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1.
bioRxiv ; 2023 Oct 25.
Article in English | MEDLINE | ID: mdl-37662284

ABSTRACT

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC), pericytes, as well as the lymphatic muscle (LMCs) cells themselves. Here we tested the extent to which these cell types are invested into the mouse cLV wall and if any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a contiguous network; spontaneous Ca2+ transients; and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreERT2 to target ICLC; PdgfrßCreERT2 to target pericytes; PdgfrαCreER™ to target CD34+ adventitial fibroblast-like cells or ICLC; and Myh11CreERT2 to target LMCs. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreERT2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrßCreERT2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreER™ labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Titrated induction of the smooth muscle-specific Myh11CreERT2 revealed a LMC population with heterogeneous morphology. Only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 by Myh11CreERT2, but not PdgfrαCreER™ or c-KitCreERT2, resulted in a propagated contraction. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.

2.
J Immunol ; 142(11): 3985-92, 1989 Jun 01.
Article in English | MEDLINE | ID: mdl-2541203

ABSTRACT

We examined the interaction between IFN-gamma, LPS, and glucocorticoids on release of oxygen radicals by human monocytes cultured in vitro. After 48 h culture, monocytes released low amounts of superoxide anion (O2-) when stimulated by PMA or FMLP. Monocytes incubated with either IFN-gamma or LPS became "primed" and released greater amounts of O2- in response to stimuli. Monocytes incubated with hydrocortisone, methylprednisolone, dexamethasone, or prednisolone alone showed decreased release of O2-. Prednisone and progesterone, which are not active glucocorticoids, had no effect. When glucocorticoids were co-incubated with IFN-gamma or LPS, the effect of hydrocortisone and other active steroids was blocked, and the monocytes released high O2-. However, when monocytes were preincubated with hydrocortisone for 24 h before addition of IFN-gamma or LPS, priming for enhanced O2- production by LPS was partially inhibited whereas there was no effect on IFN-gamma priming. We suggest that IFN-gamma and LPS can block the anti-inflammatory effects of glucocorticoids, contributing to increased inflammation at tissue sites; however, the mechanism of this effect may differ for the two macrophage activators. To investigate the mechanisms of priming by IFN-gamma and LPS, we examined the effects of these agents and of hydrocortisone on secretion of IL-1 and TNF-alpha. Both IL-1 and TNF-alpha primed monocytes for enhanced release of O2- in response to PMA. LPS caused monocytes to secrete both IL-1 beta and TNF-alpha. LPS-induced secretion of TNF-alpha and IL-1 beta was completely blocked by hydrocortisone, but the priming effect of LPS on O2- release was only partly blocked. IFN-gamma did not cause monocytes to secrete IL-1 beta or TNF-alpha, under our culture conditions (mononuclear cells cultured in Teflon in endotoxin-free modified Earle's salt solution without serum). Therefore, priming by LPS and IFN-gamma, and the inhibition of priming by glucocorticoids involve mechanisms that extend beyond regulation of secretion of IL-1 and TNF-alpha.


Subject(s)
Glucocorticoids/pharmacology , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Monocytes/metabolism , Superoxides/metabolism , Cell Separation , Cell Survival/drug effects , Centrifugation , Dose-Response Relationship, Immunologic , Free Radicals , Humans , Hydrocortisone/pharmacology , Interleukin-1/physiology , Monocytes/drug effects , Monocytes/immunology , Recombinant Proteins , Time Factors , Tumor Necrosis Factor-alpha/physiology
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