Department of Physiology, Temple University Medical School, Philadelphia, Pennsylvania 19140
Submitted 5 April 2004 ; accepted in final form 27 June 2004
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ABSTRACT |
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N-formyl-methionyl-leucyl-phenylalanine; elastase; CD11b
The acute phase of the inflammatory response involves an increase in the concentrations of different plasma proteins that include fibrinogen and multiple proinflammatory mediators. The modulatory effects of soluble fibrinogen on the process of sequential neutrophil activation have been poorly investigated (28). Neutrophil recruitment is closely related to neutrophil activation and cell survival (37). In this context, it was recently established that integrin engagement is able to trigger intracellular signaling events that lead to cytokine production (36) and exert regulatory effects in platelet-neutrophil interaction (8), phagocytosis (6, 28, 29), chemotaxis (18), production of reactive oxygen species (ROS) (22), and apoptosis (28, 29). Moreover, it has been suggested that elevated fibrinogen levels may alter vascular remodeling when blood flow is compromised and thus may lead to the development of atherosclerotic lesions (13, 19). Fibrinogen can activate intracellular signaling mechanisms in neutrophils such as intracellular Ca2+ increase and phosphorylation of focal adhesion kinases (29). In addition, enhanced exudation of fibrinogen into the perivascular space was associated with neutrophil migration at the inflammatory site in an in vivo model of acute inflammation in rats (30), strongly suggesting that fibrinogen might have well-defined roles in modulating the neutrophil responses. Passively released or actively secreted elastase from neutrophils has been linked to the pathological processes of a variety of inflammatory diseases, including idiopathic pulmonary fibrosis, rheumatoid arthritis, adult respiratory distress syndrome, and cystic fibrosis (7b). Investigators at our laboratory recently showed that fibrinogen is required for the release of primary granule release from neutrophils triggered by extracellular nucleotides, such as ATP or UTP (20).
In the present study, we investigated the effects of soluble fibrinogen on isolated human polymorphonuclear leukocytes, with particular emphasis on determining the intracellular signaling mechanisms involved in the potentiating effect of fibrinogen on the release of primary granule release from isolated human neutrophils. We also have shown that soluble fibrinogen has a potentiating effect on other neutrophil responses, such as intracellular Ca2+ increase and ROS production. We have found evidence that fibrinogen potentiation of primary granule release induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) is mediated by the integrin CD11b/CD18 through phosphatidylinositol 3-kinase (PI3K)- and tyrosine kinase-dependent pathways. In addition, we show that p44/42 mitogen-activated protein kinase (p44/42 MAPK) and p38 MAPK make no contribution to this mechanism, but it is likely that other regulatory intracellular signaling mechanisms also may be involved in the enhancing effect of fibrinogen on neutrophil degranulation.
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MATERIALS AND METHODS |
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Neutrophil isolation. Venous blood was collected in polypropylene tubes containing anticoagulant (1.5% citric acid, 2.5% sodium citrate, 2% dextrose) upon receipt of written consent from healthy volunteers. Blood was mixed with an equal volume of 3% Dextran T500 in saline. Erythrocytes were allowed to sediment for 20 min, then the leukocyte-rich plasma was subjected to centrifugation on Ficoll-Paque at 400 g for 45 min. The pellet was collected, and the contaminating erythrocytes were removed by hypotonic lysis. Isolated neutrophils were resuspended in Hanks' balanced salt solution (HBSS) containing 0.2% BSA. Neutrophils were counted using a Reichert-Jung hemacytometer (Hausser Scientific, Horsham, PA). Cell viability was checked using the Trypan blue exclusion method and was routinely found to be >96%.
Measurement of cytoplasmic free Ca2+ concentration. Isolated neutrophils were incubated for 45 min at room temperature with 1 µM fura 2-AM. The cells were washed twice and then resuspended at a concentration of 3 x 106/ml in HBSS containing 0.2% BSA, and cytoplasmic free Ca2+ concentration was measured as previously described (35). Briefly, aliquots of 0.5 ml were placed in disposable methacrylate cuvettes under stirring conditions at 37°C. Intracellular Ca2+ concentrations were recorded during agonist stimulation using excitation wavelengths of 340 and 380 nm, and the emission was monitored at 510 nm using an AB2 luminescence spectrophotometer (Spectronics Instruments, Rochester, NY). Cytoplasmic Ca2+ concentrations were calculated according to Tsien's ratiometric method (10).
Measurement of ROS production. The release of ROS was measured as described elsewhere (9). Briefly, horseradish peroxidase (1 U/ml) and isoluminol (5 µM) were added to neutrophil suspensions (3 million/ml) in a glass cuvette under stirring conditions at 37°C in a lumiaggregometer model 560-CA (Chronolog, Havertown, PA). The cells were allowed to equilibrate, reagents were added to the cell suspension, and the luminescence signal was recorded on a chart recorder. A standard solution of hydrogen peroxide was used to determine the sensitivity of the peroxidase-isoluminol system and for normalization of data.
Western blotting.
Neutrophils were isolated and stimulated with fMLP (100 nM) in the presence of the same concentration (2 mg/ml) of fibrinogen or BSA at 37°C for different intervals. The stimulation was terminated by adding to each sample an equal volume of 2x lysis buffer containing a standard mixture of protease and phosphatase inhibitors. The final concentrations of reagents in the lysis medium were 25 mM Tris·HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM sodium vanadate, 2 mM sodium pyrophosphate, and 10 mM -glycerol phosphate. Lysis was performed on ice for 20 min, followed by centrifugation of the samples at 13,000 rpm for 5 min. A volume of 200 µl of the cytosolic fraction (the supernatant) was collected and mixed with 100 µl of sample buffer containing 6% sodium dodecyl sulfate, 30% glycerol, 3 mM EDTA, and 0.03% phenol red. Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA), and incubated for 30 min in Tris-buffered saline (TBST; 20 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 2% BSA (blocking buffer). Membranes were incubated overnight at 4°C in the presence of the indicated primary antibody, which was diluted 1:1,000 in blocking buffer. Next, membranes were washed four times at room temperature in TBST. The appropriate secondary antibody conjugated with alkaline phosphatase was diluted 1:5,000 in blocking buffer. The membranes were incubated with the secondary antibody for 1 h at room temperature followed by five consecutive washes. CDP-Star with Nitro-Block II (Tropix, Bedford, MA) was used for chemiluminescence detection of the reactive proteins, and a Fujifilm LAS-1000 charge-coupled device system was used for imaging.
Measurement of elastase release. Cells were resuspended in fresh medium containing 2 mM CaCl and 1 mM MgCl and incubated with inhibitors or solvent at 37°C for 30 min. Cytochalasin B (10 µM) and either fibrinogen or BSA (2 mg/ml) were added, and cells were incubated for 10 min at 37°C. Aliquots of 150-µl cell suspension were stimulated with agonists for 20 min at 37°C in a polypropylene 96-well plate. The plate was centrifuged for 3 min at 250 g and then supernatants were collected, and the elastase activity was assayed using N-(methoxysuccinyl)-Ala-Ala-Pro-Val p-nitroanilide as a chromogenic substrate. The absorbance for each sample was determined at 405 nm every 5 min using a Polarstar Galaxy microplate reader (BMG Labtechnologies, Bristol, RI). The elastase activity was expressed as the rate of cleavage of the chromogenic substrate and normalized for the maximum degranulating effect induced by fMLP.
Statistical analysis. The probability of a statistically significant difference between the mean values of two data sets was determined using a nonparametric two-tailed Student's t-test; when two or more concentration-response curves were compared, two-way analysis of variance was performed.
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RESULTS |
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Fibrinogen has a potentiating effect on fMLP- or PMA-induced ROS production in isolated human neutrophils.
The production of ROS has a major role in bacterial killing by neutrophils. Therefore we investigated the effect of soluble fibrinogen on the ability of isolated neutrophils to generate ROS. The effect of fibrinogen (2 mg/ml) on the release of ROS was determined on neutrophil suspensions in the presence of isoluminol and horseradish peroxidase as described in MATERIALS AND METHODS. When added alone to neutrophil suspension, fibrinogen did not cause any luminescent signal (data not shown), demonstrating that the intracellular signal provided by fibrinogen alone is not sufficient to cause release of ROS. However, fibrinogen addition altered the responses induced by fMLP or PMA. In control experiments, equal concentrations of BSA were used instead of fibrinogen. An increase with 54 ± 10.7% (n = 3) of the amplitude of ROS released by fMLP (10 nM) was measured in the presence of fibrinogen and compared with BSA-treated samples (Fig. 2A). To determine whether the duration of preincubation with fibrinogen was critical for its effect, we performed experiments in which fibrinogen was added at different time points before the addition of the agonist. No significant difference was observed between the samples incubated with fibrinogen for 1 min and those incubated for longer times (10 min), providing evidence that the potentiating effect of fibrinogen occurs fast and is relatively stable in time (data not shown).
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The potentiating effect of soluble fibrinogen on fMLP-induced primary granule release is dependent on the binding to CD11b. Elastase release from the azurophilic or primary granules is an important physiological response of neutrophils to chemoattractants and is commonly used as a marker for primary granule release. Hence, we measured the elastase release from azurophilic granules using a chromogenic assay as described in MATERIALS AND METHODS. It has been shown that fMLP can cause primary granule release from human neutrophils pretreated with cytochalasin B; in the absence of cytochalasin B, a negligible amount of elastase is released from neutrophils (4). When added to neutrophil suspensions, fibrinogen alone did not cause measurable primary granule release in the presence or absence of cytochalasin B. However, when added 10 min before the addition of fMLP, fibrinogen significantly enhanced the degranulating effect of the formyl peptide.
It has been shown that fibrinogen acts as a ligand for CD11b/CD18, and this integrin is able to cause outside-in signaling in neutrophils. The possibility that the potentiating effect of fibrinogen is due to binding of fibrinogen to Mac-1 was explored. We found that the effect of fibrinogen is completely inhibited (Fig. 3) in the presence of an antibody raised against CD11b (Beckman Coulter, Miami, FL). Hence, we confirmed that the effect of fibrinogen on primary granule release depends on the binding to the integrin Mac-1, in agreement with similar findings of other effects induced by fibrinogen in neutrophils, such as antibody-dependent cytotoxicity and apoptosis (28). The anti-CD11b antibody did not have any effect on degranulation in the absence of fMLP.
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Phosphorylation of MAPK does not contribute to the effect of fibrinogen on primary granule release. We studied the phosphorylation of p44/42 MAPK and p38 MAPK in neutrophils stimulated with fMLP (Fig. 5, A and B). In parallel, we evaluated the impact of MAPK inhibition on the effect of fibrinogen on elastase release (Fig. 5C). The effect of fMLP (100 nM) on p44/42 MAPK phosphorylation was rapid and lasted longer than 5 min. The phosphorylation of p38 MAPK induced by fMLP was more transient, reaching a maximum at 1 min. Fibrinogen (2 mg/ml) enhanced the phosphorylation of both p44/42 MAPK and p38 MAPK, suggesting that MAPK might be involved in mediating the potentiating effect of fibrinogen. To investigate this hypothesis, we used U0126, a MEK1/2 inhibitor, and SB-203580, a p38 MAPK inhibitor. Both reagents were tested at concentrations that were previously shown to block MAPK activity effectively (21). U0126 (3 µM) did not cause any inhibition of fMLP-induced degranulation. Although SB-203580 (10 µM) slightly inhibited the fMLP-induced degranulation, when fibrinogen was added to the medium, SB-203580 failed to cause any inhibition to primary granule release (Fig. 5C).
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We further investigated the effect of several other PKC inhibitors on primary granule release from human neutrophils. We found that staurosporine (100 nM) had an effect similar to that observed with Ro 31-8220 (10 µM), causing a potentiating effect on elastase release but a marked inhibitory effect on ROS production induced by fMLP, consistent with the findings previously reported by Krause et al. (15). We also used GF-109203X (1 µM), a nonselective PKC inhibitor, which caused effects similar to those of Ro 31-8220 and staurosporine (Fig. 7). Finally, Gö6976 (1 µM), a selective inhibitor of classical PKC isoform, significantly inhibited fMLP-induced elastase release and abolished ROS production.
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DISCUSSION |
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Soluble fibrinogen increased the amplitude of the intracellular Ca2+ increase caused by fMLP, suggesting that fibrinogen might enhance other chemoattractant-induced effects in human neutrophils. Therefore we determined the effect of fibrinogen on ROS production and degranulation triggered by fMLP. We found that fibrinogen has potentiating effects on chemoattractant-induced production of ROS and on primary granule release.
Production of ROS is one of the major mechanisms used by polymorphonuclear leukocytes to kill bacteria, and it is expected that multiple extra- and intracellular mechanisms contribute to the modulation of ROS production. Our data provide evidence that soluble fibrinogen is one of the plasma factors that enhance ROS production induced by chemoattractants. However, this modulating effect appears to have a much smaller impact than other priming agents, such as the extracellular nucleotides (ATP, UTP), which are able to amplify the effect of fMLP three- to eightfold (16, 31). Moreover, because the effect of PMA is enhanced when soluble fibrinogen is added to the medium, it is likely that the potentiating effect of fibrinogen does not depend on the signaling events taking place upstream of PKC, but rather is more directly related to the activation of NADPH oxidase system.
The release of primary granule from isolated human neutrophils was studied by determining the concentration of elastase, a commonly used marker for primary granules, in the extracellular medium. In the presence of soluble fibrinogen, the concentration-response curve to fMLP was shifted to the left, demonstrating that fibrinogen potentiates not only the ROS production but also other microbicidal mechanisms relying on neutrophil degranulation. The intracellular signaling mechanisms responsible for the potentiating effect of fibrinogen were investigated using specific inhibitors for intracellular signaling molecules.
Wortmannin, a PI3K inhibitor, showed a strong inhibitory effect on the degranulating effect of fMLP, causing a rightward shift of the concentration-response curve to fMLP that was similar in the presence or in the absence of fibrinogen. A similar effect was caused by genistein, a nonspecific tyrosine kinase inhibitor. Thus our data strongly support the view that both the PI3K and tyrosine kinases are essential for primary granule release (21). Moreover, it appears that PI3K and tyrosine kinases may be involved in the potentiating effect of fibrinogen on fMLP-induced elastase release because the fibrinogen-induced leftward shift of the concentration-response curve was not significant in the presence of wortmannin or genistein.
Akt has been shown to be activated downstream of PI3K in various cell types. On the basis of the observations that HIMO, a specific inhibitor of Akt, caused no significant effect and PI3K inhibition caused a dramatic rightward shift of the concentration-response curve elicited by fMLP, we suggest that the effect of PI3K in mediating primary granule release from neutrophils is independent of Akt activation.
Because protein kinase C isotypes can be controlled by PI3K through the protein kinase PDK1 (17), we investigated the role of PKC on the effect of fibrinogen. We used Ro 31-8220, a widely used PKC inhibitor, to block the activation of classic and novel PKC isoforms, which were previously shown to have the most significant roles in mediating functional responses in neutrophils (14, 23, 27, 32). In the presence of Ro 31-8220, the concentration-response curve to fMLP was significantly shifted to the left, suggesting that certain classic and/or novel PKC isoforms that are inhibited by Ro 31-8220 have an inhibitory effect on primary granule release. When fibrinogen was added to cells treated with Ro 31-8220, the elastase release elicited by fMLP was not further enhanced (Fig. 6D). To further confirm the effect of PKC inhibition on primary granule release, we used staurosporine and GF-109203X, two other nonselective PKC inhibitors, as well as Gö6976, a selective inhibitor of classic PKC isoforms. While staurosporine and GF-109203X caused potentiation of elastase release in a manner similar to that of Ro 31-8220, Gö6976 significantly inhibited fMLP-induced degranulatin in the presence or absence of fibrinogen (Fig. 7).
The data obtained with the PKC inhibitors suggest that the classic PKC isoform is required for primary granule release and ROS production in human neutrophils. It is possible that one of the novel or atypical PKC isoforms might have a constitutive inhibitory effect on neutrophil degranulation. The nonselective PKC inhibitors block both the potentiating and inhibitory PKC isoforms, and the effect on the inhibitory isoforms appears to be dominant; hence the elastase release is potentiated. On the other hand, Gö6976 may block only the classic PKC isoforms that participate in elastase release, leaving the activity of the inhibitory isoforms unaffected. It is possible that fibrinogen might trigger certain intracellular signaling events that are mimicked by the nonselective PKC inhibitors; however, further investigation is required to confirm this hypothesis.
It has been established that integrin activation is able to initiate intracellular signaling events in neutrophils, leading to changes in gene expression (36), increased phagocytosis, spreading, and chemotaxis (8, 22, 28). In the present study, we have shown that fibrinogen causes a dose-dependent increase in intracellular Ca2+ concentrations. Fibrinogen potentiates fMLP- and PMA-induced ROS production as well as fMLP-induced primary granule release from isolated human neutrophils. In the presence of the anti-CD11b antibody, the potentiating effect of fibrinogen was completely inhibited. Hence it appears that fibrinogen modulates formyl peptide-induced primary granule release from neutrophils through mechanisms dependent mostly on CD11b/CD18 activation.
PI3K and tyrosine kinase-dependent mechanisms are essential for primary granule release and for the potentiating effect induced by fibrinogen. Although fibrinogen enhances the chemoattractant-induced phosphorylation of p44/42 MAPK and p38 MAPK, the lack of effect of the MAPK inhibitors (U0126 and SB-203580) on the effect of fibrinogen strongly suggests that the activation of MAPK is not responsible for the effect of fibrinogen in degranulation. We noticed that SB-203580 (10 µM) inhibited neutrophil degranulation caused by concentrations of fMLP as high as 1 µM but not by lower concentrations of fMLP, in agreement with previous reports (21, 24). We conclude that the effect of p38 MAPK inhibition on fMLP-induced primary granule release from human neutrophils depends on the concentration of fMLP used as an agonist. Concentrations of fMLP <1 µM cause an effect that is not markedly affected by p38 MAPK blockade, while at 1 µM fMLP causes a degranulating effect sensitive to p38 MAPK blockade. We suggest that the low-affinity receptor for fMLP (fMLP receptor-like 1), which is activated by concentrations of fMLP in the micromolar range and has been shown to be present in neutrophils, might have a significant role in primary granule release induced by high concentrations of fMLP. It is possible that this receptor might cause degranulation using a different pathway than the classic high-affinity fMLP receptor and that this pathway might rely on activation of p38 MAPK. Further investigations using cell lines expressing these receptors are required to confirm this hypothesis.
We conclude that fibrinogen potentiation of primary granule release induced by fMLP is mediated by the integrin CD11b/CD18 through PI3K- and tyrosine kinase-dependent pathways, but other regulatory mechanisms also might be involved.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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