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Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

Jin R, Yu S, Song Z, Zhu X, Wang C, Yan J, Wu F, Nanda A, Granger DN, Li G - PLoS ONE (2013)

Bottom Line: Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined.Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion.These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.

View Article: PubMed Central - PubMed

Affiliation: Vascular Biology and Stroke Research Laboratory, Department of Neurosurgery, Louisiana State University Health Science Center in Shreveport, Shreveport, Louisiana, United States of America.

ABSTRACT
Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.

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Effects of CD40, Mac-1, and different protein kinase inhibitors on sCD40L- stimulated neutrophil oxidative burst.A & B: CD40, but not Mac-1, is required for sCD40L- stimulated neutrophil oxidative burst. A. Representative flow cytometry plots showing ROS generation in WT, CD40−/−, and Mac-1−/− neutrophils using CM-H2DCFDA as a fluorescent probe (dashed gray line: without the probe as a control). Neutrophils were stimulated without (green line) or with (red line) rm-CD40L (100 ng/ml) for 30 min. B. Quantitation of ROS generation in the indicated groups of neutrophils. Data obtained from five independent experiments are shown. NS  =  not significant. C. Effect of the indicated protein kinase inhibitors on sCD40L-stimulated neutrophil oxidative burst. WT neutrophils were stimulated without or with rm-CD40L (100 ng/ml) for 30 min in the presence of PKCζ pseudosubstrate (PS, 5 μM), PI3-kinase (LY294002,10 µM), or NF-κB (BAY11-7082, 10 µM) inhibitors, or dimethyl sulfoxide (DMSO) as a solvent control. Data obtained from five independent experiments are shown. *P<0.01 versus bar 1, and #P<0.01 versus bar 2.
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pone-0064631-g005: Effects of CD40, Mac-1, and different protein kinase inhibitors on sCD40L- stimulated neutrophil oxidative burst.A & B: CD40, but not Mac-1, is required for sCD40L- stimulated neutrophil oxidative burst. A. Representative flow cytometry plots showing ROS generation in WT, CD40−/−, and Mac-1−/− neutrophils using CM-H2DCFDA as a fluorescent probe (dashed gray line: without the probe as a control). Neutrophils were stimulated without (green line) or with (red line) rm-CD40L (100 ng/ml) for 30 min. B. Quantitation of ROS generation in the indicated groups of neutrophils. Data obtained from five independent experiments are shown. NS  =  not significant. C. Effect of the indicated protein kinase inhibitors on sCD40L-stimulated neutrophil oxidative burst. WT neutrophils were stimulated without or with rm-CD40L (100 ng/ml) for 30 min in the presence of PKCζ pseudosubstrate (PS, 5 μM), PI3-kinase (LY294002,10 µM), or NF-κB (BAY11-7082, 10 µM) inhibitors, or dimethyl sulfoxide (DMSO) as a solvent control. Data obtained from five independent experiments are shown. *P<0.01 versus bar 1, and #P<0.01 versus bar 2.

Mentions: Oxidative burst represents as one of the most important aspects of neutrophil functions in inflammation. We measured oxidative burst by flow cytometry using CM-H2DCFDA as a fluorescent probe in neutrophils isolated from WT, CD40 −/−, and MAC-1 −/− mice. Our data showed that rm-CD40L (100 ng/ml, 30 min) significantly enhanced neutrophil CM-H2DCFDA fluorescence, with comparable levels in WT and MAC-1 −/− neutrophils (Fig. 5a, 5b), but it had no effect in CD40-deficient neutrophils (Fig. 5a, 5b). Furthermore, our data showed that this rm-CD40L-timulated oxidative burst in WT neutrophils was completely inhibited with PI3-kinase (LY294002, 10 µmol/L) or NF-κB (BAY11-7082, 10 µmol/L) inhibitors, but is was only slightly inhibited with the PKCζ pseudosubstrate inhibitor (PS, 5 µmol/L) (Fig. 5c).


Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

Jin R, Yu S, Song Z, Zhu X, Wang C, Yan J, Wu F, Nanda A, Granger DN, Li G - PLoS ONE (2013)

Effects of CD40, Mac-1, and different protein kinase inhibitors on sCD40L- stimulated neutrophil oxidative burst.A & B: CD40, but not Mac-1, is required for sCD40L- stimulated neutrophil oxidative burst. A. Representative flow cytometry plots showing ROS generation in WT, CD40−/−, and Mac-1−/− neutrophils using CM-H2DCFDA as a fluorescent probe (dashed gray line: without the probe as a control). Neutrophils were stimulated without (green line) or with (red line) rm-CD40L (100 ng/ml) for 30 min. B. Quantitation of ROS generation in the indicated groups of neutrophils. Data obtained from five independent experiments are shown. NS  =  not significant. C. Effect of the indicated protein kinase inhibitors on sCD40L-stimulated neutrophil oxidative burst. WT neutrophils were stimulated without or with rm-CD40L (100 ng/ml) for 30 min in the presence of PKCζ pseudosubstrate (PS, 5 μM), PI3-kinase (LY294002,10 µM), or NF-κB (BAY11-7082, 10 µM) inhibitors, or dimethyl sulfoxide (DMSO) as a solvent control. Data obtained from five independent experiments are shown. *P<0.01 versus bar 1, and #P<0.01 versus bar 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675111&req=5

pone-0064631-g005: Effects of CD40, Mac-1, and different protein kinase inhibitors on sCD40L- stimulated neutrophil oxidative burst.A & B: CD40, but not Mac-1, is required for sCD40L- stimulated neutrophil oxidative burst. A. Representative flow cytometry plots showing ROS generation in WT, CD40−/−, and Mac-1−/− neutrophils using CM-H2DCFDA as a fluorescent probe (dashed gray line: without the probe as a control). Neutrophils were stimulated without (green line) or with (red line) rm-CD40L (100 ng/ml) for 30 min. B. Quantitation of ROS generation in the indicated groups of neutrophils. Data obtained from five independent experiments are shown. NS  =  not significant. C. Effect of the indicated protein kinase inhibitors on sCD40L-stimulated neutrophil oxidative burst. WT neutrophils were stimulated without or with rm-CD40L (100 ng/ml) for 30 min in the presence of PKCζ pseudosubstrate (PS, 5 μM), PI3-kinase (LY294002,10 µM), or NF-κB (BAY11-7082, 10 µM) inhibitors, or dimethyl sulfoxide (DMSO) as a solvent control. Data obtained from five independent experiments are shown. *P<0.01 versus bar 1, and #P<0.01 versus bar 2.
Mentions: Oxidative burst represents as one of the most important aspects of neutrophil functions in inflammation. We measured oxidative burst by flow cytometry using CM-H2DCFDA as a fluorescent probe in neutrophils isolated from WT, CD40 −/−, and MAC-1 −/− mice. Our data showed that rm-CD40L (100 ng/ml, 30 min) significantly enhanced neutrophil CM-H2DCFDA fluorescence, with comparable levels in WT and MAC-1 −/− neutrophils (Fig. 5a, 5b), but it had no effect in CD40-deficient neutrophils (Fig. 5a, 5b). Furthermore, our data showed that this rm-CD40L-timulated oxidative burst in WT neutrophils was completely inhibited with PI3-kinase (LY294002, 10 µmol/L) or NF-κB (BAY11-7082, 10 µmol/L) inhibitors, but is was only slightly inhibited with the PKCζ pseudosubstrate inhibitor (PS, 5 µmol/L) (Fig. 5c).

Bottom Line: Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined.Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion.These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.

View Article: PubMed Central - PubMed

Affiliation: Vascular Biology and Stroke Research Laboratory, Department of Neurosurgery, Louisiana State University Health Science Center in Shreveport, Shreveport, Louisiana, United States of America.

ABSTRACT
Recent work has revealed an essential involvement of soluble CD40L (sCD40L) in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40), i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ) is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b) and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better understanding of the underlying mechanisms by which sCD40L/CD40 pathway contributes to inflammation and vascular diseases.

Show MeSH
Related in: MedlinePlus