A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration
Abstract
We designed a 48-well chemotaxis chamber to minimize manipulation time and amount of material required by the larger blindwell or Boyden chemotaxis chamber. Cell and chemoattractant dose-response curves showed that results were comparable to or better than those obtained with blindwell chambers. The volume of chemoattractant per well is 25 μl; the number of cells can be as low as 10,000. The time needed for setting up this multiwell unit and for staining the membrane filter sheet is negligible. Combined with the use of an image analyzer to count the number of migrated cells, the method is suitable for clinical research on the functional state of monocytes in large groups of patients.
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We sought to test our hypothesis that MC proteases can influence the functionality of human lung fibroblasts (HLFs).
Primary HLFs were treated with MC chymase or tryptase, followed by assessment of parameters related to fibroblast function.
HLFs underwent major morphologic changes in response to chymase, showing signs of cellular contraction, but were refractory to tryptase. However, no effects of chymase on HLF viability or proliferation were seen. Chymase, but not tryptase, had a major impact on the output of extracellular matrix–associated compounds from the HLFs, including degradation of fibronectin and collagen-1, and activation of pro–matrix metalloprotease 2. Further, chymase induced the release of various chemotactic factors from HLFs. In line with this, conditioned medium from chymase-treated HLFs showed chemotactic activity on neutrophils. Transcriptome analysis revealed that chymase induced a proinflammatory gene transcription profile in HLFs, whereas tryptase had minimal effects.
Chymase, but not tryptase, has a major impact on the phenotype of primary airway fibroblasts by modifying their output of extracellular matrix components and by inducing a proinflammatory phenotype.
Regulation of monoamine oxidase A (MAO-A) expression, activity, and function in IL-13–stimulated monocytes and A549 lung carcinoma cells
2018, Journal of Biological ChemistryMonoamine oxidase A (MAO-A) is a mitochondrial flavoenzyme implicated in the pathogenesis of atherosclerosis and inflammation and also in many neurological disorders. MAO-A also has been reported as a potential therapeutic target in prostate cancer. However, the regulatory mechanisms controlling cytokine-induced MAO-A expression in immune or cancer cells remain to be identified. Here, we show that MAO-A expression is co-induced with 15-lipoxygenase (15-LO) in interleukin 13 (IL-13)-activated primary human monocytes and A549 non-small cell lung carcinoma cells. We present evidence that MAO-A gene expression and activity are regulated by signal transducer and activator of transcription 1, 3, and 6 (STAT1, STAT3, and STAT6), early growth response 1 (EGR1), and cAMP-responsive element–binding protein (CREB), the same transcription factors that control IL-13–dependent 15-LO expression. We further established that in both primary monocytes and in A549 cells, IL-13–stimulated MAO-A expression, activity, and function are directly governed by 15-LO. In contrast, IL-13–driven expression and activity of MAO-A was 15-LO–independent in U937 promonocytic cells. Furthermore, we demonstrate that the 15-LO–dependent transcriptional regulation of MAO-A in response to IL-13 stimulation in monocytes and in A549 cells is mediated by peroxisome proliferator–activated receptor γ (PPARγ) and that signal transducer and activator of transcription 6 (STAT6) plays a crucial role in facilitating the transcriptional activity of PPARγ. We further report that the IL-13–STAT6–15-LO–PPARγ axis is critical for MAO-A expression, activity, and function, including migration and reactive oxygen species generation. Altogether, these results have major implications for the resolution of inflammation and indicate that MAO-A may promote metastatic potential in lung cancer cells.
Determining whether observed eukaryotic cell migration indicates chemotactic responsiveness or random chemokinetic motion
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The roles of a ribosomal protein S19 polymer in a mouse model of carrageenan-induced acute pleurisy
2017, ImmunobiologyC5-deficient mice usually present moderate neutrophil activation during the initiation phase of acute inflammation. Conversely, C5a receptor (C5aR)-deficient mice show unusually excessive activation of neutrophils. We identified the ribosomal protein S19 (RP S19) polymer, which is cross-linked at Lys122 and Gln137 by transglutaminases in apoptotic neutrophils, as a second C5aR ligand during the resolution phase of acute inflammation. The RP S19 polymer promotes apoptosis via the neutrophil C5aR and phagocytosis via the macrophage C5aR. To confirm the roles of the RP S19 polymer, we employed a carrageenan-induced acute pleurisy mouse model using C57BL/6J mice with a knock-in of the Gln137Glu mutant RP S19 gene and replaced the RP S19 polymer with either an S-tagged C5a/RP S19 recombinant protein or the RP S19122–145 peptide monomer and dimer (as functional C5aR agonists/antagonists) and the RP S19122–145 peptide trimer (as a functional C5aR antagonist). Neutrophils and macrophages were still present in the thoracic cavities of the knock-in mice at 24 h and 7 days after carrageenan injection, respectively. Knock-in mice showed structural organization and severe hemorrhaging from the surrounding small vessels of the alveolar walls in the lung parenchyma. In contrast to the RP S19122–145 peptide monomer and trimer, the simultaneous presence of S-tagged C5a/RP S19 and the RP S19122–145 peptide dimer completely improved the physiological and pathological acute inflammatory cues. The RP S19 polymer, especially the dimer, appears to play a role at the resolution phase of carrageenan-induced acute pleurisy in C57BL/6J model mice.
Mutation of the key residue for extraribosomal function of ribosomal protein S19 cause increased grooming behaviors in mice
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Rate laws of the self-induced aggregation kinetics of Brownian particles
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