Regulation of phagocyte migration and recruitment by Src-family kinases.

Src-family kinases (SFKs) regulate different granulocyte and monocyte/macrophage responses. Accumulating evidence suggests that members of this family are implicated in signal transduction pathways regulating phagocytic cell migration and recruitment into inflammatory sites. Macrophages with a genetic deficiency of SFKs display marked alterations in cytoskeleton dynamics, polarization and migration. This same phenotype is found in cells with either a lack of SFK substrates and/or interacting proteins such as Pyk2/FAK, c-Cbl and p190RhoGAP. Notably, SFKs and their downstream targets also regulate monocyte recruitment into inflammatory sites. Depending on the type of assay used, neutrophil migration in vitro may be either dependent on or independent of SFKs. Also neutrophil recruitment in in vivo models of inflammation may be regulated differently by SFKs depending on the tissue involved. In this review we will discuss possible mechanisms by which SFKs may regulate phagocytic cell migratory abilities.

Role of Src kinases and Syk in Fcgamma receptor-mediated phagocytosis and phagosome-lysosome fusion.

Phagocytosis is increased by Fcgamma receptors (FcgammaRs), and studies with syk(-/-) macrophages demonstrated that Syk kinase is required for FcgammaR phagocytosis. Similar studies with macrophages lacking the Src family kinases Hck, Fgr, and Lyn showed that these kinases are not required for phagocytosis but that they enhance the rate of particle engulfment. In this report we show that both wild-type and hck(-/-)fgr(-/-) macrophages expressed Fyn, Src, and Yes and that these kinases were activated on ingestion of immunoglobulin G (IgG)-coated particles and redistributed, together with Syk, to actin-rich phagocytic cups and the phagosomal membrane. At doses blocking IgG-dependent phagocytosis, the tyrosine kinase inhibitors PP1 and piceatannol inhibited both Src family kinase and Syk activities, as well as their redistribution to actin-rich phagocytic cups. Hck, Fgr, and Lyn were dispensable for lysosome-phagosome fusion (PLF) induced by IgG-coated particles. However, PP1 or piceatannol hampered unopsonized yeast-induced PLF despite the fact that they did not block yeast internalization.

Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages.

Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.

Glucose kinetics in interstitial fluid can be predicted by compartmental modeling.

The purposes of this study were to define in sheep a compartmental model for glucose kinetics in the basal condition and to test the hypothesis that interstitial fluid obtained by sampling thoracic duct lymph (TDL) represents one or more peripheral compartments of the glucose model. A bolus of [6,6-(2)H]glucose was injected in nine animals, followed by frequent sampling of blood and TDL. Linear kinetic modeling has been applied to plasma data, indicating that a three-compartment model adequately describes glucose kinetics. Both catenary and mammillary models were identified, and their predictions for the tracer behavior in nonaccessible pools were evaluated. In all experiments, regardless of the model structure, predictions in the compartment most rapidly exchanging with blood (pool 2) well matched the measured tracer-to-tracee ratio in TDL. Furthermore, modeling analysis showed that 90-95% of the tracer in TDL comes from this pool. This supported the physiological identification of a peripheral pool of glucose kinetics as extracellular fluid represented by TDL measurements.

Bicarbonate kinetics in humans: identification and validation of a three-compartment model.

A model of bicarbonate kinetics is crucial to a correct interpretation of experiments for measuring oxidation in vivo of carbon-labeled compounds. The aim of this study is to develop a compartmental model of bicarbonate kinetics in humans from tracer data by devoting particular attention to model identification and validation. The data base consisted of impulse-dose studies of 14C-labeled bicarbonate in nine normal subjects. The decay curve of specific activity of CO2 in expired air (saRCO2) was frequently sampled for 4-7 h. In addition, endogenous production of CO2, VCO2, was measured by indirect calorimetry. A model of data, i.e., an exponential model, analysis of decay curves of saRCO2 showed first that three compartments are necessary and sufficient to describe bicarbonate tracer kinetics. Compartmental models were then used as models of system. To correctly describe the input-output configuration, labeled CO2 flux in the expired air, phi RCO2 (= saRCO2.VCO2), has been used as measurement variable in tracer model identification. A mammillary three-compartment model with a respiratory and a nonrespiratory loss has been studied. Whereas there is good evidence that respiratory loss takes place in the central compartment, whether nonrespiratory loss is taking place in the central compartment or in one of the two peripheral compartments is uncertain. Thus three competing tracer models were considered. Using a model-independent analysis of data, based on the body activity variable, to calculate mean residence time in the system, we have been able to validate a specific model structure, i.e., with the two irreversible losses taking place in the central compartment. This validated tracer model was then used to quantitate bicarbonate masses in the system. Because there is uncertainty about where endogenous production enters the system, lower and upper bounds of masses of bicarbonate in the body are derived.

A study of plasma disposition kinetics of lacidipine after single oral ascending doses.

This study was designed to assess the plasma disposition kinetics of single oral 4-, 6-, and 8-mg doses of lacidipine, a new once-daily calcium-entry blocker. Seventeen healthy volunteers attended the study. In almost all cases, detectable levels were found up to 24 h after the drug administration, using a new HPLC-RIA assay. The usual pharmacokinetic parameters were calculated: Cmax, tmax, AUCinf, lambda z and t1/2. The half-life was similar after all doses, around 7 h, whereas Cmax and AUCinf did not show a linear correlation with the doses.