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Mitogen-activated protein kinase activation during IgG-dependent phagocytosis in human neutrophils: inhibition by
S J Suchard1, P J Mansfield, L A Boxer
1Department of Pediatrics, University of Michigan, Ann Arbor 48109, USA.
Abstract:
In FMLP-activated polymorphonuclear leukocytes (PMNs) challenged with IgG-opsonized erythrocytes (EIgG), the termination of phagocytosis correlates with an accumulation of ceramide, a product of sphingolipid metabolism. Furthermore, the exogenous addition of short chain ceramides inhibits EIgG-mediated phagocytosis. In the present study, we identified p42 and p44 mitogen-actived protein (MAP) kinases, referred to as extracellular signal-regulated kinases ERK2 and ERK1, respectively, as intracellular targets of ceramide action during Fc gammaR-mediated phagocytosis. The tyrosine phosphorylation of ERK1 and ERK2 increased within 30 s of addition of EIgG, with maximal phosphorylation by 1 to 5 min. By 30 min, ERK1 and ERK2 were almost completely dephosphorylated. The kinetics of ERK1 and ERK2 tyrosine phosphorylation indicated that MAP kinase activation preceded target ingestion. N-Acetylsphingosine (C2-ceramide) inhibited phagocytosis, reduced ERK1 and ERK2 phosphorylation to basal levels, and reduced ERK1 and ERK2 activity by 85 to 90% and 70 to 80%, respectively. In contrast, N-acetyldihydrosphingosine (dihydro-C2-ceramide) had no effect on either tyrosine phosphorylation or activity of ERK1 and ERK2. In the presence of the MAP kinase kinase (MEK) inhibitor, PD 098059, phagocytosis was reduced by approximately 50%, while ERK1 and ERK2 activity was reduced by 85 to 90%. Thus, engagement of Fc gammaRs led to ERK1 and ERK2 phosphorylation and activation, and the activation of these enzymes was critical for phagocytosis. Furthermore, the inhibition of phagocytosis by C2-ceramide correlated with the inhibition of tyrosine phosphorylation and activation of ERK1 and ERK2. These results suggest that ceramides generated during phagocytosis act on the MAP kinase signaling pathway, ultimately "turning off" the phagocytic response.
Insights
Ceramides, generated during phagocytosis, inhibit the process by targeting the mitogen-activated protein (MAP) kinase signaling pathway. This research identifies extracellular signal-regulated kinases (ERK1 and ERK2) as key targets, revealing how ceramide accumulation turns off phagocytosis.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Phagocytosis, a critical immune process, is regulated by complex intracellular signaling pathways.
- Ceramide, a sphingolipid metabolite, accumulates during phagocytosis and has been shown to inhibit this process.
- The precise molecular mechanisms by which ceramide terminates phagocytosis remain incompletely understood.
Purpose of the Study:
- To identify the intracellular targets of ceramide action during Fc gamma receptor (FcγR)-mediated phagocytosis.
- To elucidate the role of mitogen-activated protein (MAP) kinases, specifically ERK1 and ERK2, in ceramide-mediated regulation of phagocytosis.
- To investigate the signaling cascade linking ceramide accumulation to the inhibition of phagocytic activity.
Main Methods:
- Utilized FMLP-activated polymorphonuclear leukocytes (PMNs) challenged with IgG-opsonized erythrocytes (EIgG) to model FcγR-mediated phagocytosis.
- Assessed tyrosine phosphorylation and activity of ERK1 and ERK2 in response to EIgG and ceramide treatment.
- Employed the MAP kinase kinase (MEK) inhibitor PD 098059 to evaluate the role of ERK activation in phagocytosis.
Main Results:
- Tyrosine phosphorylation and activation of ERK1 and ERK2 were rapidly induced upon FcγR engagement, preceding target ingestion.
- Exogenous addition of N-Acetylsphingosine (C2-ceramide) inhibited phagocytosis and significantly reduced ERK1/ERK2 phosphorylation and activity.
- Inhibition of MAP kinase kinase (MEK) with PD 098059 reduced phagocytosis and ERK1/ERK2 activity, confirming their critical role.
- N-acetyldihydrosphingosine (dihydro-C2-ceramide) had no effect, indicating the specificity of ceramide's action.
Conclusions:
- Ceramides generated during phagocytosis act on the MAP kinase signaling pathway, specifically targeting ERK1 and ERK2.
- The activation of ERK1 and ERK2 is essential for the phagocytic response.
- Ceramide accumulation serves as a negative feedback mechanism, inhibiting phagocytosis by downregulating ERK1/ERK2 activation.