Phagocytic capacity of cytokineplasts from human blood polymorphonuclear leukocytes

Cytokineplasts (CKP) are membrane-bounded anucleate cytoplasmic fragments, induced from polymorphonuclear leukocytes (PMN) by the brief application of heat; derived from the cortical cytoplasm that gathers at the leading front of migrating PMN; and endowed with many of the motile properties of the parent cell. In this study we examine their phagocytic capacity by quantitative methods. CKP ingest Staphylococcus aureus and Serratia marcescens somewhat less avidly than do the corresponding intact PMN, yet rather impressively when one considers how restricted a portion of the parent cell they represent. Under the conditions employed, CKP killed about half as many of the bacteria presented to them as did their parent PMN. Thus, despite a heat-associated loss of demonstrable respiratory burst oxidase activity and a paucity of cytoplasmic granules, the organelle-depleted CKP deals with bacteria in a way that mimics its parent PMN.     

Phagocytic leukocytes and reactive oxygen species

Phagocytic leukocytes, when appropriately stimulated, display a respiratory burst in which they consume oxygen and produce superoxide anions. Superoxide is produced by the phagocyte NADPH-oxidase system which is a multiprotein complex that is dissociated in quiescent cells and is assembled into the functional oxidase following stimulation of these cells. Also associated with the respiratory burst is the generation of other reactive oxygen species. The identity of components of the NADPH-oxidase system and their interactions are known in considerable molecular detail. Understanding of the regulation of superoxide production is less well known. This review also points out the important role of microscopy in complementing biochemical studies to understand better the cell biology of the phagocyte respiratory burst. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Phagocytic labeling of leukocytes with 99mTc-albumin colloid for nuclear imaging

A procedure is described for the phagocytic labeling of white blood cells (WBC) with high specific activity ^99mTc-albumin colloid (TAC). The preparation contains approximately equal activities of granulocytes and monocytes. Heparinized whole blood (40 cm³) yields a preparation containing a total of 148–222 MBq (4–6 mCi) TAC-WBC including about 20% free TAC. The complete preparation time is 75 min. Imaging is completed 30 min to 4 h post administration of the TAC-WBC. Quality control methods and imaging protocols are described.     

Down-regulation of proinflammatory capacity during apoptosis in human polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMNs) are essential to innate immunity in humans and contribute significantly to inflammation. Although progress has been made, the molecular basis for termination of inflammation in humans is incompletely characterized. We used human oligonucleotide microarrays to identify genes encoding inflammatory mediators that were differentially regulated during the induction of apoptosis. One hundred thirty-three of 212 differentially expressed genes encoding proinflammatory factors, signal transduction mediators, adhesion molecules, and other proteins that facilitate the inflammatory response were down-regulated during the induction of apoptosis following PMN phagocytosis. Among these, 42 genes encoded proteins critical to the inflammatory response, including receptors for IL-8 beta, IL-10 alpha, IL-13 alpha 1, IL-15 alpha, IL-17, IL-18, C1q, low-density lipoprotein, IgG Fc (CD32), and formyl peptide, Toll-like receptor 6, platelet/endothelial cell adhesion molecule-1 (CD31), P-selectin (CD62), IL-1 alpha, IL-16, and granulocyte chemoattractant protein-2 were down-regulated. Many of these genes were similarly down-regulated during Fas-mediated or camptothecin-induced apoptosis. We used flow cytometry to confirm that IL-8R beta (CXCR2) and IL-1 alpha were significantly down-regulated during PMN apoptosis. We also discovered that 23 genes encoding phosphoinositide and calcium-mediated signal transduction components, which comprise complex pathways essential to the inflammatory response of host cells, were differentially regulated during PMN apoptosis. Importantly, our data demonstrate that PMNs down-regulate proinflammatory capacity at the level of gene expression during induction of apoptosis. These findings provide new insight into the molecular events that resolve inflammation following PMN activation in humans.     

Phagocytic and bactericidal activities of leukocytes in whole blood from atomic bomb survivors

This study evaluated the phagocytic and bactericidal activities of peripheral blood leukocytes from Hiroshima and Nagasaki atomic bomb survivors for Staphylococcus aureus. The data were analyzed by multiple linear regression for age, sex, radiation exposure, city of exposure, and neutrophil counts. No significant radiation effect was observed for either blood phagocytic or bactericidal activities. The only significant variable for these functions was the neutrophil count.     

Phagocytic capacity of invasive malignant cells in three-dimensional culture

To study the phagocytic capacity of invasive malignant cells, fragments of the hypoblast from chick blastoderms were confronted in three-dimensional culture with spheroidal aggregates of 1) malignant virally transformed C3H mouse cells (MO4), 2) HeLa cells and 3) embryonic chick heart cells. The hypoblast was used because it contains yolk, a marker that is absent in the confronting cells and that can be identified histologically and ultrastructurally. The confronting tissues were incubated on semi-solid agar-agar medium or in fluid medium on a gyrotory shaker. Cultures were followed for 1 to 7 days by stereomicroscopy, cinemicrophotography, light and transmission electron microscopy. Confrontation with MO4 cells of HeLa cells, known to be invasive in vitro, led to complete disappearance of the hypoblast. The fragments of hypoblast were well conserved when cultured alone or confronted with aggregates of chick heart cells. Degeneration of the hypoblast is shown at the area of contact with MO4-cell or HeLa-cell aggregates, in contrast to heart cells. Filopodia-like extensions from the MO4 or HeLa cells penetrate intercellularly, transcellularly and intracellularly into the hypoblast. Phagosomes, containing yolk and unidentified debris are observed in MO4 cells and in HeLa cells, but not in heart cells. These observations demonstrate the phagocytic capacity of invasive malignant cells.