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First published online May 13, 2004
Journal of Experimental Biology 207, 2147-2155 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01001

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The sea urchin complement homologue, SpC3, functions as an opsonin

Lori A. Clow^1,*,, David A. Raftos^3,*, Paul S. Gross⁴ and L. Courtney Smith^1,2,

¹ Graduate Program in Genetics, Institute of Biomedical Sciences, George Washington University, Washington DC, USA
² Department of Biological Sciences, George Washington University, Washington DC, USA
³ Department of Biological Sciences, Macquarie University, Sydney, Australia
⁴ Department of Biochemistry, Medical University of South Carolina, Charleston SC, USA

View larger version (17K): [in a new window]   Fig. 1. Phagocytic activities (yeast phagocytosed per 100 coelomocytes) of coelomocytes harvested from sea urchins at various times before and after the injection of lipopolysaccharide (LPS) or artificial seawater (ASW). Arrows show the days on which sea urchins were injected. Prior to phagocytosis, yeast were incubated for 40 min in either ASW (non-opsonized) or coelomic fluid (CF; opsonized) taken from the same sea urchin that donated coelomocytes. Phagocytosis was allowed to proceed for 45 min.Values are means ± S.E.M.(N3).

View larger version (10K): [in a new window]   Fig. 2. Opsonic activities of different concentrations of coelomic fluid (CF). Sea urchins received two injections of LPS at 2-day intervals and CF was collected 3 days after the second LPS injection and diluted with ASW (% CF) before being used to opsonize yeast for 40 min. Phagocytosis was allowed to proceed for 45 min. PSI, phagocytic stimulation index (see Materials and methods). Values are means ± S.E.M.(N=8).

View larger version (12K): [in a new window]   Fig. 3. SpC3 concentrations in coelomic fluid (CF) collected from sea urchins at various times before and after the injection of LPS or ASW. Data are shown as relative intensities of SpC3 bands determined by western blotting and densitometry. Arrows indicate the days on which animals received injections of either LPS or ASW. Values are means ± S.E.M.(N4).

View larger version (15K): [in a new window]   Fig. 4. Opsonic activities of coelomic fluid (CF; number of yeast phagocytosed per 100 coelomocytes) plotted against the SpC3 concentration of CF (relative intensities) collected from the same sea urchin. Sea urchins (N=18) were injected with either LPS or ASW on day 0 and day 4. Coelomic fluid was collected on day –1, day 1 or day 5. SpC3 titers were determined by densitometric analysis of western blots on which SpC3 was stained with anti-SpC3-6H. Values are means ± S.E.M.(N=3).

View larger version (109K): [in a new window]   Fig. 5. Polygonal phagocytes take up opsonized yeast. Confocal microscopic image of S. purpuratus coelomocytes that had been incubated for 45 min with CF-opsonized yeast. Yeast were stained with RITC (red) and SpC3 was detected with anti-SpC3'pep and GRIg-A (green). pp, polygonal phagocyte; dp, discoidal phagocyte. Scale bar, 50 µm.

View larger version (12K): [in a new window]   Fig. 6. Opsonic activities are influenced by SpC3 and blocked by anti-SpC3-6H. Coelomic fluid (CF) was collected from sea urchins that had received two injections at 2-day intervals of LPS and was harvested 1 day after the second LPS injection. Yeast were opsonized for 40 min and phagocytosis was allowed to proceed for 45 min. Yeast were opsonized with either CF (yeast+CF), CF incubated for 2 h with anti-SpC3-6H (1:20 dilution; yeast+CF+-SpC3-6H), CF incubated for 2 h with anti-profilin (1:20 dilution; yeast+CF+-profilin) or ASW (yeast+ASW). Some of the yeast that had been opsonized with CF+anti-SpC3-6H were acid-washed prior to use in phagocytosis (yeast+CF+-SpC3-6H washed). PSI, phagocytic stimulation index. Values are means ± S.E.M. (N=6). *P<0.05, yeast+CF+-SpC3-6H vs. yeast+CF.

View larger version (12K): [in a new window]   Fig. 7. SpC3 binds to yeast surfaces. Coelomic fluid was obtained from sea urchins that had received two injections of LPS at 2-day intervals and was collected 2 days after the second injection. Yeast were incubated with ASW or CF, and SpC3 was detected on the yeast surface with anti-SpC3'pep (primary; 1°) and G RIgAP (secondary; 2°) followed by substrate incubation in 4-nitrophenol phosphate prior to absorbance readings at 415 nm. Yeast+ASW, yeast were incubated with ASW but antibodies were omitted prior to analysis; yeast+CF, yeast were opsonized with CF but antibodies were omitted prior to analysis; yeast+CF+2°, yeast were opsonized with CF but only the 2° antibody was used prior to analysis; yeast+CF+1°+2°, yeast were opsonized with CF and incubated with 1° and 2° antibodies prior to analysis; yeast+1°+2°, yeast were incubated with ASW and 1° and 2° antibodies were added prior to analysis. Values are means ± S.E.M.(N=3).