Majeske AJ et al. (2013), Aggregation of sea urchin phagocytes is augment...

ECB-ART-42856

PLoS One 2013 Apr 16;84:e61419. doi: 10.1371/journal.pone.0061419.

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Aggregation of sea urchin phagocytes is augmented in vitro by lipopolysaccharide.

Majeske AJ , Bayne CJ , Smith LC .

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Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ~10% of the cells were positive for Sp185/333 proteins. At 24 hr, ~90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.

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Genes referenced: 185-333 LOC100887844 LOC100893907 LOC115919910 LOC115925415 LOC590297
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	Figure 1. Viability of phagocytes from the purple sea urchin in short-term cultures.Viability decreases from 91.6 to 57.0% over six days. Standard error of the mean (SEM) are shown.
	Figure 2. Clusters of phagocytes are present in cultures after 1 hr.Phagocytes are labeled for actin (green, A), Sp185/333 (red, B) and DNA (blue, C). Merged images are shown in D and E. Arrows in A indicate the direction of the actin cables for polygonal phagocytes labeled 1 and 2. The large cell with Sp185/333+ perinuclear vesicles (indicated by the arrow in B) is most likely a polygonal phagocyte. Small phagocytes that are not associated with clusters are shown in E. Scale bar is 10 µm.
	Figure 3. Phagocytes aggregate into syncytia-like structures after 3 hr of incubation.Settled phagocytes were incubated for 3 hr in ECCM, fixed and labeled for actin (green, A, E), Sp185/333 (red, B, F) and DNA (blue, C, G). Merged images are shown in D and H. The morphology of different types of phagocytes is no longer recognizable (compare to Figure 2). Sp185/333 proteins (arrow in B, F) within syncytia-like structures are presumably in perinuclear vesicles, as described previously [24]. Scale bars are 10 µm.
	Figure 4. Syncytia-like structures are present after 5 hr of incubation.Settled phagocytes were processed for immunocytology and stained and labeled as in figure 3. Structures contain clustered nuclei associated with Sp185/333 proteins. Arrows in B indicate Sp185/333 proteins (red) in syncytia-like structures. In C, a group of tightly packed nuclei is circled, which are associated with Sp185/333 proteins (D, merged). Scale bar is 10 µm.
	Figure 5. A syncytium-like structure contains both tightly packed and more evenly dispersed nuclei at 24 hr.Settled phagocytes were processed for immunocytology and stained as in Figure 3. Merged images are shown in D and H. Arrows in A mark the direction of actin cables that cross the structure. Sp185/333 proteins (red) associated with syncytia-like structures are present in the center of B. Tightly packed nuclei (smaller circle in C) and more evenly dispersed nuclei (larger circle in C) are both present within a syncytium-like structure. Sp185/333 proteins are associated with the clustered nuclei in a syncytium-like structure (E–H). Scale bars are 10 µm.
	Figure 6. Phagocyte aggregation rate increases with exposure to LPS in culture.Settled phagocytes from Iq animal #2 were incubated with 0 (A), 10 µg (B), or 100 µg (C) LPS/ml ECCM and the number of cells within aggregates was compared to non-aggregated cells. D. All results are shown together for direct comparisons. The dashed lines represent cells that are not incorporated into syncytia-like structures and the solid lines represent the number of nuclei incorporated per syncytium-like structure. There are significantly more aggregated phagocytes than non-aggregated phagocytes after 3 hr and 24 hr of exposure to 10 (B) or 100 (C) µg LPS/ml compared to shorter incubation times (asterisks; P<0.05). Lines marked in D with the same number (1 or 2) are not significantly different. SEM are shown.
	Figure 7. The proportion of nuclei associated with Sp185/333 proteins in cultured phagocytes increases after exposure to LPS.Phagocytes from Iq animals (n = 4) were incubated with 0 µg (A), 10 µg (B), 50 µg (C), or 100 µg (D) LPS/ml in ECCM over time. The percentage of nuclei associated with Sp185/333 proteins is significantly higher after incubation with 100 µg LPS/ml (D) compared to 0 (A), 10 (B) or 50 (C) µg LPS/ml (P<0.05). There is a significant increase in the percentages of nuclei associated with Sp185/333 proteins when cultures are incubated with LPS (all concentrations) for ON to 48 hr compared to cultures incubated for shorter periods of up to 3 hr (brackets with asterisks; P<0.05). ON indicates 16–21 hr. SEM are shown.
	Figure 8. The proportion of nuclei associated with Sp185/333 proteins in cultured phagocytes from Ch animals increases after in vitro exposure to LPS.Phagocytes from Ch animals (n = 3) were incubated with 0 (A), 10 (B), 50 (C) or 100 (D) µg LPS/ml ECCM over time. There is a significant increase in the percentage of nuclei associated with Sp185/333 proteins when exposed to 100 µg/ml LPS (D) compared to 0 µg/ml LPS (A; P<0.05; asterisks) regardless of incubation time. SEM are shown.
	Figure 9. The proportion of nuclei associated with Sp185/333 proteins in cultured phagocytes from N-Ac animals does not change after exposure to LPS in vitro.Phagocytes from N-Ac animals (n = 2) were incubated with 0 µg LPS/ml (A), 10 µg LPS/ml (B), 50 µg LPS/ml (C), or 100 µg LPS/ml (D) in ECCM. There are no significant differences among cultures treated with or without LPS. SEM are shown.
	Figure 10. The proportion of nuclei associated with Sp185/333 proteins in cultured phagocytes from Iq, Ch and N-Ac animals is variable when coelomocytes are exposed to LPS.The data in Figures 7–9 are presented together for direct comparisons. Phagocytes were exposed to 0 (A), 10 (B), 50 (C) or 100 µg LPS/ml (D). Significant differences are indicated by numbers (1 or 2; P<0.05) associated with the data points. Lines marked with the same number are not significantly different. SEM are shown.
	Figure 11. The percentage of nuclei associated with Sp185/333 proteins increases when phagocytes are exposed to LPS in culture.Coelomocytes from Iq animal #2 were settled for 1 hr onto culture well plates and incubated with 0 µg (A) 10 µg (B) or 100 µg (C) LPS/ml ECCM for different times. D shows combined data from A, B and C. There is a significant increase in the proportion of nuclei associated with Sp185/333 proteins within syncytia-like structures vs. non-aggregated Sp185/333+ phagocytes after 3 h and 24 hr exposure to 10 (B) or 100 (C) µg LPS/ml, compared to results for 1 hr (asterisks; P<0.05). Significant differences are shown in D where data points are marked with a different number (1 or 2). Lines in D with the same number are not significantly different. SEM are shown.

References [+] :

BOOLOOTIAN, Clotting of echinoderm coelomic fluid. 1959, Pubmed, Echinobase