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Comparisons of CapG and gelsolin- macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing.

Witke W, Li W, Kwiatkowski DJ, Southwick FS - J. Cell Biol. (2001)

Bottom Line: However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function.These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages.These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a Capg allele engineered into mice. Both CapG- mice and CapG/gelsolin double- mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG- macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

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Phagocytic rates of wild-type and Capg−/− macrophages. (A) Line graph quantitating the number of IgG-opsonized zymosan particles ingested over time. Macrophages were exposed to the opsonized zymosan particles and at the times depicted were cooled to 4°C. Cells were overlaid with Trypan blue to quench extracellular particles and the number of particles inside each cell were counted. Brackets represent the SEM of 90–100 cells counted per time point. The slope of the Capg−/− cells was half that of wild-type cells. (B) Line graph quantitating the number of complement-opsonized zymosan particles ingested over time. Brackets represent the SEM of 100 cells per time point. The slope was reduced by approximately 35% in the Capg−/− cells. Wild-type cells failed to ingest additional particles at 22.5 min (mean particles/cell 8.8 ± 0.3; n =100) Therefore, meaningful comparisons between wild-type and Capg−/− cells could not be made at this time point. (C) Line graph quantitating the number of unopsonized zymosan particles ingested over time. No significant ingestion was observed at 7.5 min. Brackets represent the SEM of 100 cells/time point.
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fig5: Phagocytic rates of wild-type and Capg−/− macrophages. (A) Line graph quantitating the number of IgG-opsonized zymosan particles ingested over time. Macrophages were exposed to the opsonized zymosan particles and at the times depicted were cooled to 4°C. Cells were overlaid with Trypan blue to quench extracellular particles and the number of particles inside each cell were counted. Brackets represent the SEM of 90–100 cells counted per time point. The slope of the Capg−/− cells was half that of wild-type cells. (B) Line graph quantitating the number of complement-opsonized zymosan particles ingested over time. Brackets represent the SEM of 100 cells per time point. The slope was reduced by approximately 35% in the Capg−/− cells. Wild-type cells failed to ingest additional particles at 22.5 min (mean particles/cell 8.8 ± 0.3; n =100) Therefore, meaningful comparisons between wild-type and Capg−/− cells could not be made at this time point. (C) Line graph quantitating the number of unopsonized zymosan particles ingested over time. No significant ingestion was observed at 7.5 min. Brackets represent the SEM of 100 cells/time point.

Mentions: The ability of bone marrow macrophages to ingest complement- and IgG-opsonized, as well as unopsonized fluorescein-labeled zymosan particles was also examined. The rates of IgG-mediated phagocytosis were decreased to ∼1/2 that of wild-type cells (Fig. 5 A, P < 0.0001 at 15 and 22.5 min). Similarly, complement-mediated phagocytosis was decreased, although to a lesser degree (Fig. 5 B, P < 0.0001 at 7.5 and 15 min). Capg−/− macrophages also demonstrated significantly slower rates of ingestion of unopsonized particles compared with wild-type macrophages (P = 0.005 at 15 min and P < 0.0001 at 22.5 min) (Fig. 5 C). The reduction in phagocytic rate of IgG-coated particles could not be accounted for by a difference in particle adherence. The mean number of IgG-opsonized particles attached to CapG- macrophages (0.7 ± 0.1 particles/cell SEM, n = 100 cells) after incubation at 37°C for 22 min was not significantly different than wild-type macrophages (0.9 ± 0.1 particles/cell; n = 100, P = 0.14).


Comparisons of CapG and gelsolin- macrophages: demonstration of a unique role for CapG in receptor-mediated ruffling, phagocytosis, and vesicle rocketing.

Witke W, Li W, Kwiatkowski DJ, Southwick FS - J. Cell Biol. (2001)

Phagocytic rates of wild-type and Capg−/− macrophages. (A) Line graph quantitating the number of IgG-opsonized zymosan particles ingested over time. Macrophages were exposed to the opsonized zymosan particles and at the times depicted were cooled to 4°C. Cells were overlaid with Trypan blue to quench extracellular particles and the number of particles inside each cell were counted. Brackets represent the SEM of 90–100 cells counted per time point. The slope of the Capg−/− cells was half that of wild-type cells. (B) Line graph quantitating the number of complement-opsonized zymosan particles ingested over time. Brackets represent the SEM of 100 cells per time point. The slope was reduced by approximately 35% in the Capg−/− cells. Wild-type cells failed to ingest additional particles at 22.5 min (mean particles/cell 8.8 ± 0.3; n =100) Therefore, meaningful comparisons between wild-type and Capg−/− cells could not be made at this time point. (C) Line graph quantitating the number of unopsonized zymosan particles ingested over time. No significant ingestion was observed at 7.5 min. Brackets represent the SEM of 100 cells/time point.
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Related In: Results  -  Collection

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fig5: Phagocytic rates of wild-type and Capg−/− macrophages. (A) Line graph quantitating the number of IgG-opsonized zymosan particles ingested over time. Macrophages were exposed to the opsonized zymosan particles and at the times depicted were cooled to 4°C. Cells were overlaid with Trypan blue to quench extracellular particles and the number of particles inside each cell were counted. Brackets represent the SEM of 90–100 cells counted per time point. The slope of the Capg−/− cells was half that of wild-type cells. (B) Line graph quantitating the number of complement-opsonized zymosan particles ingested over time. Brackets represent the SEM of 100 cells per time point. The slope was reduced by approximately 35% in the Capg−/− cells. Wild-type cells failed to ingest additional particles at 22.5 min (mean particles/cell 8.8 ± 0.3; n =100) Therefore, meaningful comparisons between wild-type and Capg−/− cells could not be made at this time point. (C) Line graph quantitating the number of unopsonized zymosan particles ingested over time. No significant ingestion was observed at 7.5 min. Brackets represent the SEM of 100 cells/time point.
Mentions: The ability of bone marrow macrophages to ingest complement- and IgG-opsonized, as well as unopsonized fluorescein-labeled zymosan particles was also examined. The rates of IgG-mediated phagocytosis were decreased to ∼1/2 that of wild-type cells (Fig. 5 A, P < 0.0001 at 15 and 22.5 min). Similarly, complement-mediated phagocytosis was decreased, although to a lesser degree (Fig. 5 B, P < 0.0001 at 7.5 and 15 min). Capg−/− macrophages also demonstrated significantly slower rates of ingestion of unopsonized particles compared with wild-type macrophages (P = 0.005 at 15 min and P < 0.0001 at 22.5 min) (Fig. 5 C). The reduction in phagocytic rate of IgG-coated particles could not be accounted for by a difference in particle adherence. The mean number of IgG-opsonized particles attached to CapG- macrophages (0.7 ± 0.1 particles/cell SEM, n = 100 cells) after incubation at 37°C for 22 min was not significantly different than wild-type macrophages (0.9 ± 0.1 particles/cell; n = 100, P = 0.14).

Bottom Line: However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function.These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages.These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

View Article: PubMed Central - PubMed

Affiliation: Hematology Division, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

ABSTRACT
Capping the barbed ends of actin filaments is a critical step for regulating actin-based motility in nonmuscle cells. The in vivo function of CapG, a calcium-sensitive barbed end capping protein and member of the gelsolin/villin family, has been assessed using a Capg allele engineered into mice. Both CapG- mice and CapG/gelsolin double- mice appear normal and have no gross functional abnormalities. However, the loss of CapG in bone marrow macrophages profoundly inhibits macrophage colony stimulating factor-stimulated ruffling; reintroduction of CapG protein by microinjection fully restores this function. CapG- macrophages also demonstrate approximately 50% impairment of immunoglobulin G, and complement-opsonized phagocytosis and lanthanum-induced vesicle rocketing. These motile functions are not impaired in gelsolin- macrophages and no additive effects are observed in CapG/gelsolin double- macrophages, establishing that CapG function is distinct from, and does not overlap with, gelsolin in macrophages. Our observations indicate that CapG is required for receptor-mediated ruffling, and that it is a major functional component of macrophage phagocytosis. These primary effects on macrophage motile function suggest that CapG may be a useful target for the regulation of macrophage-mediated inflammatory responses.

Show MeSH
Related in: MedlinePlus