Expression of the protein kinase C substrate pleckstrin in macrophages: association with phagosomal membranes.
(17/1146)
Despite evidence suggesting that protein kinase C (PKC) isoforms are important in phagocytosis by Fcgamma receptors, the mechanisms by which the substrates of these kinases act are largely unknown. We have investigated the role of one PKC substrate, pleckstrin, in cells of the monocyte/macrophage lineage. Pleckstrin expression in mouse macrophages was induced severalfold in response to bacterial LPS and IFN-gamma. In unstimulated cells, the protein was largely confined to the cytosol. Upon ingestion of IgG-opsonized zymosan particles (OPZ), however, pleckstrin accumulated on the phagosomal membrane. This association was transient, being maximal after 15 min and declining thereafter. Similar kinetics of association was also seen for both filamentous actin and the delta isoform of PKC. Ingestion of OPZ was found to induce phosphorylation of pleckstrin. To examine whether phosphorylation was required for phagosomal association, pleckstrin was expressed in CHO-IIA cells that stably express the FcgammaRIIA receptor and are competent for phagocytosis of OPZ. In these cells, both wild-type pleckstrin and mutants in which the phosphoacceptor sites had been mutated to either alanine (nonphosphorylatable) or glutamine (pseudophosphorylated) were found to accumulate on OPZ phagosomes. Thus, association of pleckstrin with phagosomes is independent of its phosphorylation. Our findings suggest that pleckstrin may serve as an intracellular adaptor/targeting protein in response to particulate stimuli. By targeting interacting ligands to the phagosomal compartment, pleckstrin may serve to regulate phagocytosis and/or early steps during maturation of the phagosome. (+info)
Peritoneal lymphatic absorption and solute exchange during zymosan-induced peritonitis in the rat.
(18/1146)
Lymph flow is elevated in most inflammatory conditions. However, a few previous studies have indicated that peritoneal lymph flow may actually fall during acute peritonitis. This study was performed to explore this issue further and to study the pathophysiology of peritoneal exchange during peritonitis. Therefore, we wanted to assess the total peritoneal clearance (Cl) and the clearance from peritoneum to plasma (Cl --> P) of 125I-labeled albumin (125I-albumin) as well as plasma-to-dialysate clearance (Cl --> D) of Evans blue-labeled albumin together with peritoneal ultrafiltration (UF) profiles and mass transfer area coefficients of 51Cr-EDTA and glucose in rats after acute peritonitis induced by zymosan. Zymosan incubation of the peritoneal cavity (120 mg) for 4 h generally led to a 4- to 10-fold increase in peritoneal fluid white blood cell count, indicating that acute peritonitis had been induced. Then 16 ml of 3.86% Dianeal and 125I-albumin were instilled intraperitoneally, whereas Evans blue-labeled albumin and 51Cr-EDTA were given as infusions intravenously. Compared with control, mass transfer area coefficients for glucose and 51Cr-EDTA increased markedly from 0.43 +/- 0.06 and 0.25 +/- 0.04 to 0.91 +/- 0.06 and 0.59 +/- 0.05 (SE) ml/min, respectively, during peritonitis, whereas Cl and Cl --> D increased from 32.8 +/- 5.6 and 8.6 +/- 1.6 to 74.5 +/- 7.3 and 12.9 +/- 1.0 microl/min, respectively. The UF profile in peritonitis indicated type I loss of UF (resulting from the increases in permeability-surface area product for glucose). However, the Cl --> P declined to 5.9 +/- 1.0 microl/min from 7.9 +/- 0.8 microl/min (P < 0.05) in control. In conclusion, despite marked effects on peritoneal solute transport and on UF, conceivably resulting from vasodilatation and increases in capillary permeability, zymosan-induced peritonitis did not cause any acute increases in direct peritoneal lymphatic absorption. (+info)
Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma.
(19/1146)
Post-traumatic cystic cavitation, in which the size and severity of a CNS injury progress from a small area of direct trauma to a greatly enlarged secondary injury surrounded by glial scar tissue, is a poorly understood complication of damage to the brain and spinal cord. Using minimally invasive techniques to avoid primary physical injury, this study demonstrates in vivo that inflammatory processes alone initiate a cascade of secondary tissue damage, progressive cavitation, and glial scarring in the CNS. An in vitro model allowed us to test the hypothesis that specific molecules that stimulate macrophage inflammatory activation are an important step in initiating secondary neuropathology. Time-lapse video analyses of inflammation-induced cavitation in our in vitro model revealed that this process occurs primarily via a previously undescribed cellular mechanism involving dramatic astrocyte morphological changes and rapid migration. The physical process of cavitation leads to astrocyte abandonment of neuronal processes, neurite stretching, and secondary injury. The macrophage mannose receptor and the complement receptor type 3 beta2-integrin are implicated in the cascade that induces cavity and scar formation. We also demonstrate that anti-inflammatory agents modulating transcription via the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma may be therapeutic in preventing progressive cavitation by limiting inflammation and subsequent secondary damage after CNS injury. (+info)
Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils.
(20/1146)
Leukocyte chemoattractants are known to stimulate signaling pathways that involve Rho family GTPases. Direct evidence for the regulation of the leukocyte cytoskeleton by Rho GTPases and their effector targets is limited. The p21-activated kinases (PAKs) are specific targets of activated GTP-bound Rac and Cdc42, and have been proposed as regulators of chemoattractant-driven actin cytoskeletal changes in fibroblasts. PAK1 colocalizes with F-actin to cortical actin structures in stimulated fibroblasts, and activated PAK1 mutants induce membrane ruffling and polarized cytoskeletal rearrangements. We investigated whether PAK1 was associated with remodeling of the actin cytoskeleton in activated human neutrophils. We monitored the redistribution of PAK1 and F-actin into the actin cytoskeleton after stimulation of human neutrophils with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) or the particulate stimulus, opsonized zymosan (OZ). PAK1 exhibited a similar distribution as F-actin in fMLP-stimulated leukocytes, localizing in membrane ruffles and to lamellipodia at the leading edge of polarized cells. Addition of OZ induced phagocytic uptake of this particulate stimulus, and PAK1 re-localized to the F-actin-rich pseudopodia and phagocytic cups associated with this process. Once the OZ was internalized, there was little PAK1 localized around the ingested particles, suggesting that PAK1 may be regulating the cytoskeletal extensions and events required for engulfment of bacteria, but not the subsequent steps of internalization. Localization of PAK1 and F-actin in cytoskeletal structures was abolished by the actin polymerization inhibitor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin. Our data suggest that PAK1 may regulate a subset of cytoskeletal dynamics initiated by chemoattractant and phagocytic stimuli in human neutrophils. (+info)
Acute in vivo toxicity of heat-sterilized glucose peritoneal dialysis fluids to rat peritoneal macrophages.
(21/1146)
OBJECTIVE: To evaluate the in vivo effects of heat-sterilized peritoneal dialysis (PD) fluids on the respiratory burst response of rat peritoneal leukocytes. DESIGN: Rats were exposed to intraperitoneal injections of a laboratory-made PD fluid that was either heat-sterilized (H-PD) or filtered (F-PD). Control groups of animals were given Hank's buffer (HBSS) or saline (NaCl). Leukocytes were harvested by intraperitoneal lavage at different times in different animals and analyzed with respect to cell numbers, differential counts, and production of superoxide (chemiluminescence) in response to opsonized zymosan. The chemiluminescence responses of the macrophage and the neutrophil populations, respectively, were obtained by curve-fitting techniques from the responses of the mixed populations. RESULTS: All fluids induced a recruitment of neutrophils, the PD fluids causing a cell number increase that was more transient than that caused by NaCl and HBSS. Macrophage numbers were only slightly influenced, but were generally higher after NaCl and HBSS injections than after PD fluid injections. The H-PD exposure induced a significant inhibition of the macrophage chemiluminescence response after 2 and 12 hours, compared with the exposure to F-PD. The neutrophil chemiluminescence response was not significantly affected. CONCLUSION: The toxins produced by heat-sterilization of glucose-containing PD fluids inhibit in vivo the respiratory burst response of peritoneal macrophages. (+info)
Neutrophil chemotaxis on silicone and polyurethane surfaces.
(22/1146)
Silicone vascular catheters have a greater risk of infection and produce greater inflammation in vivo and greater complement activation in vitro than other vascular catheter polymer materials. This study investigated whether polymorphonuclear leukocyte (PMNL) chemotaxis under agarose on silicone surfaces is different than on polyurethane (PU). Glass slides were coated with silicone and PU by use of a constant-speed dipping apparatus. Chemotaxis (3 h) in response to (10-7 mL) FMLP, zymosan-activated serum, and fresh serum (100%) was greater on silicone than on PU (P<.05). Polyclonal antibody to C5a blocked >50% of the movement toward serum (P<.05). Serum in the PMNL well significantly decreased chemotaxis toward FMLP on silicone (P<.05) but not on PU. These findings suggest that excessive complement activation by silicone may interfere with chemotaxis, but further work is necessary to determine whether this is relevant to an increased risk of catheter-related infection. (+info)
IL-13 induces serine phosphorylation of cPLA2 in mouse peritoneal macrophages leading to arachidonic acid and PGE2 production and blocks the zymosan-induced serine phosphorylation of cPLA2 and eicosanoid production.
(23/1146)
In a recent investigation, we demonstrated that long-term treatment of macrophages with IL-13 enhances cPLA2 expression and modulates zymosan-stimulated AA mobilization. In the present study, we examine the ability of IL-13 to modify the cPLA2 activity and the AA mobilization of macrophages after a short-period of treatment. We demonstrate that in resting macrophages, IL-13 induces, through a MAP kinase-dependent process, (1) an increase of free AA release within 15 min, followed by increased PGE2 production and (2) a time-dependent serine phosphorylation of cPLA2. Conversely, in macrophages stimulated by zymosan, IL-13 added 30 min before zymosan inhibited the AA release and the serine phosphorylation of cPLA2 induced by the phagocytic agonist. In conclusion, these findings show for the first time that a Th2-type cytokine can upregulate cPLA2 activity and downregulate zymosan-induced AA metabolism. Thus, establishment of the connection between these two events may help to understand the complex regulatory role of IL-13 on the macrophage AA metabolism. (+info)
The role of polymorphonuclear leukocytes and oxygen-derived free radicals in experimental acute pancreatitis: mediators of local destruction and activators of inflammation.
(24/1146)
Using a retrograde infusion sodium taurocholate pancreatitis model in the rat treatment with oxygen radical scavengers or monoclonal anti-ICAM-1 antibody decreased tissue damage and polymorphonuclear leukocytes (PMN) infiltration. Scavengers or anti-ICAM-1 treatment attenuated the activating capacity of blood PMNs following zymosan stimulation. The local production of oxygen free radicals in the pancreas by systemic infusion of hypoxanthine and regional infusion of xanthine oxidase did not induce acute pancreatitis, although an increase of infiltrating PMNs was observed. Our data suggest that oxygen free radicals and infiltrating PMNs aggravate acute pancreatitis and that both are important mediators of local destruction and systemic activation of PMNs. (+info)