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Adherence regulates macrophage signal transduction and primes tumor necrosis factor production
G J Bauer1, S Arbabi, I A Garcia
1Department of Surgery, University of Washington Medical School, Seattle, USA.
Abstract:
While monocyte/macrophage (Mphi) adherence to a matrix is necessary for differentiation and prolonged survival, the effect of adherence on the signaling mechanisms responsible for Mphi activation is unknown. Lipopolysaccharide (LPS) activates Mphi by signaling through members of the mitogen activated protein kinase (MAPK) family thereby inducing transcription of proinflammatory cytokines, such as TNF-alpha. Since adherence has been shown to affect different activities of various myeloid phagocytes, we investigated whether adherence affects intracellular signaling and modulates activation of the Mphi proinflammatory phenotype. We assessed the effect of adherence on activation of rabbit alveolar Mphi by measuring LPS-induced TNF-alpha mRNA and TNF-alpha secreted product in adherent versus nonadherent cells, in vitro. The effect of adherence on LPS-induced activation of MAPK was assessed by western analysis using a dual phosphospecific antibody against p38MAPK, p42,44ERK, and p54SAPK. LPS is known to induce activation of NF-kappaB and AP-1. Modulation of these two transcription factors by LPS under adherent versus nonadherent conditions was evaluated by gel-shift analyses. The results were that adherent cells treated with LPS, 10 ng/mL or 1 microg/ml, elicited a 26- and 132-fold increase, respectively, in TNF-alpha production. Nonadherent cells did not elicit significant TNF-alpha in response to LPS. Adherence alone induced significant ERK and AP-1 activation, but did not stimulate a significant TNF-alpha response and no further activation of ERK and AP-1 was observed with LPS stimulation. Adherence alone did not activate p38MAPK or NF-kappaB, but primed Mphi for an augmented response to LPS in activation of p38, NF-kappaB and in production of TNF-alpha. We conclude that adherence primes Mphi for activation and regulates MAPK signal transduction pathways.
Insights
Cell adherence primes macrophages (Mphi) for activation, significantly increasing TNF-alpha production in response to lipopolysaccharide (LPS) by regulating MAPK signaling pathways.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Monocyte/macrophage (Mphi) adherence is crucial for differentiation and survival.
- Lipopolysaccharide (LPS) activates Mphi via mitogen-activated protein kinase (MAPK) pathways, inducing proinflammatory cytokines like TNF-alpha.
- The impact of adherence on Mphi activation signaling remains largely unknown.
Purpose of the Study:
- To investigate if Mphi adherence affects intracellular signaling pathways.
- To determine if adherence modulates Mphi activation and proinflammatory cytokine production.
- To elucidate the role of adherence in LPS-induced MAPK and transcription factor activation.
Main Methods:
- Rabbit alveolar Mphi were cultured under adherent and nonadherent conditions.
- TNF-alpha mRNA and secreted protein levels were measured after LPS stimulation.
- MAPK activation (p38MAPK, ERK, SAPK) was assessed by Western analysis.
- NF-kappaB and AP-1 activation was evaluated using gel-shift assays.
Main Results:
- Adherent Mphi showed significantly higher LPS-induced TNF-alpha production compared to nonadherent cells.
- Adherence alone activated ERK and AP-1, but not p38MAPK or NF-kappaB.
- Adherent Mphi exhibited primed activation of p38MAPK, NF-kappaB, and augmented TNF-alpha production upon LPS stimulation.
Conclusions:
- Mphi adherence primes the cells for enhanced activation.
- Adherence regulates MAPK signal transduction pathways, influencing the Mphi proinflammatory response.
- This priming effect is critical for robust Mphi activation by stimuli like LPS.