In vivo validation of signaling pathways regulating human monocyte chemotaxis

Bhattacharjee, A.; Mishra, Ravi Shankar; Cathcart, Martha K.

doi:10.1016/j.jim.2007.11.011

Cited by 6 publications

(5 citation statements)

References 29 publications

(29 reference statements)

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“…The impaired in vivo migration of adoptively transferred mouse monocytes that contain either inactive PLA 2 (by AACOCF 3 ) or were rendered defi cient in PLA 2 by AS-ODN validates our in vitro observations ( 14 ) in a dynamic in vivo milieu. We now have adapted this in vivo assay to monitor human monocyte chemotaxis ( 48 ). Our reports show the importance of in vitro studies for identifi cation of potential signaling components.…”

Section: Discussionmentioning

confidence: 98%

iPLA2β: front and center in human monocyte chemotaxis to MCP-1

Mishra

Carnevale

Cathcart

2008

The Journal of Experimental Medicine

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Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca2+-independent phospholipase (iPLA2β) and cytosolic phospholipase (cPLA2α), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA2β is recruited to the membrane-enriched pseudopod. In contrast, cPLA2α is recruited to the endoplasmic reticulum. Although iPLA2β or cPLA2α antisense oligodeoxyribonucleotide (ODN)–treated monocytes display reduced speed, iPLA2β also regulates directionality and actin polymerization. iPLA2β or cPLA2α antisense ODN–treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA2β and cPLA2α regulate monocyte migration from different intracellular locations, with iPLA2β acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

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Section: Discussionmentioning

confidence: 98%

iPLA2β: front and center in human monocyte chemotaxis to MCP-1

Mishra

Carnevale

Cathcart

2008

The Journal of Experimental Medicine

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“…Primary monocytes were either nucleofected with GFP-PKCb-wild type (WT) or GFP-PKCb-dominant negative (DN) using the method providing optimal transfection efficiency as described earlier (25) using Human Monocyte Nucleofector Solution (Amaxa, Gaithersburg, MD) and the program Y-01 (in monocyte nucleofector solution). The GFP-tagged PKCb WT (GFP-PKCb-WT, pBK-CMV-GFP-PKCbII) and the DN mutant form of PKCb (GFP-PKCb-DN, pBK-CMV-GFP-PKCbII K371R) employed in our experiments were previously reported (25) and were a kind gift from Y. Hannun (Medical University of South Carolina, Charleston, SC). After nucleofection, the monocytes were carefully transferred to six-well plates containing Human Monocyte Nucleofector medium and incubated for 48 h.…”

Section: Transfection Of Pkcb Wild Type and Dominant-negative Mutant Into Primary Monocytesmentioning

confidence: 99%

A PKCβ–LYN–PYK2 Signaling Axis Is Critical for MCP-1–Dependent Migration and Adhesion of Monocytes

Das

Pal

Oldfield

et al. 2021

The Journal of Immunology

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MCP-1-induced monocyte chemotaxis is a crucial event in inflammation and atherogenesis. Identifying the important signal transduction pathways that control monocyte chemotaxis can unravel potential targets for preventive therapies in inflammatory disease conditions. Previous studies have shown that the focal adhesion kinase Pyk2 plays a critical role in monocyte motility. In this study, we investigated the MCP-1-mediated activation of Pyk2 (particularly by the phosphorylation of Tyr 402 ) in primary human peripheral blood monocytes. We showed that MCP-1 induces Src phosphorylation in a similar time frame and that the MCP-1-induced Pyk2 tyrosine phosphorylation is controlled by the Src family kinase. We also report, in this study, that PKCb, an isoform of PKC, is required for both Src and Pyk2 activation/phosphorylation in response to MCP-1 stimulation. We identified Lyn as the specific Src kinase isoform that is activated by MCP-1 and acts upstream of Pyk2 in primary monocytes. Furthermore, Lyn is found to be indispensable for monocyte migration in response to MCP-1 stimulation. Moreover, our coimmunoprecipitation studies in monocytes revealed that PKCb, Pyk2, and Lyn exist constitutively in a molecular complex. To our knowledge, our study has uncovered a novel PKCb-Lyn-Pyk2 signaling cascade in primary monocytes that regulates MCP-1-induced monocyte adhesion and migration.

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“…The adoptive transfer model system was performed using tagged mouse monocytes transferred to recipient animals. This model has also been used to monitor the trafficking of human monocytes in mice during peritonitis, and human monocytes were shown to traffic similarly to mouse monocytes (37). Therefore, this adoptive transfer model can be used to assess the relevance of a particular enzyme for regulating human monocyte chemotaxis.…”

Section: In Vivo Validationmentioning

confidence: 99%

Signal-activated phospholipase regulation of leukocyte chemotaxis

Cathcart

2009

Journal of Lipid Research

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Signal-activated phospholipases are a recent focus of the rapidly growing field of lipid signaling. The extent of their impact on the pathways regulating diverse cell functions is beginning to be appreciated. A critical step in inflammation is the attraction of leukocytes to injured or diseased tissue. Chemotaxis of leukocytes, a requisite process for monocyte and neutrophil extravasation from the blood into tissues, is a critical step for initiating and maintaining inflammation in both acute and chronic settings. Recent studies have identified new important and required roles for two signal-activated phospholipases A 2 (PLA 2 ) in regulating chemotaxis. The two intracellular phospholipases, cPLA 2 a (Group IVA) and iPLA 2 b (Group VIA), act in parallel to provide distinct lipid mediators at different intracellular sites that are both required for leukocytes to migrate toward the chemokine monocyte chemoattractant protein-1. This review will summarize the separate roles of these phospholipases as well as what is currently known about the influence of two other classes of intracellular signal-activated phospholipases, phospholipase C and phospholipase D, in regulating chemotaxis in eukaryotic cells, but particularly in human monocytes. The contributions of these phospholipases to chemotaxis both in vitro and in vivo will be highlighted. Studies exploring the mechanisms regulating cell movement have often focused on protein receptors, protein kinase cascades, and protein-protein interactions, paying less attention to the importance of lipids in controlling migration. The exception to this has been the broad acceptance of the importance of phosphatidyl inositol phosphates and their kinases/phosphatases in regulating cell migration. A relatively recent focus of studies characterizing the roles of lipids in signaling has been on the role of signal-activated phospholipases, enzymes that metabolize glycerophospholipids to generate bioactive products capable of regulating downstream effector pathways. These studies have made us realize how little we understand about the importance of lipid mediators in regulating cell function.Primary blood leukocytes are one of the most commonly employed cell types used for investigating chemotaxis of human cells. Leukocytes are white blood cells that are major participants in inflammatory responses and are central mediators of inflammation, with neutrophils and monocyte/macrophages serving as the prime effector cells of acute and chronic inflammation, respectively. In response to an inflammatory stimulus, blood leukocytes adhere to and cross the endothelium of the blood vessel wall and then home to various tissue sites or participate in inflammatory reactions in the vascular wall, depending on the location and nature of the particular tissue injury or inflammatory stimulus. The migration of leukocytes from the blood into tissue is driven by chemotactic factors called chemokines, a group of small molecular weight cytokines that function as cell attractants in a gradientdepende...

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In vivo validation of signaling pathways regulating human monocyte chemotaxis

Cited by 6 publications

References 29 publications

iPLA2β: front and center in human monocyte chemotaxis to MCP-1

iPLA2β: front and center in human monocyte chemotaxis to MCP-1

A PKCβ–LYN–PYK2 Signaling Axis Is Critical for MCP-1–Dependent Migration and Adhesion of Monocytes

Signal-activated phospholipase regulation of leukocyte chemotaxis

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