75° National Congress of Italian Zoological Union

75° National Congress of Italian Zoological Union – Bari, 22 - 25 september 2014
Dipartimento di Scienze e Tecnologie Biologiche,
Istituto Euro Mediterraneo di Scienze e Tecnologie
Chimiche e Farmaceutiche (STEBICEF) - Palermo
(I.E.ME.S.T.) - Palermo
Marco Chiaramonte, Debora Russo, Vincenzo Arizza
The coelomic fluid of the sea urchin Paracentrotus lividus contains numerous
coelomocyte types, including the phagocytes (Ph) and uncoloured
spherulocytes (US), involved in immune defence systems of the animal.
Previously we demonstrated that the Ph were able to engulf foreign cells such
as yeasts, while the US had cytotoxic activity against mammalian
erythrocytes and tumour cells. We already shown that the cytotoxic ability of
the US decreased significantly if they were separated from Ph through a
Iodixinol discontinuous gradient fractionation and the activity was recovered
if, in the cytotoxic reaction, were present Ph, their short culture supernatant
Coelomic fluid
removal
(SCF) or their lysate. This cooperative effect would depend by the molecules
contained in the phagocytes.
In this study, we show that the fractionated US are able to recover the
cytotoxic activity against rabbit erythrocytes when in the reaction mixture
were present exosomes extracted from SCF. Exosomes are vesicles released
from the endosomal system of the cells with a exocytotic mechanism during
the formation of multi-vesicular bodies, and are involved in cytotoxic
mechanisms and in the spreading of cytokines.
Exosome isolation
Cell separation
Iodixinol %
B1: 89.2% Ph; 7.5 vibratile cells; 2.6% US
10
25
30
B2: 84.3% vibratile cells; 8.2% % Ph; 3.9% US; 3.1% red cells
B3: 90.2% US; 6.2% vibratile cells; 5.5% red cells; 1.5% Ph
B4: 91.4% red cells; 3.3% US; 2.0% vibratile cells; 1.1% Ph
Coelomocyte cytotoxic
activity assay against
rabbit erythrocytes (RE)
CELL CULTURE SUPERNATANT
300g – 10 min
To eliminate dead cells
2,000g – 20 min
Plaque forming cells
(PFC) assay against RE
To remove debris
10,000g – 30 min
Ultracentrifugation at 100,000g – 70 min – 4°C
PURIFIED EXOSOMES
Electrophoresis
Densitometric analysis
Western-blot
Plaque forming cells (PFC) assay
Coelomocytes (E:T ratio 1:3)
N. PFC / 5 × 104 cells
Unfractionated
62 ± 3,4
Ph
0
US
5 ± 1,0
Ph + US
63 ± 1,5
Phagocytes release + US
30 ± 2,3
Phagocytes release ads with anti-hrIL-1α + US
6 ± 2,5
Immunoblotting of coelomocytes lysate supernatant (CLS)
of Ph and US with anti-hrIL-1RI
Human IL-1 Receptor - like is present
only in the lysate supernatant of US
180
116
90
The interaction between Ph and US would be due to one
or more soluble factors released by phagocytes
Coelomocyte immunocytochemistry with anti-hrIL-1α
Anti-hrIL-1α
58
48.5
36.5
26.6
Analysis of exosomes isolated by ultracentrifugation
Western blot analysis revealed the
presence of exosome markers
Ph
Coelomocyte cytotoxic activity against rabbit erythrocytes
Control
!
Molecules in the Ph cross-react
with anti-hrIL-1α
Immunoblotting of phagocytes release with anti-hrIL-1α
90
58
48.5
36.5
26.6
Hemolysis degree (%)
Unfractionated
85,9 ± 7,15
Ph
26,8 ± 3,74
US
56,7 ± 5,53***
Exosomes
0
Ph + US
78,2 ± 7,11**
US + Exosomes
69 ± 1,4**
US: *** P < 0.001 compared with Unfractioned
180
116
Coelomocytes (E:T ratio 1:3)
SCF contains a protein component
that cross-reacts with specific
antibodies for anti-hrIL-1α
Ph + US: ** P < 0.01 compared with US
US + Exosomes: ** P < 0.01 compared with US
* Data are expressed as mean ± SD. p - value (* = p < 0.05; ** = p < 0.01; *** = p < 0,001)
are indicators of statistical significance(Student’s t test).

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