Labelling of neutrophils and endothelial cells for 24h time lapse imaging.
Endothelial cells were seeded onto 3cm glass-bottomed iWAKI dishes and cultured until
they reached 90% confluence. Following optimisation, endothelial cells were incubated with
calcein-AM (2.5µM) for 1h. Freshly isolated neutrophils were incubated with 100nM
mitotracker red for 15min. Neutrophils were washed and resuspended in microvascular
endothelial cell medium supplemented with 2mM glutamine at 5 x 10 5 cells/ml. Endothelial
cells were washed and medium was replaced with 1.5ml of neutrophil suspension and 500µl
of either control or SSc serum.
24h time lapse movies were taken on a Pascal confocal microscope (Zeiss). FITC and
Mitotracker Red were excited using the 488nm and 543nm lasers respectively. Images were
taken from 6 fields continuously over a period of 24h and processed using the LSM510
software.
Male:Female
1:16
Median disease duration/mths (IQR)
37 (25.5,58)
Median Raynaud’s duration/yrs (IQR)
6.5 (4, 10.5)
Median rodnan skin score (IQR)
6 (4,11)
Clinical feature
Number (%)
Limited subtype
13/17 (76)
Diffuse subtype
4/17 (24)
Lung involvement
6/17 (35)
Pulmonary artery hypertension
1/17(6)
Renal involvement
0/17 (0)
ANA positive
12/17 (71)
Anticentromere positive
6/17 (35)
Anti Scl70 positive
5/17 (29)
Anti RNP positive
3/17 (18)
DMARDs
6/17 (35)
Mycophenolate mofetil
5/17 (29)
Sildenafil
2/17 (12)
Methotrexate
1/17 (6)
Supplementary table 1. Patient characteristics.
Supplementary Fig.1. Apoptosis and E-selectin expression in endothelial cell (A) and
neutrophil (B) single cell cultures after 24h. There was no difference in apoptosis or E-selectin
expression in endothelial cell cultures exposed to control or SSc serum (25%). There was no
difference in neutrophil apoptosis on exposure to SSc or control serum however, there was a
small but significant increase in E-selectin expression (p=0.02).
Supplementary Fig. 2. There was no difference in E-selectin expression in neutrophils following
24h co-culture with endothelial cells with control or SSc serum. Supernatants were removed
from co-cultures of HDMECs and healthy control neutrophils in the presence of 25% control or
SSc serum after 24h. E-selectin expression was measured using an APC-conjugated E-selectin
antibody by flow cytometry. Mean±SD shown n=3.
Median [Cytokine] pg/ml (IQR)
GMCSF
IFNγ
IL1-β
IL2
IL4
IL5
IL6
IL8
IL10
TNFα
GCSF
IL1-RA
IL17
Control serum
0 (0, 23.48)
0 (0)
0 (0, 16.28)
0 (0, 2.0)
0 (0)
4.69 (4.69, 4.69)
9.87 (8.41, 10.24)
42.56 (40.28, 46.72)
4.34 (4.06, 4.62)
0 (0)
0 (0)
404.35 (273.9, 626.79)
5.37 (3.4, 18.10)
SSc serum
2466.11 (1233.05, 2629.08)
0 (0)
309.73 (157.26, 1162.02)
87.54 (43.77, 464.72)
0 (0, 531.30)
9.47 (7.08, 36.89)
171.94 (92.38, 184.27)
66.05 (60.74, 108.33)
275.78 (140.06, 390.46)
7.28 (5.49, 539.55)
350.11 (175.05, 7049.78)
4898.15 (2976.15, 74914.15)
8.74 (7.05, 322.99)
Supplementary Table 2. Serum cytokine concentration in control and SSc serum as measured by
luminex. (N=3)
Supplementary Fig.3. Serum IL-6 concentrations were elevated in SSc (median and
interquartile range shown in A. p=0.1, N=3) and correlated with apoptosis (B) and
endothelial cell activation (C) in neutrophils: endothelial cell co-cultures.
Supplementary Fig.4. SSc serum increased apoptosis and ICAM-1 expression in neutrophil:
endothelial cell co-cultures compared to control serum. RA serum also increased ICAM-1
expression, though to a lesser extent, but did not increase apoptosis.