The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific β- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of/3-glucuronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasmalemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and ~20% less actin than extracts of cells sheared in the absence of nylon wool fibers. Nearly all of the actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change.We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis. 956

The subcellular localization, kinetics of activation, and substrate specificity of the guinea pig granulocyte superoxide (O2-) generating system was investigated. Membrane-enriched particles (podosomes) were made from granulocytes by mild sonication and differential centrifugation. These podosomes are enriched threefold for known plasma membrane markers, 5′-nucleotidase, and adenylate cyclase. Podosomes made from resting granulocytes have very little NAD(P)H-dependent O2- production. Podosomes made from cells stimulated with digitonin are equally enriched for membrane markers but have a 15- to 20-fold increase in NAD(P)H-dependent O2- production. The KmAPP for NADPH is one-tenth that for NADH, but the Vmax is the same. The kinetics of digitonin-stimulated whole-cell O2- production parallel the changes in enzyme activity in these podosomes. Temperature affects both the rate and extent of activation of this enzyme. The pH optimum for the enzyme, the pH optimum for activation, and the pH optimum for whole-cell O2- production are all 7.5. Enzyme activity is increased if the cells are treated with glucose and cyanide, inhibited in cells treated with 2- deoxyglucose (2-DOG), and requires the presence of calcium for activation. These effects are similar to those found for granulocyte O2- production. Thus, the granulocyte O2- generating enzyme system is located on a fraction enriched for plasma membrane markers, and the kinetics of granulocyte production are directly related to the rate and amount of activation of this enzyme.

The effects of UV irradiation on double-stranded PBS-2 phage DNA, which contains uracil in place of thymine, were studied. Irradiation by 254nm light, or by 313nm light in the presence of the triplet sensitizer acetophenone, resulted in production of uracil-containing pyrimidine photodimers. DNA chain breaks were also produced under both conditions. Uracil-DNA glycosylase released free uracil, but no uracil-containing dimers from such irradiated DNA. The rate of enzymatic release of uracil from irradiated PBS-2 DNA was decreased as the number of photodimers per DNA molecule was increased.

A proposed mechanism for nonspecific immunity to Listeria monocytogenes in rats based on the existence of an activatable lysin is described. Using a deoxyribonucleic acid release assay, we found lysin activity in serum made from whole blood but not in serun made from platelet-free plasma. Washed platelets and platelet lysates exhibited only partial activity as compared with that in serum. This activity was amplified by the addition of platelet-free plasma serum. The activity of the lysin was unaffected by heparin, dialysis, a serine esterase inhibitor, or heating to 56°C for 30 min. Effective inhibitors were ethylenediaminetetraacetic acid and stronger heating (to 65°C). Listeria organisms were found to reduce the recalcified clotting time of platelet-rich plasma in a dose-dependent fashion, indicating that the organisms can exhibit procoagulant activity. The susceptibility of rats to Listeria infection was enhanced by anticoagulant treatment. Rats were infected with Listeria organisms with and without administration of heparin. Heparin-treated rats developed bacteremia, and some died. None of the control rats developed bacteremia or died. These results suggest that natural immunity to Listeria infection is partly due to a platelet-dependent lysin which is activated during clotting and is, in turn, promoted by the Listeria organisms themselves.