. In summary, our results show that SP-A and DOPG play a role in the surfactant-mediated inhibition of sPLA2-IIA expression in alveolar macrophages and that this inhibition occurs via a downregulation of NF-kappaB activation.

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.

During acute lung inflammation, the airspaces are invaded by circulating neutrophils. These may then injure tissues through the release of elastase. Different natural specific inhibitors such as alpha1-proteinase inhibitor, secretory leukocyte proteinase inhibitor, and elafin are nonetheless able to counteract the enzymatic activity of elastase. The present study was undertaken to assess the role of these different inhibitors in the intrinsic antielastase barrier during lipopolysaccharide-induced lung inflammation in mice. Upon intranasal administration of lipopolysaccharide to mice, the antielastase activity recovered from bronchoalveolar lavage fluids (BALF) increases progressively up to 48 h (7-fold) and returns to the basal level within 72 h. By contrast, when the same experiments are performed with neutropenic mice (pretreatment with an antigranulocyte antibody, or vinblastine), the increase is almost totally absent. Ultrafiltration of BALF through 100 kD cutoff membranes shows that the activity remains in the retentate, thus ruling out a role for native alpha1-proteinase inhibitor, secretory leukocyte proteinase inhibitor, and elafin. Gel filtration and fraction analysis show that the material eluted with a Mr of 600 kD. Agarose gel electrophoresis and ethidium bromide staining reveal that the activity corresponds to the presence a large amount of DNA. Interestingly, DNase treatment of the active fraction suppresses the antielastase activity. Analysis of BALF from patients with acute lung inflammation shows the presence of DNA with antielastase activity. We therefore concluded that during acute lung inflammation, the recruitment of neutrophils in the airspaces accounts for the increased presence of DNA, which in turn contributes to the antielastase barrier.

We previously demonstrated that resident alveolar macrophages from naive mice do not synthesize interleukin (IL)-10, whereas mononuclear phagocytes (MP) recruited during the lung inflammatory process are transiently competent for IL-10 production when exposed to lipopolysaccharide (LPS) in vitro. As surfactant protein A (SP-A), a member of the collectin family, inhibits LPS-induced in vitro IL-10 formation by bone marrow-derived macrophages, we studied its effect on MP under in vivo inflammatory conditions. When mice with LPS-induced inflamed lungs were given a second intranasal LPS administration, IL-10 concentration recovered in the bronchoalveolar lavage fluids varied as a function of the time interval between the two LPS doses. Thus, IL-10 concentration increased with the number of MP up to Day 3, and then decreased to undetectable values within 24 h, despite a continued increase in the number of MP. Analysis of IL-10 mRNA from purified MP indicated that gene expression correlated with the IL-10 level in the bronchoalveolar lavage fluid. In contrast to IL-10 production, SP-A concentrations during LPS-induced inflammation decreased with a nadir at Day 3, and then increased significantly within 24 h. Furthermore, intranasal administration of exogenous SP-A to mice with LPS-induced inflamed lungs led to a repression of the IL-10 production. In summary, this study demonstrates for the first time an in vivo inhibitory role of SP-A on the anti-inflammatory activity of MP, through inhibition of IL-10 production.

Proteinase-activated receptor (PAR)-2 is cleaved within its aminoterminal extracellular domain by serine proteinases such as trypsin, unmasking a new aminoterminus starting with the sequence SLIGKV, which binds intramolecularly and activates the receptor. PAR-2 has been reported to be involved in inflammation within the lungs. We show that PAR-2 is expressed not only by human alveolar (A549), but also by bronchial (16HBE) epithelial cell lines, using RT-PCR and flow cytometry with a PAR-2 antibody whose epitope maps over the trypsin cleavage site. PAR-2 activation by trypsin and by the activating peptide SLIGKV-NH(2) leads to intracellular calcium mobilization in both lung epithelial cells. During lung inflammation, airspaces are burdened by neutrophils that release elastase and cathepsin G, two serine proteinases. We demonstrate that these proteinases do not activate PAR-2, but rather disarm the receptor, preventing activation by trypsin but not by SLIGKV-NH(2). Preincubation of a PAR-2-transfected cell line, as well as 16HBE and A549 cells, with either proteinase led to the disappearance of the cleavage/activation epitope recognized by the PAR-2 antibody. We hypothesize that elastase and cathepsin G disarm PAR-2 by proteolysis of the extracellular domain downstream from the trypsin cleavage/activation site, while leaving unmodified the SLIGKV-NH(2)-binding site. These findings suggest that the neutrophil serine proteinases may play a role in PAR-2-mediated lung inflammation.

Lipopolysaccharide (LPS) derived from enterobacteria elicit in several cell types cellular responses that are restricted in the use of Toll-like receptor 4 (TLR4) as the principal signal-transducing molecule. A tendency to consider enterobacterial LPS as a prototypic LPS led some authors to present this mechanism as a paradigm accounting for all LPSs in all cell types. However, the structural diversity of LPS does not allow such a general statement. By using LPSs from bacteria that do not belong to the Enterobacteriaceae, we show that in bone marrow cells (BMCs) the LPS of Rhizobium species Sin-1 and of three strains of Legionella pneumophila require TLR2 rather than TLR4 to elicit the expression of CD14. In addition, exposure of BMCs from TLR4-deficient (C3H/HeJ) mice to the lipid A fragment of the Bordetella pertussis LPS inhibits their activation by the Legionella lipid A. The data show selective action of different LPSs via different TLRs, and suggest that TLR2 can interact with many lipid A structures, leading to either agonistic or specific antagonistic effects.

The collectin surfactant protein-A (SP-A) is involved in the innate host defense and the regulation of inflammatory processes in the lung. In this work we investigated the molecular mechanisms related to the immunostimulatory activity of SP-A using macrophages from C3H/HeJ mice, which carry an inactivating mutation in the Toll-like receptor (TLR)4 gene, and TLR4-transfected Chinese hamster ovary cells. We demonstrate that SP-A-induced activation of the NF-kappaB signaling pathway and up-regulation of cytokine synthesis such as TNF-alpha and IL-10 are critically dependent on the TLR4 functional complex. These findings support the concept that TLR4 is a pattern recognition receptor that signals in response to both foreign pathogens and endogenous host mediators.

Activation of CXCR4 by the CXC chemokine stromal cell-derived factor-1 (SDF-1) requires interaction of the amino-terminal domains of both molecules. We report that proteinases released from either mononucleated blood cells or polymorphonuclear neutrophils degranulated by inflammatory stimuli generate an SDF-1 fragment that is deleted from amino-terminal residues Lys(1)-Pro(2)-Val(3), as characterized by mass spectrometry analysis. The proteolyzed chemokine fails to induce agonistic functions and is unable to prevent the fusogenic capacity of CXCR4-tropic human immunodeficiency viruses. Furthermore, we observed that exposure of CXCR4-expressing cells to leukocyte proteinases results in the proteolysis of the extracellular amino-terminal domain of the receptor, as assessed by flow cytometry analysis and electrophoretic separation of immunoprecipitated CXCR4. Blockade of SDF-1 and CXCR4 proteolysis by the specific leukocyte elastase inhibitor, N-methoxysuccinyl-alanine-alanine-proline-valine-chloromethyl ketone, identified elastase as the major enzyme among leukocyte-secreted proteinases that accounts for inactivation of both SDF-1 and CXCR4. Indeed, purified leukocyte elastase generated in either SDF-1 or CXCR4 a pattern of cleavage indistinguishable from that observed with leukocyte-secreted proteinases. Our findings suggest that elastase-mediated proteolysis of SDF-1/CXCR4 is part of a mechanism regulating their biological functions in both homeostatic and pathologic processes.

Upon LPS exposure, mononuclear phagocytes produce TNF-alpha and IL-10, two cytokines with pro- and anti-inflammatory activities, respectively. We previously described that murine resident alveolar macrophages, which play a central role in the immunosurveillance of the lung alveoli, do not synthesize IL-10 in vivo or in vitro when exposed to LPS. In the present report we demonstrate that during lung inflammation induced by the intranasal administration of LPS, bronchoalveolar cells collected between days 3 and 5 are able to synthesize IL-10 when exposed to LPS. We also show that depletion of resident alveolar macrophages by an intratracheal instillation of liposome-encapsulated clodronate is followed by subsequent replenishment of the airspaces by mononuclear phagocytes. This is accompanied by the transient competence of cells for IL-10 production. The cell capacity to produce IL-10 is evident up to 3 days and then decreases. This led us to hypothesize that the alveolar environment contains a down-regulator of LPS-induced IL-10 synthesis by recently emigrating mononuclear phagocytes. We show that the surfactant protein A, an airspace protein that has known immunomodulatory activities, dramatically inhibits LPS-induced IL-10 formation by bone marrow-derived macrophages. These data show a difference between resident and inflammatory macrophages with respect to IL-10 synthesis. Moreover, this study highlights for the first time the inhibitory role of surfactant protein A in the anti-inflammatory activity of macrophages through inhibition of IL-10 production.

The intranasal administration of lipopolysaccharide (LPS) to mice triggers a huge influx of polymorphonuclear neutrophils (PMNs) into the airway spaces, with a peak at 48 h. The increase in protein concentration, an index of microvascular permeability, displayed a different pattern, i.e., a first increase with a plateau between 3 and 24 h followed by a second increase peaking at 72 h. When mice were depleted of circulating PMNs, the increase in protein concentration was inhibited at 3 h but not at 24 h. The lack of PMN involvement at 24 h was confirmed by 1) in situ activation of exudated PMNs present in the air spaces on intranasal administration of LPS and 2) induction of the migration of PMNs sequestered in lung vessels on intraperitoneal administration of LPS. These findings show that the increase in microvascular permeability during lung inflammation is due to at least two distinct mechanisms, an early one related to the neutrophil influx and a delayed one occurring even under neutropenic conditions.

Neutrophil elastase (NE) upregulates the fibrinogen binding activity of the platelet integrin alpha(IIb)beta(3) through proteolysis of the alpha(IIb) subunit. This cleavage allows a strong potentiation of platelet aggregation induced by low concentrations of cathepsin G (CG), another neutrophil serine proteinase. During this activation process, we observed a strong fibrinogen binding and aggregation-dependent phosphatidylinositol 3,4-bis-phosphate (PtdIns(3,4)P(2)) accumulation. PtdIns(3,4)P(2) has been suggested to play a role in the stabilization of platelet aggregation, possibly through the control of a maintained alpha(IIb)beta(3) integrin activation. Here we show that inhibition of phosphoinositide 3-kinase (PI 3-K) by very low concentrations of wortmannin or LY294002 transformed the irreversible platelet aggregation induced by a combination of NE and low concentrations of CG into a reversible aggregation. However, although inhibition of PI 3-K was very efficient in inducing platelet disaggregation, it did not modify the level of alpha(IIb)beta(3) activation as assessed by binding of an activation-dependent antibody. These results indicate that PI 3-K activity can control the irreversibility of platelet aggregation even under conditions where alpha(IIb)beta(3) integrin remains activated.

A major property of monocytes/macrophages is to recognize and to be activated by bacterial wall components such as LPS, through membrane receptors including the key element CD14. We demonstrate that CD14 expression is down-regulated, as judged by flow cytometry analysis, upon incubation of human monocytes with purified cathepsin G (CG), a releasable neutrophil serine proteinase. The progressive decrease of CD14 expression due to increasing concentrations of CG highly correlates (P < 0.0001) with the decreased synthesis of tumor necrosis factor alpha (TNF-alpha) in response to lipopolysaccharide (LPS). This effect is dependent on the enzymatic activity of CG but is not exerted through an activation of monocytes. Immunoblot analysis reveals that CD14 (M(r) = 57,000) is directly cleaved by CG and released into the extracellular medium as a high-M(r) species (M(r) = 54,000). In this context, incubation of monocytes with activated neutrophils leads to a down-regulation of CD14 expression, a process blocked by a serine proteinase inhibitor. These data suggest a paradoxical anti-inflammatory property for CG.

The central role of alveolar macrophages in the establishment of lipopolysaccharide (LPS)-induced lung inflammation is well demonstrated. They produce and release numerous proinflammatory molecules, among which is tumor necrosis factor alpha (TNF-alpha), a cytokine responsible in part for the neutrophilic alveolitis. Interleukin-10 (IL-10) produced by LPS-activated mononuclear phagocytes is a major anti-inflammatory cytokine that down-regulates TNF-alpha synthesis. We studied the ability of murine alveolar macrophages to produce IL-10 in vivo and in vitro, in response to LPS. Unexpectedly, the IL-10 protein was not detected in the whole lung and airspaces after LPS intranasal instillation. In addition, no IL-10 protein was found in supernatants of isolated and LPS-stimulated alveolar macrophages. The lack of IL-10 synthesis was confirmed by the absence of specific RNA transcripts. By contrast and as expected, autologous peritoneal macrophages produced IL-10 upon LPS challenge. Drugs that usually modify the TNF-alpha/IL-10 balance in favor of IL-10 were used without success. Thus, maneuvers allowing an increase in intracellular cAMP concentrations did not reverse this unexpected phenotype. Moreover, direct activation of protein kinase C with PMA was unable to trigger IL-10 formation by alveolar, by contrast to peritoneal, macrophages. The current findings describe a specific phenotype for murine alveolar macrophages during LPS-induced inflammation.

Human leukocyte elastase (HLE), a polymorphonuclear neutrophil (PMN) serine proteinase, is proteolytically active on some membrane receptors at the surface of immune cells. The present study focused on the effect of HLE on the expression of CD14, the main bacterial lipopolysaccharide (LPS) receptor at the surface of monocytes. HLE exhibited a time- and concentration-dependent downregulatory effect on CD14 surface expression. A 30-minute incubation of 3 microM HLE was required to display 95% disappearance of the receptor. This downregulation resulted from a direct proteolytic process, not from a shedding consecutive to monocyte activation as observed upon challenge with phorbol myristate acetate (PMA). To confirm that CD14 is a substrate for HLE, this enzyme was incubated with recombinant human CD14 (Mr approximately 57,000), and proteolysis was further analyzed by immunoblot analysis. Cleavage of the CD14 molecule was directly evidenced by the generation of short-lived fragments (Mr approximately 47,000 and 30,000). As a consequence of the CD14 proteolysis, a decrease in the responsiveness of monocytes to LPS was observed, as assessed by measuring tumor necrosis factor-alpha (TNF-alpha) formation. This inhibition was only observed with 1 ng/ml of LPS, i.e., when only the CD14-dependent pathway was involved. At a higher LPS concentration, such as 10 microgram/ml, when CD14-independent pathways were operative, this inhibition was overcome. The direct proteolysis by HLE of the membrane CD14 expressed on monocytes illustrates a potential anti-inflammatory effect of HLE through inhibition of LPS-mediated cell activation.

Bronchopulmonary hyperreactivity (BHR), an increased responsiveness to nonspecific bronchoconstrictor agents, is a well-known characteristic of bronchial asthma. It has been recently suggested that the severity of this disease is related to the endotoxin content of house dust. In the present report, it is shown that the i.p. administration of bacterial LPS to mice is followed by a marked early dose-dependent BHR in response to methacholine. The microscopic examination showed no ultrastructural lesions of the lungs or of the airways, but a marked neutrophil accumulation in the capillaries, as confirmed by an increase of the lung content in the neutrophil enzyme marker myeloperoxidase. In parallel, high levels of TNF-alpha were found in plasma as well as its transcripts in the lung tissues. Using immunologic (anti-TNF-alpha and anti-granulocyte Abs), and pharmacologic (dexamethasone and vinblastine) tools, it is demonstrated that BHR is apparently neither related to the presence of neutrophils in the pulmonary microvasculature nor to the synthesis of TNF-alpha.

Glycopeptides derived from the GRGDS sequence were synthesized to study the effect of the sugar residue on the activity of these peptides. The peptides were tested as inhibitors of cell adhesion to fibronectin and of platelet aggregation. The sugar moiety was found to reduce the biological activity of the parent compounds except for the cyclic derivatives P37 and P38 where the inhibition of platelet aggregation was increased. Some interesting differences were observed between the peptides bearing sugar residues with free hydroxyl groups and those with peracetylated sugars.

1. Our previous work demonstrated that bacterial lipopolysaccharide (LPS), administered by aerosol, induced tumour necrosis factor (TNF-alpha) synthesis leading to the infiltration of neutrophils into mice lungs. The treatment of animals with prostaglandin E2 or dibutyryl cyclic AMP impaired both processes. In this study, the target cell for LPS and the modulation by cyclic AMP of TNF-alpha production and neutrophil recruitment were investigated. 2. One hour after inhalation of 2 ml of 0.3 mg ml(-1) LPS, TNF-alpha levels measured by an ELISA method increased in the bronchoalveolar lavage fluid (BALF) of BALB/c mice, reaching a maximal level 3 h after inhalation. The immunocytochemistry assay demonstrated that 1 h after inhalation, 21.2% of alveolar macrophages collected in the BALF were immunopositive for TNF-alpha. 3. When mice were pretreated, i.p., with 20 mg kg(-1) rolipram, a selective inhibitor of phosphodiesterase type 4, TNF-alpha levels in the BALF were significantly reduced and only 7.3% of alveolar macrophages were immunopositive for TNF-alpha. 4. Alveolar macrophages from rolipram-treated mice collected 30 min after inhalation of LPS had a significant increase in the intracellular concentrations of cyclic AMP. This was accompanied by a marked reduction of TNF-alpha levels in the BALF that were associated with a suppression of TNF-alpha mRNA expression. 5. Systemic treatment with 20 mg kg(-1) rolipram almost completely inhibited the LPS-induced neutrophil recruitment, whereas it did not significantly reduce the recruitment induced by rmTNF-alpha. 6. Our results indicate that alveolar macrophages may be the target cells for both the induction and control of the lung inflammatory response to LPS. They also suggest that systemic treatment with cyclic AMP-elevating agents may be useful to control local inflammation resulting from inhalation of bacterial endotoxin.

Secretory leukocyte proteinase inhibitor (SLPI) is the main neutrophil elastase (HLE) inhibitor found in the upper airways during pulmonary inflammation. It has been shown to be synthesized and secreted in vitro by epithelial cells and has been localized in tracheal glands and bronchiolar epithelial cells by immunocytochemistry. In this study, using immunodetection and immunopurification techniques with specific anti-SLPI immunoglobulin G (IgG), we show that SLPI is present as a native 14-kDa molecule in neutrophil cytosol. In addition, we demonstrate that SLPI is the major inhibitor of HLE present in neutrophil cytosol because pre-incubation with specific anti-SLPI IgG was able to inhibit completely the anti-HLE activity of the cytosol. SLPI can be secreted (probably in an inactive form) by neutrophils and its secretion is enhanced when the cells are stimulated with phorbol myristate acetate (PMA). Elafin, an elastase-specific inhibitor, is also present in minute amounts in neutrophil cytosol and its secretion can be up-regulated. The presence of SLPI in the cytosol of neutrophils may serve as a protective screen against proteinases spilling from azurophilic granules. An alternative or supplementary role may be the maintenance of a differentiated phenotype.

Neutrophil elastase (NE) and cathepsin G are two serine proteinases released concomitantly by stimulated polymorphonuclear neutrophils. We previously demonstrated that while NE by itself does not activate human platelets, it strongly enhances the weak aggregation induced by a threshold concentration of cathepsin G (threshold of cathepsin G) (Renesto, P., and Chignard, M. (1993) Blood 82, 139-144). The aim of this study was to delineate the molecular mechanisms involved in this potentiation process. Two main pieces of data prompted us to focus on the activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. First, previous studies have shown this integrin to be particularly prone to proteolytic regulation of its function. Second, we found that the potentiating activity of NE on the threshold of cathepsin G-induced platelet aggregation was strictly dependent on the presence of exogenous fibrinogen. Using flow cytometry analysis, NE was shown to trigger a time-dependent binding of PAC-1 and AP-5, two monoclonal antibodies specific for the activated and ligand-occupied conformers of alphaIIbbeta3. Furthermore, the potentiated aggregation was shown to result from an increased capacity of platelets to bind fibrinogen. Indeed, the combination of NE and threshold of cathepsin G increased the binding of PAC-1 approximately 5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by approximately 3-fold in threshold of cathepsin G-stimulated platelets (p < 0.05). By contrast, phosphatidic acid accumulation, pleckstrin phosphorylation, and calcium mobilization produced by the combination of NE and threshold of cathepsin G were not significantly different from those measured with threshold of cathepsin G alone (p > 0.05), indicating that the phospholipase C/protein kinase C pathway is not involved in the potentiation of aggregation. The foregoing data, as well as the requirement of catalytically active NE to trigger alphaIIbbeta3 activation and potentiate threshold of cathepsin G-initiated platelet aggregation, led us to examine whether the structure of this integrin was affected by NE. Immunoblot and flow cytometry analysis revealed a limited proteolysis of the carboxyl terminus of the alphaIIb subunit heavy chain (alphaIIbH), as judged by the disappearance of the epitope for the monoclonal antibody PMI-1. Mass spectrometry studies performed on a synthetic peptide mapping over the cleavage domain of alphaIIbH predicted the site of proteolysis as located between Val837 and Asp838. Treatment by NE of ATP-depleted platelets or Chinese hamster ovary cells expressing human recombinant alphaIIbbeta3 clearly established that activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of alphaIIbH. In support of this latter observation, a close correlation was observed between the kinetics of proteolysis of alphaIIbH on platelets and that of expression of the ligand binding activity of alphaIIbbeta3 (r2 = 0.902, p

Suramin, a hexasulfonated naphtylurea recently used as an anti-tumor drug, is a potent inhibitor of human neutrophil elastase, cathepsin G, and proteinase 3. The complexes it forms with these enzymes are partially active on synthetic substrates, but full inhibition takes place when elastase activity is measured with fibrous elastin or when cathepsin G activity is measured using platelet aggregation. One molecule of elastase binds four molecules of suramin with a Ki of 2 x 10(-7) M as determined by enzyme inhibition or intrinsic fluorescence enhancement of suramin. The binding curves show no sign of cooperativity or anticooperativity. The Ki for the complexes with cathepsin G and proteinase 3 are 8 x 10(-8) and 5 x 10(-7) M, respectively. Ionic strength increases the Ki of the elastase-suramin complex in a way that suggests that four of the six sulfonate groups of suramin form ionic interactions with basic residues of the enzyme and that at saturation almost all arginines of elastase form salt bridges with suramin. The neutrophil proteinase-inhibitory activity of suramin might be used to prevent tissue destruction and thrombus formation in diseases where massive infiltration and activation of neutrophils take place.

The aim of this study was to investigate the inhibitory effects of human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR3) on the activation of endothelial cells (ECs) and platelets by thrombin and to elucidate the underlying mechanisms. Although preincubation of ECs with HLE or Cat G prevented cytosolic calcium mobilization and prostacyclin synthesis induced by thrombin, these cell responses were not affected when triggered by TRAP42-55, a synthetic peptide corresponding to the sequence of the tethered ligand (Ser42-Phe55) unmasked by thrombin on cleavage of its receptor. Using IIaR-A, a monoclonal antibody directed against the sequence encompassing this cleavage site, flow cytometry analysis showed that the surface expression of this epitope was abolished after incubation of ECs with HLE or Cat G. Further experiments conducted with platelets indicated that not only HLE and Cat G but also PR3 inhibited cell activation induced by thrombin, although they were again ineffective when TRAP42-55 was the agonist. Similar to that for ECs, the epitope for IIaR-A disappeared on treatment of platelets with either proteinase. These results suggested that the neutrophil enzymes proteolyzed the thrombin receptor downstream of the thrombin cleavage site (Arg41-Ser42) but left intact the TRAP42-55 binding site (Gln83-Ser93) within the extracellular aminoterminal domain. The capacity of these proteinases to cleave five overlapping synthetic peptides mapping the portion of the receptor from Asn35 to Pro85 was then investigated. Mass spectrometry studies showed several distinct cleavage sites, i.e., two for HLE (Val(72)-Ser73 and Ile74-Asn75), three for Cat G (Arg41-Ser42, Phe55-Trp56 and Tyr69-Arg70), and one for PR3 (Val(72)-Ser73). We conclude that this singular susceptibility of the thrombin receptor to proteolysis accounts for the ability of neutrophil proteinases to inhibit cell responses to thrombin.

In the present study we investigated the modulation of the polymorphonuclear neutrophil (PMN)-endothelial cell adhesion process by the two main proteinases released from activated PMN during their adhesion to endothelium. Our results showed that, in contrast with elastase, cathepsin G was a powerful inhibitor of PAIN adhesion to interleukin-1 (IL-1)-treated human umbilical vein endothelial cells. This inhibitory effect was linked to the enzymatic activity of the proteinase and was selectively directed against PMN. Because the viability and the reactivity of PMN were not modified by cathepsin G, we looked for a possible effect on adhesion molecules. L-selectin was not cleaved by cathepsin G, whereas it was by chymotrypsin, a closely related proteinase. Cathepsin G blocked PMN adhesion to activated endothelial cells, but also to serum- or fibrinogen-coated plates, three adhesion processes mediated by CD11b/CD18. However, by FACScan analysis or by immunoprecipitation, we failed to find evidence of modifications of CD11b/CD18 expression. Although the precise molecular target(s) of cathepsin G remain(s) to be defined, these data indicate that this proteinase, which is known as an inflammatory mediator, can also be considered as a potential down-regulator of adhesion reactions involved in the inflammatory process.

The serine-proteinase cathepsin G (CG) is a potent agonist of platelet aggregation inducing the release and surface expression of alpha-granule adhesive proteins such as fibrinogen (Fg) and thrombospondin-1 (TSP-1). Because Fg and TSP-1 are potential substrates for the enzymatic activity of CG, we investigated the fate of these proteins during CG-induced platelet aggregation using an immunoblot technique. Only a small proportion of secreted Fg was proteolyzed by CG and platelet aggregation was efficiently inhibited by anti-fibrinogen Fab fragments. In contrast, TSP-1 was extensively proteolyzed on aggregated platelets releasing in the milieu a fragment with Mr approximately 28 000, corresponding to the amino-terminal heparin-binding domain (HBD). Several antibodies, directed against the cell-associated carboxy-terminal TSP-1f fragment (Mr approximately 165000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD.

1. The adult respiratory distress syndrome (ARDS) is an acute lung inflammation developed after direct or indirect contact with pathogenic agents. In the present study, a mouse model was developed to mimic this condition using aerosolized bacterial lipopolysaccharide (LPS) and to investigate the mechanisms involved in the lung inflammatory response. 2. Inhalation of LPS led to a time and dose-dependent increase in tumour necrosis factor-alpha (TNF-alpha) production and neutrophil recruitment into the bronchoalveolar lavage fluid (BALF) of Balb/c mice. Under the same conditions, neutrophil infiltration was also found in the BALF of the LPS-sensitive mouse strain C3H/HeN, but was absent in the LPS-resistant strain C3H/HeJ. Intranasal administration of murine recombinant TNF-alpha also triggered neutrophil recruitment. 3. One hour after inhalation of LPS, half of the maximal level of TNF-alpha was measured in the BALF, but only a few neutrophils were detected at this time. The peak TNF-alpha concentration was reached at 3 h, when the neutrophil amount started to increase. At 24 h, maximal neutrophil number was found in the BALF and TNF-alpha was no longer present. 4. Pretreatment of mice under different experimental conditions demonstrated that: (a) cycloheximide almost completely blocks both neutrophil recruitment and TNF-alpha production; (b) anti TNF-alpha antibodies block neutrophil recruitment; (c) indomethacin or aspirin enhance by two fold neutrophil recruitment; (d) indomethacin significantly increases TNF-alpha production 1 h after inhalation of LPS; (e) dibutyryl cyclic AMP and prostaglandin E2 (PGE2) block both neutrophil recruitment and TNF-alpha production. 5. It is concluded that aerosolized LPS in mice triggers an acute lung inflammation which can be used as a potential model of inhalational ARDS and that, strategies leading to the elevation of cyclic AMP levels in vivo can be effective in modulating LPS-induced TNF-alpha synthesis and neutrophil recruitment.

1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates.

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 microM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 microM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

Upon activation, human polymorphonuclear neutrophils (PMN) release two serine proteinases, cathepsin G (Cat.G) and elastase (HLE), which in turn synergize to activate nearby platelets. We looked for the inhibitory effect of plasma and the involvement of alpha 1 antichymotrypsin (alpha 1 ACT) and alpha 1 antitrypsin (alpha 1 AT), on this cell-to-cell cooperation. It was observed that inhibition by plasma of PMN-mediated platelet activation was rather correlated with an effect on HLE (r = 0.95) than on Cat.G (r = 0.65) enzymatic activity. Purified alpha 1 AT suppressed in a concentration-dependent manner HLE activity present in the supernatant of activated PMN. When HLE was fully blocked, alpha 1 AT started to inhibit Cat.G activity. By contrast and as expected, purified alpha 1 ACT inhibited only Cat.G activity. Using specific blocking polyclonal antibodies against alpha 1 AT and alpha 1 ACT, it was demonstrated that the inhibitory effect of plasma vs. HLE was entirely mediated by alpha 1 AT. By contrast, blockade of Cat.G activity was only partly due to plasma alpha 1 ACT and around 50% was attributable to alpha 1 AT. When plasma from patients with an acute inflammatory state was used in place of plasma from normal subjects, the inhibitory effect was more pronounced, while plasma depleted in alpha 1 AT and alpha 1 ACT was less effective. These data indicate a predominant role of alpha 1 AT in the inhibition by plasma of the PMN-mediated platelet activation.

The proteolytic activity of the neutrophil serine-proteinase cathepsin G (CG) on platelet adherence receptors, the glycoprotein (GP) Ib-IX complex and the integrin alpha IIb beta 3, has been investigated. In the range 50 to 200 nmol/l, CG is a potent platelet agonist which induces shape change, granule exocytosis and aggregation. Investigation of the proteolysis of the receptors' subunits during the course of platelet activation by CG was performed by immunoblot analysis of platelet proteins using a panel of specific antibodies. Exposure of platelets for 3 min at 37 degrees C to CG at a concentration that induces full cell activation resulted in an extensive cleavage of the N-terminal region of the extracellular domain of GPIb alpha, the largest (relative molecular mass, M(r), 143,000) of the three subunits constituting the GPIb-IX complex. In contrast, no detectable proteolytic modification of the two other subunits, GPIb beta and GPIX, was detected. Similarly, we observed that neither of the two subunits of the alpha IIb beta 3 receptor were proteolytically modified by CG. Cleavage of GPIb alpha by CG leaves a remnant of the polypeptide chain with M(r) approx. 106,000 in the plasma membrane, while releasing into the extracellular milieu the N-terminal domain with M(r) in the range 40,000 to 46,000. N-terminal sequencing of the CG-derived fragments of GPIb alpha indicated that the Leu275-Tyr276 peptide bond was the primary cleavage site for this proteinase. Proteolysis of GPIb alpha was already detectable at concentrations of CG as low as 25 nmol/l, while with 200 nmol/l the cleavage was detected as soon as 10 s after exposure of platelets to the proteinase. Comparison of the kinetics and concentration dependency for the proteolysis of GPIb alpha and for the activation of platelets by CG showed that cleavage of the GPIb-IX receptor is an early event that accompanies exocytosis and aggregation. Quantitative evaluation of the conversion of GPIb alpha into its membrane fragment indicated that, under optimal conditions, a maximum of approx. 50% of the total GPIb alpha can be affected by proteolysis. However, this proteolysis was > 90% complete when platelets were in the presence of the potent antagonist prostacyclin, suggesting that cellular redistribution of the GPIb-IX receptor may also occur during activation by CG. These results thus indicate that the very early phase of platelet activation by CG is accompanied by extensive modifications in the structure and expression of the GPIb-IX receptor, an effect that might be of functional significance for the interaction of platelets with the vessel wall.

We previously showed that two polymorphonuclear neutrophil (PMN)-derived proteinases, namely cathepsin G (Cat. G) and elastase (HLE), acting in synergy activated nearby platelets in vitro. This cellular interaction could result in a pathology such as the adult respiratory distress syndrome (ARDS). Since elevated levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-8 (IL-8) were detected in these patients and therefore could be involved in this disease, we looked for their effects in the PMN-platelet cooperation system. Addition of IL-8 to mixed suspensions of PMN and platelets induced weak but significant platelet aggregations. Upon preincubation with TNF-alpha, aggregations triggered by IL-8 were significantly increased. The targets of these cytokines were not the platelets but the PMNs. This was shown by following beta-glucuronidase release and more interestingly by measuring the enzymatic activities of Cat. G and HLE in the supernatant. Inhibition of the platelet response upon addition of a serine proteinase inhibitor, eglin C, clearly demonstrated the involvement of these two enzymes. Taken together, these results constitute an additional argument for the role of the PMN-platelet interaction in ARDS.

Addition of platelets to activated human polymorphonuclear neutrophils (PMNs) led to their aggregation and degranulation, with these responses decreasing as a function of the time interval between PMN activation and platelet addition. Thus, for a 15-second interval platelet aggregation and serotonin release reached 51.3% +/- 6.7% (n = 11) and 64.3% +/- 4.9% (n = 8), respectively, but after a 5-minute interval they were totally absent. This effect was correlated with the decrease in enzymatic activities of elastase (HLE) and cathepsin G (CAT-G) that were released on PMN activation and responsible for the activation of nearby platelets (r = 0.86 and 0.90 for CAT-G and HLE, respectively; p < 0.05). Because it has been recently shown that PMNs express an alpha 1-proteinase inhibitor (alpha 1-Pl) gene and secrete this antiproteinase at their surface, we investigated whether the PMN alpha 1-Pl could regulate the biologic activities of both proteinases. Although superoxide anions oxidize alpha 1-Pl and reduce its affinity for CAT-G and HLE, maneuvers aimed at modifying their concentrations did not modify the loss of CAT-G and HLE. In fact, the progressive decrease of the two proteinase enzymatic activities followed the same pattern whether PMNs were present or not. It is concluded that PMN alpha 1-Pl is not involved in the time-dependent inactivation of CAT-G and HLE released from activated PMNs.

Purified proteinase 3 (PR3) devoid of any elastase (HLE) and cathepsin G (Cat.G) contaminants, was prepared from azurophilic granules of human polymorphonuclear neutrophils by using a novel procedure. Although unable to induce platelet activation (up to 25 micrograms/ml) by itself, PR3 at a concentration as low as 2.5 micrograms/ml enhanced the platelet response to a concomitantly added threshold concentration of Cat.G, a recognized platelet agonist. In the presence of 10 micrograms/ml PR3, aggregation and degranulation of platelets induced by Cat.G were 43.2 +/- 5.9% and 27.1 +/- 1.9% as compared with 5.5 +/- 2.9% and 4.2 +/- 1.5% (n = 4) for Cat.G alone. This enhancing effect by PR3 was also observed with collagen and a cyclic endoperoxide analogue, and was inhibited by eglin C and elafin, two PR3 inhibitors. Associated with the removal of activity by a anti-PR3 mAb and the lack of effect of the secretory leukocyte proteinase inhibitor, these data demonstrated that the effect is specifically related to the enzymatic activity of PR3. It is hypothesized that this mechanism could play a role in the polymorphonuclear neutrophil-mediated platelet activation, an event already known to be dependent on Cat.G and HLE. This is supported by the fact that the association of PR3 and HLE, at concentrations ineffective by themselves, was able to potentiate Cat.G-induced platelet activation.

Two major membrane receptors implicated in the adhesive properties of blood platelets are the GPIb-IX complex, a receptor for subendothelial von Willebrand factor, and the alpha IIb beta 3 integrin, the receptor for plasma fibrinogen. We have evaluated how the biological activities of these receptors can be potentially modulated through limited proteolysis when platelets are exposed to the serine-proteinases secreted by activated polymorphonuclear neutrophils, i.e., leukocyte elastase (EL) and cathepsin G (CG). CG can activate the alpha IIb beta 3 integrin through intracellular metabolic pathways, but has no direct proteolytic activity on the receptor subunits. By contrast, EL does not activate the platelet metabolism, but specifically cleaves a short peptide sequence within the alpha IIb subunit, and this cleavage occurs in parallel with an up-regulation of the activity of the fibrinogen receptor. On another hand, both EL and CG cleave the amino-terminal portion of the GPIb alpha subunit of the GPIb-IX receptor, eliminating the binding site for von Willebrand factor and diminishing the capacity of platelets to interact with this adhesion protein. Thus, neutrophil proteinases have the potential to regulate the activity of platelet adhesion receptors, and such experimental observations may prove to be relevant in vivo in various pathological conditions.

When human polymorphonuclear neutrophils and platelets were incubated with human leukocyte elastase before N-formyl-Met-Leu-Phe (FMLP) challenge, a time- and concentration-dependent inhibition of the resulting platelet activation was observed. Thus, when the mixed cell suspension was preincubated for 6 min with 1 microM elastase before stimulation of neutrophils with 0.5 microM FMLP, resulting aggregations and serotonin releases were respectively only 4.4 +/- 4.1% (n = 4) and 1.6 +/- 2.4% (n = 4) as compared to 41.6 +/- 5.2% (n = 9) and 71.3 +/- 16.0 (n = 9) for controls. A direct inhibitory action of elastase on neutrophil activation was ruled out, as well as a breakdown of cathepsin G, a mediator involved in neutrophil-mediated platelet activation. In fact, we demonstrated that the target for the inhibitory effect of elastase in such a cell-to-cell cooperation system was the platelet. This phenomenon is likely to play a role under in vivo conditions in pathologies in which a significant granulocytic proteolytic activity has been detected in the plasma.

We have focused our interest on the platelet-activating properties of two polymorphonuclear neutrophil (PMN)-derived proteinases, namely elastase (HLE) and cathepsin G (Cat.G). First of all, we observed that whereas HLE was unable to trigger platelet activation by itself, it enhanced platelet activation induced by Cat.G when both proteinases were added simultaneously. It has been recently described that, upon stimulation, PMN released Cat.G, which in turn activated surrounding platelets. Thus, we looked for a combined effect of Cat.G and HLE during this cell-to-cell interaction. When PMN (5 x 10(6)/mL) were stimulated by 0.5 mumol/L N-formyl-Met-Leu-Phe, they released 237.9 +/- 49.1 nmol/L Cat.G and 381.7 +/- 28.0 nmol/L HLE. Such a concentration of purified Cat.G (240 nmol/L) induced only a moderate platelet activation when added to a PMN-platelet mixture. However, when Cat.G (240 nmol/L) and HLE (380 nmol/L) were added together, the resulting platelet activation was strictly comparable to that corresponding to the addition of N-formyl-Met-Leu-Phe (P > .05) in terms of aggregation, dense and alpha granule secretion, and thromboxane B2 production. In fact, Elafin, a specific HLE inhibitor, when added to the PMN-platelet cooperation system triggered by N-formyl-Met-Leu-Phe, prevented platelet activation within the same range of concentrations as for inhibition of HLE activity. In conclusion, we now show that not only Cat.G, but also HLE is involved in the PMN-mediated platelet activation.

The aim of this study was to investigate a possible activation of human endothelial cells in monolayer culture by a purified neutrophil-derived proteinase, i.e. elastase. Cells were isolated from human umbilical cord veins, and incubated either in primary culture or after two passages, in the presence of various concentrations of this proteinase. Although a lack of prostacyclin formation was noted, elastase induced a large release of von Willebrand factor (vWf) in a concentration-dependent manner. Thus, upon incubation with 1 microgram.ml-1 elastase for 30 min, 70 mU.ml-1 vWf were detected in the incubation medium, as compared to 5 mU.ml-1 for control. Using cells in primary culture, a fivefold higher concentration of vWf was recovered following incubation with 10 micrograms.ml-1 elastase than with 0.5 IU.ml-1 thrombin. This effect was linked to the enzymatic activity of elastase and not to its cationic charge, as deduced from the inhibition by eglin C, and the lack of effect of the phenylmethylsulphonyl fluoride (PMSF) treated proteinase. We conclude that vWf release was not due to cell activation, since cytoplasmic calcium mobilization was absent, and inhibition of protein kinase C did not modify the response. In fact, this release was the consequence of cell damage, since concentrations of vWf recovered correlated with cell lysis. These results support the hypothesis that the high level of plasma vWf in patients with sepsis or adult respiratory distress syndrome could result from damage to the endothelial cells by elastase released from activated neutrophils.

1. The capacity of recombinant human secretory leukocyte proteinase inhibitor (SLPI) to inhibit human leukocyte elastase (HLE) and cathepsin G (Cat G) was investigated and compared with a recombinant truncated form (carboxyl-terminal domain, Asn55-Ala107) called 1/2 SLPI. 2. Both compounds were efficient when tested against enzymatic activities of purified HLE and Cat G indicating that the HLE- and Cat G-inhibitory sites were preserved in the truncated form. SLPI and 1/2 SLPI also affected platelet activation induced by 0.2 microM Cat G (IC50 = 112 +/- 13 nM for SLPI and 280 +/- 12 nM for 1/2 SLPI). 3. The effects of SLPI and 1/2 SLPI were then tested against polymorphonuclear neutrophil (PMN)-mediated platelet activation, a cell-to-cell interaction mediated by HLE and Cat G released from PMN. In this experimental system, addition of SLPI or 1/2 SLPI before N-formyl-Met-Leu-Phe (fMLP) led to the inhibition of the resulting platelet activation. As was the case for Cat G enzymatic activity and Cat G-induced platelet activation, SLPI was more efficient than 1/2 SLPI (IC50 = 676 +/- 69 nM vs 1121 +/- 150 nM). 4. The ratio of the IC50 against PMN-mediated platelet activation compared to purified Cat G-mediated platelet activation was 6.03 for SLPI and 4.32 for 1/2 SLPI. This difference may be due to the smaller size of the truncated form which could allow this molecule to diffuse more easily between PMN and platelets. 5. In conclusion, 1/2 SLPI could be a promising candidate in the treatment of pathological states linked to inflammation in which participation of HLE and Cat G has been evoked.

Accumulation of polymorphonuclear neutrophils (PMN) and epithelium damage have often been described during airway inflammation. We studied the effects of two PMN-derived proteinases, namely elastase and cathepsin G, on guinea-pig tracheal epithelial cells in culture. Both proteinases activated tracheal epithelial cells in terms of prostaglandin (PG) E2 production. A concentration- and time-dependent effect was observed with 10 micrograms/ml and 6 h as the optimal conditions for both enzymes. Optical microscopic studies confirmed an effect on tracheal epithelial cells as intercellular gaps were observed upon incubation of the monolayers with proteinases. A small cytotoxic effect was observed after 1 h incubation but remained stable up to 6 h. This cytotoxic effect, more pronounced with elastase than with cathepsin G, was dissociated from PGE2 formation.

Activated human polymorphonuclear neutrophils (PMNs) induce platelet stimulation via cathepsin G (cat G), a platelet-activating cationic serine proteinase released from the azurophilic granules. Heparin inhibited up to 100% of aggregation and serotonin release triggered by 180 nmol/L of purified cat G in a concentration-dependent manner between 10 and 70 mU/ml. When tested against the enzymatic activity of 180 nmol/L cat G (hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), inhibition by heparin never exceeded 60% (up to 100 U/ml). Inhibition was most probably related to electrostatic interactions between the cationic cat G and the anionic heparin, because addition of 180 mU/ml of protamine sulfate restored platelet activation. Low molecular weight heparin, CY 216, used between 5 and 50 mU/ml, also inhibited cat G-induced platelet activation. When purified PMNs and washed platelets were mixed together and challenged with a PMN agonist (FMLP at 1 mumol/L), platelet activation was observed. Pretreatment of the mixed cell population with 300 mU/ml heparin prevented platelet activation. This was illustrated by electron microscopy studies. The present data, apart from confirming a participation of cat G in the PMN-platelet interaction, bring evidence that heparin has potent antiproteinase effect in a biologic model.

In this paper we have studied the combined effects on platelet activation, of two polymorphonuclear neutrophil (PMN)-derived agonists, namely platelet-activating factor (PAF) and cathepsin G (Cat.G), used at threshold concentrations. Our results showed that the order of agonist addition was a determinant factor since the addition of Cat.G prior to PAF induced a full platelet activation while the reverse combination had no effects. The successive challenge of platelets by Cat.G and then PAF induced a strong aggregation accompanied by an enhancement of alpha and dense granule secretion. The observed phenomenon was also dependent on the time interval between agonist addition. It was significant at 30 s (P less than 0.05) and plateaued over 1-2 min. Platelet activation resulting from the combination Cat.G-PAF can be described as a function of PAF concentrations, the synergism being significant between 10 nM and 1 microM. The mechanism by which Cat.G primes platelets remains to be elucidated. However, some points have been examined and have led us to conclude that an increase in expression and/or affinity of PAF receptors, [Ca2+]i movements, protein kinase C activation and phospholipase A2 pathway are not involved. Whatever the biochemical mechanism underlying this synergism which involved PMN and platelets, it may constitute a link between the inflammatory and haemostatic processes in response to tissue damage.

1. The capacity of various drugs (acetylsalicylic acid (ASA), ketoprofen, diclofenac, piroxicam, BW 755C, BW A4C, nedocromil sodium and azelastine) to inhibit human polymorphonuclear neutrophil (PMN)-mediated platelet activation was investigated. In this model, stimulated PMN release cathepsin G (Cat G), a serine proteinase which, in turn, induces platelet activation. 2. Among the different tested drugs, azelastine (100 microM for 1 min) was the only one able to prevent platelet aggregation. The cyclo-oxygenase inhibitors were all inactive, although used at effective concentrations as judged by inhibition of thromboxane B2 (TxB2) formation. Inhibition of platelet aggregation by azelastine was concentration-dependent, the range of active concentrations being of 20-70 microM. Release from platelets of 5-hydroxytryptamine was also inhibited at 30 microM and above, but never reached 100%. 3. The inhibition by azelastine is due to an effect on both cells. Indeed, beta-glucuronidase release from activated PMN and platelet activation by purified Cat G were both affected. 4. However, used at high concentrations (greater than 100 microM) azelastine was toxic since it released significant amounts of lactate dehydrogenase (LDH) from PMN and platelets. 5. These results show the capacity of azelastine, an anti-allergic and anti-asthmatic compound, to inhibit the cell-to-cell communication between PMN and platelets, an effect which may be relevant for its therapeutic efficacy or for a new application in diseases in which PMN and platelets are involved.

In this paper we show that TNF-alpha enhances platelet activation. Experiments were performed on a human polymorphonuclear neutrophil (PMN)-platelet cooperation system in which PMN, stimulated by FMLP, release cathepsin G (Cat.G), a serine proteinase responsible for the activation of nearby platelets. Pretreatment of the mixed cell suspension with 5 ng/ml TNF-alpha resulted in a strong platelet activation (37.7 +/- 3.2% aggregation; 46.0 +/- 14.4% serotonin release) in response to a weak concentration of FMLP (1.25 x 10(-8) M) inducing by itself only 7.7 +/- 4.0% of aggregation and 3.8 +/- 4.1% of serotonin release (mean +/- SD; n = 10). This effect was concentration dependent (maximum between 5 and 10 ng/ml) and was optimal for a brief preincubation time (5 min). Under these experimental conditions the target of TNF-alpha was PMN, as shown by beta-glucuronidase release. The observed potentiation was modified neither by 0.1 mM acetyl salicylic acid (a cyclo-oxygenase inhibitor) nor by 0.1 mM BN 52021 (a platelet-activating factor antagonist), while such a phenomenon was fully inhibited by 20 micrograms/ml eglin C, a strong and specific inhibitor of the human granulocytic proteinases, elastase and Cat.G. In fact, full inhibition was also observed with 300 nM alpha-1-antichymotrypsin, a specific inhibitor of Cat.G. This clear-cut evidence of Cat.G involvement was substantiated by the enhancement of Cat.G release from FMLP-activated PMN primed with TNF-alpha. These results demonstrate that the priming of PMN by TNF-alpha may modulate the activation of other inflammatory cells, particularly of platelets. It is hypothesized that this phenomenon could contribute to pulmonary pathologies, and more specifically to the adult respiratory distress syndrome, a disease for which PMN, platelet and TNF-alpha involvement has been proposed.

Human platelets can be stimulated by recombinant human fifth component of complement (rhC5a) in the presence of human neutrophils. After challenge with N-formyl-Met-Leu-Phe or rhC5a, concentrated neutrophils release cathepsin G into the supernatant. The concentrations of cathepsin G recovered by titration of the enzymatic activity correlate with the capability of these supernatants to induce platelet stimulation as measured by serotonin release. Cathepsin G purified from neutrophil granules triggered platelet aggregation and serotonin release independent of arachidonic acid metabolites and platelet-activating factor formation. A concentration of 100 nM of cathepsin G, which was reached in the surrounding space of activated neutrophils, induced a 50% platelet stimulation. Three distinct antiproteinases were tested against cathepsin G-induced platelet activation. Z-Gly-Leu-Phe-CH2Cl, a specific inhibitor of cathepsin G enzymatic activity, proved to be nonspecific in our biological system. By contrast, alpha 1-antichymotrypsin and alpha 1-antitrypsin displayed specific activities. The physiological specific inhibitor of cathepsin G, alpha 1-antichymotrypsin, was the most potent and was used in the rhC5a-induced neutrophils-mediated platelet activation. A complete inhibition was achieved, showing that release of cathepsin G from neutrophils accounts for platelet activation. Such a chain of events involving C5a, neutrophils, cathepsin G, and platelets may be of relevance in certain inflammatory states, particularly the adult respiratory distress syndrome.

Eglin C is an inhibitor of two serine proteinase-derived polymorphonuclear leucocytes (PMN) i.e., elastase and cathepsin G. Since the latter has recently been shown to be involved in the activation of platelets by stimulated PMN, the effects of recombinant eglin C in the PMN-platelet cooperation model were studied. First, the inhibitory capacity of eglin C against purified cathepsin G was measured spectrophotometrically by following hydrolysis of a specific synthetic substrate, N-succinyl-ala-ala-pro-phe-para-nitroanilide. The inhibition of the enzymatic activity of 180 nM (5 micrograms/ml) cathepsin G was directly proportional to eglin C concentration and reached 100% with 2 micrograms/ml (240 nM). Platelet activation generated by a submaximal concentration of cathepsin G (200 nM) was also totally suppressed by 2 micrograms/ml of eglin C. Inhibition was specific (a 100 times higher concentration of eglin C did not alter platelet activation induced by thrombin), and surmountable (an increase of cathepsin G concentration reduced the eglin C effect). Thus, the mechanism of inhibition by eglin C of cathepsin G-induced platelet activation could be explained by a stoichiometric relation between eglin C and cathepsin G as previously described. Investigations were then performed with the PMN-platelet cooperation model, using two distinct stimuli, N-formylmethionylleucylphenylalanine (FMLP) or recombinant human C5a, at submaximal concentrations, 2.10(-7) M and 10(-7) M, respectively. A concentration-dependent inhibition of platelet activation aggregation and serotonin release-lambda was observed. Eglin C used at 10 micrograms/ml and 25 micrograms/ml totally blocked the platelet responses induced by recombinant human C5a and FMLP, respectively. Leucotriene B4, but also thromboxane B2 production measured by radioimmunoassays, were observed under FMLP activation. In the presence of eglin C, thromboxane B2 formation was totally suppressed, whereas leucotriene B4 synthesis was still effective. In fact, the mechanism of inhibition of eglin C is located neither on PMN (leucotriene B4 formation by FMLP-activated PMN was not affected), nor on platelets (response to thrombin was unchanged). The target is most probably cathepsin G since eglin C suppressed thromboxane B2 formation by platelets challenged by this serine proteinase. These results constitute an argument in favor of the implication of cathepsin G in the PMN-mediated platelet activation. Moreover, they reinforce the hypothesis that this mechanism could be operating under in vivo pathologic conditions, since eglin C is capable of preventing or ameliorating some experimental pulmonary diseases.

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 10(6)/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 +/- 2.5 ng/ml paf-acether. Human washed platelets (3 x 10(8)/ml) stimulated with thrombin (1 IU/ml) formed 0.60 +/- 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 +/- 4.25 ng/ml, n = 6, P less than .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 +/- 3.81 ng/ml paf-acether vs. 5.30 +/- 2.23, P less than .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 +/- 2.26 ng/ml paf-acether vs. 5.76 +/- 0.38, P less than .05, n = 3). These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.