3. Results
3
.
1
. Design of the experiments Smooth muscle cell lines from human uterine arteries
were cultured for 4 days with media containing active substances exchanged and collected each day. At the
end, the cell layers were collected and homogenized. EC-SOD was analyzed in the culture media and all
SOD isoenzymes in the cell homogenates. To compen- sate for effects of the cytokines on overall cell growth
and protein synthesis, the contents of protein and DNA were measured in the cell homogenates. In a set of
separate experiments the effects of the cytokines on incorporation of
35
S-methionine into protein were also determined, and in another set of experiments the ef-
fects of cytokines on the level of EC-SOD mRNA was determined. Since smooth muscle cell lines are hetero-
geneous and show heterogeneous responses [38], effects of the cytokines were mostly tested on four different
cell lines. Experiments were carried out in media sup- plemented both with 15 FCS or 1 BSA. Up-regulat-
ing effects on EC-SOD synthesis were generally less pronounced in media supplemented with 15 FCS.
Down-regulating effects were equally pronounced with the two media supplements.
Experiments were generally carried out on two wells for each data point. Based on 132 sets of 2-wells-per-
datapoint measurements, the following relative stan- dard deviations for the analyses were calculated:
EC-SOD in media and cell homogenates 13, CuZn- SOD 8, Mn-SOD 15, protein 14 and DNA 7.
Based on the highest relative standard deviation found 15 it was calculated that a factor between groups in
2-well experiments of 1.35 or more is statistically signifi- cant P B 0.05.
To determine the uptake of EC-SOD by cultured cells, we added medium conditioned by smooth muscle
cells to contain EC-SOD to cultures of four different human cell lines that do not produce EC-SOD. The
cultures contained the following levels of cell protein: Du 145 285 mgwell, MG-251 135 mgwell, Hep G2 140
mgwell and PL-3 50 mgwell and were found to inter- nalize 33, 21, 24 and 18 of the EC-SOD present in the
medium in 24 h, c.f. Section 2. It cannot be excluded that some of the tested factors to a certain extent
influence the internalization of EC-SOD by the SMCs. Based on these considerations and the statistical calcu-
lations, we decided only to consider differences larger than 50 in the content of this enzyme in the culture
media between groups.
3
.
2
. Effects of cytokines on EC-SOD expression IFN-g up-regulated the EC-SOD expression in all
smooth muscle cell lines. Half maximal effect was seen at 50 Uml and maximal expression near 5000 Uml,
increasing the level of secreted protein about 5-fold in the Au-2 cell line Fig. 1A and Table 1. The response
to IFN-g was slow, and appeared to evolve during the entire 4-day long experiment. There was also a similar
increase in EC-SOD content of the cells homogenized at the end of the experiment. The increase in the level
of EC-SOD synthesis was not due to stimulation of cell growth, since there was no significant difference be-
tween controls and IFN-g-treated cultures with regard to protein and DNA content in the cell layer.
35
S-me- thionine incorporation studies suggested a minor reduc-
tion in protein synthesis by the IFN-g Table 1. Very similar patterns were seen in all cell lines tested. North-
ern blot analysis revealed a relative increase in EC-SOD mRNA as compared to GAPDH mRNA on IFN-g
stimulated cells, similar in magnitude to the relative increase in secreted EC-SOD protein Fig. 2.
TNF-a down-regulated the expression of EC-SOD in all cell lines tested Fig. 1B, Table 1. Maximal re-
sponses, depressing the level to about 30 in the Au-1 cell line, were reached near 10 ngml. TNF-a appeared
not to influence the general protein synthesis in our cell lines Table 1. Northern blot analysis revealed a rela-
tive decrease in EC-SOD mRNA as compared to GAPDH mRNA on TNF-a stimulated cells similar in
magnitude to the relative decrease in secreted EC-SOD protein Fig. 2.
IL-1a was found to influence the EC-SOD expression to some extent, but the direction varied between the cell
lines Table 1. IL-4 up-regulated the EC-SOD synthesis in all cell
lines tested Table 1, Fig. 1C. As with IFN-g, the response was slow in all cell lines. The maximal re-
sponse, reaching around a 3-fold stimulation in the culture media, was seen at 1 ngml and half maximal at
Fig. 2. Effects of IFN-g, IL-4 and TNF-a on EC-SOD mRNA levels. Smooth muscle cells of the Au-1 line were cultured in media supple-
mented with 1 BSA and indicated cytokines for 4 days. Single samples were employed for active substances, but double for the
control. RNA was extracted and mRNA levels determined by North- ern blot analysis as described in Section 2. The following concentra-
tions of cytokines were used: IFN-g: 500 Uml, IL-4: 1ngml, TNF-a: 10 ngml. The EC-SODGAPDH ratios relative to the mean of the
controls are indicated in the figure.
P .
Stra ˚lin
, S
.L .
Marklund Atherosclerosis
151 2000
433 –
441
Table 1 Collection of data for cytokine treatments influencing SOD expression
a
Concentration Medium
Concentration Cell line
Concentrations Content in cultures, change -fold versus controls
additive at half
at maximum tested
maximum effect Culture media
Cell homogenates effect
EC-SOD EC-SOD
Mn-SOD CuZn-SOD
DNA Protein
35
S count 1.5
2.11 1.3
0.84 0.8
IFN-g Uml 0.95
0.5;5;50;500;5000 Au-1 15 FCS
5000 50
1.4 1.42
2.4 0.91
0.76 0.85
50 5000
15 FCS Au-2
0.5;5;50;500;5000 0.5;5;50;500;5000
50 2.9
3.61 3.7
1.2 1.0
1.0 0.65
Au-1 1 BSA
5000 4.8
5.08 4.0
1.1 1.0
1.1 50
0.68 5000
1 BSA Au-2
0.5;5;50;500;5000 5.3
6.0 2.9
0.88 1.0
1.0 500
Au-4 1 BSA
5.8 5.5
12.3 1.2
1.1 1.2
500 1 BSA
Au-5 0.01;0.1;1;10;30
1 0.29
0.21 5.4
0.85 0.93
0.8 Au-1
15 FCS TNF-a ngml
10 0.59
0.56 11
1.1 1.2
1.0 \
1 0.01;0.1;1;10;30
10 15 FCS
Au-2 \
1 0.01;0.1;1;10;30
0.37 0.41
6.7 1.0
1.0 1.0
1.1 Au-1
1 BSA 10
0.63 0.45
30 0.86
0.9 0.01;0.1;1;10;30
0.69 0.84
Au-2 1 BSA
10 \
1 0.47
0.71 7.1
1.0 1.1
1.3 30
Au-4 1 BSA
0.35 0.14
22.4 1.0
1.1 1.1
30 Au-5
1 BSA 5
0.35 0.27
1.7 1.0
0.93 15 FCS
0.93 50
Au-1 IL-1a Uml
0.005;0.05;0.5;5;50 50
0.005;0.05;0.5;5;50 5
1.0 1.39
7.6 1.1
1.1 1.6
Au-2 15 FCS
5 1.1
1.37 4.2
1.1 1.1
1 BSA 1.3
0.005;0.05;0.5;5;50 1.0
50 Au-1
0.005;0.05;0.5;5;50 50
5 1.8
1.93 16.3
0.89 1.0
1.1 1.2
Au-2 1 BSA
0.77 1.0
5.4 0.89
1.1 1.2
50 Au-4
1 BSA 0.49
0.32 13.2
1.2 1.2
1.7 50
1 BSA Au-5
1.7 1.1
n.d. n.d.
1.0 IL-4 ngml
1.2 0.03;0.1;0.3;1;3
Au-1 15 FCS
3.0 3.6
1.4 1.1
1.2 1.2
0.1 1.2
3 1 BSA
Au-1 0.03;0.1;0.3;1;3
2.1 5.11
1.4 0.03;0.1;0.3;1;3
0.84 1.1
1.2 1.3
Au-2 1 BSA
3 0.3
1.6 2.2
1.6 1.1
1.0 1.1
3 Au-4
1 BSA 1.7
1.6 0.98
3 1.3
1.0 1.1
Au-5 1 BSA
a
The effect of cytokines on smooth muscle cell SOD expression was determined as detailed in Section 2, see also Fig. 1A–C. The table presents the concentrations of cytokines tested, cell lines tested, additives to the culture media FCS or BSA, cytokine concentrations at maximal up- or down-regulation of EC-SOD expression, generally also applicable to Mn-SOD, approximate
concentrations at half maximal up- or down-regulation, maximal changes -fold compared to control cultures in EC-SOD contents in the fourth day culture media and in cell homogenates, and in Mn-SOD, CuZn-SOD, DNA and protein contents in cell homogenates. The incorporation of
35
S-methionine was determined in separate experiments, using 500 Uml IFN-g, 30 ngml TNF-a, 50 Uml IL-1a and 3 ngml IL-4. In these experiments, the responses in EC-SOD synthesis were similar to those presented in the table. n.d., not determined.
0.1 – 0.3 ngml Table 1. Effects on cell growth and general protein synthesis were small and not significant.
As with IFN-g, Northern blot analysis revealed a rela- tive increase in EC-SOD mRNA as compared to
GAPDH mRNA on IL-4 stimulated cells similar in magnitude to the relative increase in secreted EC-SOD
protein Fig. 2.
3
.
3
. Regulation of CuZn-SOD and Mn-SOD expression by cytokines
The CuZn-SOD activity of the smooth muscle cell lines was not significantly affected by any of the cytoki-
nes. The Mn-SOD activity was up-regulated about 4-fold by IFN-g. The effect was half-maximal at 50
Uml and maximal at 5000 Uml, similar to the effect on the EC-SOD Table 1, Fig. 1A. TNF-a markedly
up-regulated the Mn-SOD activity in all cell lines. The effect was half-maximal at around 0.5 ngml and maxi-
mal at 10 ngml, reaching about a 6-fold stimulation. IL-1a also markedly up-regulated the Mn-SOD activity
in all cell lines, though less than TNF-a. The effect was half-maximal at 3 Uml and near maximal at 50 Uml
Table 1.
3
.
4
. Other factors tested The Au-1 and Au-2 cell lines were also exposed to
the following factors: IFN-a 500 Uml, IL-2 1000 Uml, IL-3 30 ngml, IL-6 2 and 20 ngml, IL-8 60
and 800 ngml, TGF-b 0.005; 0.05; 0.5; 5; and 25 ngml, E. coli lipopolysacharide 1 mgml, granulo-
cyte-macrophage colony-stimulating factor 10 ngml, human growth hormone 100 and 1000 ngml, platelet
activating factor 20 and 200 ngml, and complement 5A 0.1 and 1 mmoll. None of these factors influenced
the EC-SOD, the CuZn-SOD or the Mn-SOD activities of the cells.
4. Discussion
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