Katherine B. Pryzwansky

Early degranulation of human neutrophils: Immunocytochemical studies of surface and intracellular phagocytic events

Abstract Degranulation of azurophil and specific granules after phagocytic challenge with E. coli for 5 sec to 10 min was investigated in the human polymorphonuclear neutrophil (PMN). PMN were stained simultaneously with fluorescein and rhodamine-labeled monospecific antisera to myeloperoxidase (MPO) and lactoferrin (LF) to identify azurophil and specific granules, respectively, within single cells. Fixation was designed to preserve or disrupt differential permeability of cell membrane to fluorescent conjugates in order to study granule translocation. Within 5 sec after phagocytic challenge, MPO and LF appeared on the cell surface coating the bacteria as granule contents leaked from the incompletely formed phagolysosomes. The phagocytic cup, shown by scanning electron microscopy as large and circular, appeared by immunofluorescent markers to be outlined by curvilinear staining for both granule markers, and was always coincident with bacterial localization. MPO and LF appeared singly or simultaneously on the cell surface, suggesting that degranulation to the surface was random. Sequential phagocytic events were demonstrated by comparing staining intensities for each granule marker on the surface and intracellularly within single cells. LF sometimes appeared on the cell surface independent of the nascent phagosome, suggesting that perturbation of the cell membrane by bacteria may cause some specific granule extrusion not limited to the phagosome. These results imply that bacteria make contact with granule-associated anti-microbial substances within 5 sec after phagocytosis is initiated and that free communication of granule constituents occurs between the newly forming phagolysosome and the extracellular space.

Localization of cyclic GMP-dependent protein kinase in human mononuclear phagocytes

The presence and physiological role of cGMP‐dependent protein kinase (G‐kinase) was investigated in human mononuclear phagocytes. Western blots of mono cyte extracts revealed a single polypeptide band that comigrated with purified bovine lung G‐kinase. G‐kinase was localized by immunofluorescence microscopy in freshly isolated adherent human monocytes, monocyte‐derived macrophages cultured from 4 to 14 days, and al veolar macrophages. In monocytes, G‐kinase was local ized in granules or vesicles in the cytoplasm, at the microtubule organizing center, on filaments, and in the nucleus. In monocyte‐derived macrophages, intense staining for G‐kinase was found in the vicinity of the Golgi, in vesicles throughout the cytoplasm, and diffusely in the nucleus. Dual‐label confocal laser scanning microscopy demonstrated that G‐kinase was colocalized with the endoplasmic reticulum. For comparison, G‐kinase was localized in alveolar macrophages that were adhered from 3 to 30 min. In these cells, G‐kinase was prominent within the organelle‐rich area pericortical to the nucleus. However, a well‐defined area of intense staining was also observed at the cell periphery at early time points during adherence and spreading. Rhodamine‐labeled phalloidin showed that this peripheral area was rich in F‐actin. Cytochalasin D, but not nocodazole, in hibited G‐kinase targeting to the cell margin. Further more, the guanylate cyclase inhibitor LY83583 inhibited alveolar macrophage spreading and staining for G‐kinase at the cell periphery. These data suggest that G‐kinase may play an important role in cGMP‐mediated regulation involved in protein processing and cell motility. J. Leukoc. Biol. 57: 670–678; 1995.

The Vasodilator-Stimulated Phosphoprotein Is Regulated by Cyclic GMP-Dependent Protein Kinase During Neutrophil Spreading

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.

Compartmentalization of PDE-4 and cAMP-dependent protein kinase in neutrophils and macrophages during phagocytosis.

The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils, and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D, and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4 at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization.

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice.

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI−/−) mice. We demonstrate that murine neutrophils expressed cGKIα. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI−/− bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI−/− than in WT. Enhanced chemotaxis was also observed with cGKI−/− peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI−/− vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI−/− than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI−/− (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI−/− (∼60%) than in WT (∼90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIα down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIα on the secretagogue responses are complex, depending on the granule subtype.

Scanning electron microscopic observations of early stages of phagocytosis of E. coli by human neutrophils

SummaryChanges in surface morphology, as observed by scanning electron microscopy, appear rapidly when human polymorphonuclear neutrophils (PMN) are challenged with bacteria. Monolayers of PMN adhering to glass were incubated with opsonized E. coli from 5 sec to 10 min, and then fixed and prepared for SEM. As early as 5 sec after phagocytic challenge, E. coli are found in contact with PMN and in the process of engulfment into open cavities formed by lamellipodia. The shape of the mouth of the forming phagocytic vacuole is related to the orientation of bacteria during entry. Bacteria engulfed into early forming phagosomes are surrounded by a large open space between the bacteria and the phagosome wall. As phagocytosis proceeds, the space is reduced and the loose fit around the entering bacteria becomes tight. By 30 sec, bacteria may be completely internalized and by 1 min phagocytized E. coli are packed into bulging PMN. The observations reveal the variability and rapidity of the phagocytic response and confirm the presence of sensitive mechanisms for host defense by PMN.

Control of glucan-induced systemic granulomatosis by cyclosporine A

This study has shown that cyclosporine A (CyA), under certain conditions, is a powerful inhibitor of intravascular and extravascular monocyte/macrophage accumulation. Experiments were carried out in Lewis rats in which intravenous injection of particulate glucan calls forth a striking granulomatous response in lung, liver, and spleen and produces a marked stimulation of splenic erythro- and myelopoiesis. In agreement with the results of others, there was also a considerable elevation in monocyte/macrophage chemoattractant levels in the bronchoalveolar lavage fluid, which is held to be a key reaction in the pathogenesis of the histologic lesions. Treatment of the animals with subcutaneous injections of CyA prevented the rise in the chemoattractant activity and suppressed the granulomatous organ infiltration as well as the splenic hemopoiesis. The findings supply new insights into the activities of CyA and would support its clinical use in macrophage-dominated diseases.

Phagocytosis of zymosan by human neutrophils

A rapid cellular response occurs when human neutrophils are challenged with the zymosan particle. Monolayers of neutrophils adhering to glass were incubated with opsonized zymosan at 37°C for 15 sec to 3 min, fixed and prepared for observation by SEM, immunofluorescence or TEM. At 15 sec zymosan particles are seen by SEM in contact with the cell surface and by 30 sec partial engulfment into open cavities was apparent. The cavity, into which a zymosan particle is engulfed, forms by extension of lamellipodia around and over the particle. The zymosan particle is sequestered in an intracellular vacuole or phagosome by 1 min. At 3 min neutrophils often show bulging profiles of the internalized zymosan and a loss of surface projections. During early phagocytosis, simultaneous staining with contrasting fluorescent antisera to granule constitutents, myeloperoxidase and lactoferrin, reveals granule antigens within the cavity of the forming phagosome. Sections through the cells examined by TEM after 1–3 min incubation with zymosan show granule contents in the phagosome peripheral space which retains connection to the extracellular environment.