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Neutrophils isolated from the blood were compared to those from inflammatory exudates in the peritoneal cavity of guinea pigs. Inflammatory neutrophils were shown to have 10-fold more glycogen than blood neutrophils. This was also reflected in the morphology of these cells. The large accumulations of glycogen in inflammatory neutrophils exists in ordered arrays of beta-granules. Other morphological changes including accumulations of lipid droplets and a decrease in the number of lysosomal granules also accompany the change from blood neutrophils to inflammatory neutrophils. These results show that there are major metabolic differences in the two types of neutrophils.

A mouse cell line (LM), which grows predominantly as spindle-shaped cells with numerous filopodia, was employed in this study. These filopodial projections appear to be important as sites of attachment to the substratum in LM cells. Morphologically the filopodia are slender projections from the cell body which usually attach to the substrate at their distal ends (filopodial footpads). Freeze-fracture of monolayer cultures in situ preserves the spatial relationship of filopodial processes to that of the cell body. Examination of these freeze-fracture preparations reveals a striking difference in the density of intramembrane particles (IMP) in the filopodial-footpad plasmalemma compared with the plasmalemma of the cell body (number of IMP in footpad > cell body). Additionally, there is a marked difference in the number of filipin-sterol complexes on the cell body, compared with the filopodial footpad, implying a difference in the cholesterol content in these regions (filipin-sterol complexes in footpad < cell body). These data suggest a structural and functional specialization of the filopodial-footpad plasma membrane which may be related to cell adhesion.

The distribution of intramembrane particles (IMP) as revealed by freeze-fracture electron microscopy has been analyzed following treatment of mouse L cells and fusion-deficient L cell derivatives with several concentrations of polyethylene glycol (PEG). In cell cultures treated with concentrations of PEG below the critical level for fusion, no aggregation of IMP was observed. When confluent cultures of the parental cells are treated with 50% PEG, greater than 90% of the cells fuse, and cold-induced IMP aggregation is extensive. In contrast, identical treatment of fusion-deficient cell lines shows neither extensive fusion nor IMP redistribution. At higher concentrations of PEG, however, the PEG-resistant cells fuse extensively and IMP aggregation is evident. Thus the decreased ability of the fusion-deficient cells to fuse after treatment with PEG is correlated with the failure of IMP aggregation to occur. A technique for quantifying particle distribution was developed that is practical for the accurate analysis of a large number of micrographs. The variance from the mean number of particles in randomly chosen areas of fixed size was calculated for each cell line at each concentration of PEG. Statistical analysis confirms visual observation of highly aggregated IMP, and allows detection of low levels of aggregation in parental cells that were less extensively fused by exposure to lower concentrations of PEG. When low levels of fusion were induced in fusion-deficient cells, however, no IMP aggregation could be detected.

Unusual tubular structures have been observed in rat aortic smooth muscle cells (SMC) grown in culture. These tubular structures have several characteristics that strongly suggest that they are lysosomes: they are bounded by a single membrane bilayer, contain densely staining material, and acid phosphatase activity. Furthermore, these structures are present in living cells, as demonstrated by their ability to accumulate the membrane-impermeable fluorescent dye lucifer yellow CH. In ultrastructural preparations they are best seen in samples that are cryofixed by rapid freezing and then freeze-substituted in osmium-acetone solutions. Conventional chemical fixation did not appear to preserve these structures to as great an extent as did rapid freezing. Comparison of SMC in vitro to the same cells in situ revealed differences in lysosome number as well as morphological appearance. Thus, the culturing of rat SMC leads to the formation of unusual tubular lysosomes whose ultrastructural appearance is particularly sensitive to the methods employed for examination.

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'-Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.

The ability of phorbol derivatives to function as stimulating agents for superoxide (O2-) release by guinea pig neutrophils has been evaluated and compared to the known ability of each compound to activate protein kinase C. Those that activate the kinase also stimulate O2- release, while those that are inactive with respect to the kinase have no effect on O2- release. The same correlation was observed with respect to the ability of phorbol esters to induce morphological changes in neutrophils, i.e., vesiculation and reduction in granule content. Certain phenothiazines and naphthalene sulfonamides that are known antagonists of calcium-binding proteins blocked both phorbol ester-induced O2- release and morphological changes in these cells.

Neutrophils undergo rapid morphological changes as well as metabolic perturbations when stimulated with certain phorbol esters. Stimulated cells initially exhibit pronounced projections emanating from the cell bodies, followed by rounding of the cells, reduction in granule number, and the appearance of intracellular vesicles. We show these vesicles to be derived, at least in part, from the plasmalemma. The experimental approach involved labeling stimulated and unstimulated cells with native ferritin and cationized ferritin, along with the cytochemical localization of ecto-5'-nucleotidase. The labeling patterns of the vesicles indicate that these structures are involved in both phorbol ester-stimulated adsorptive and fluid-phase endocytosis. Neutrophils stimulated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) exhibit two distinct rates of superoxide release in which the second, prolonged level is approximately 50% of the initial rate. All-trans-retinal, which we have recently shown to stimulate O2- release but not granule exocytosis or cell vesiculation, induces a single prolonged rate of maximal O2- release. Neutrophils treated with both all-trans-retinal and TPA exhibit only a single sustained rate of maximal O2- release similar to that observed with all-trans-retinal alone. Moreover, treatment of cells with all-trans-retinal blocks the vesiculation of neutrophils induced by TPA in a dose-dependent manner. This observation provides a possible explanation for the differences in the kinetics of superoxide release.

Human neutrophils contain a novel intracellular compartment that is distinct from the previously characterized azurophil and specific granules. This compartment is distinguished by the presence of cytochemically detectable alkaline phosphatase activity. The alkaline phosphatase-containing compartments are short rod-shaped organelles that rapidly undergo a dramatic reorganization upon cell stimulation with either a chemoattractant or an active phorbol ester. Biochemical analysis shows that in unstimulated neutrophils the majority of the alkaline phosphatase activity is intracellular, but after stimulation essentially all of this activity becomes associated with the cell surface. The exocytotic pathway is unusual in that these small organelles fuse to form elongated tubular structures before their association with the plasmalemma.