Chapter 8 Material and Methods (2)
8.1 Material
8.2.5 Human peripheral blood mononuclear cells isolation
Human peripheral blood mononuclear cells were separated from white blood cells
concentrate of healthy donors by Ficoll-Paque PLUS (GE Healthcare. Chicago, IL.) The
operation processes are stated briefly below. The fresh human white blood cells
concentrate (pre-treated with anticoagulant) were well mixed with the same volume of
PBS. Then the diluted blood sample were carefully layered on Ficoll-Paque PLUS and
centrifuged at 2000 rpm for 20 mins at 18℃. The upper layer was drawn off carefully.
The white layer (mainly lymphocytes) and yellow Ficoll-Paque PLUS layer (contained
monocytes and neutrophils) were carefully transferred to a clean centrifuge tube without
removing the lower layer. A minimal 3 volumes of PBS was added to gently suspend the
cells and centrifuged at 1500 rpm for 10 min at 18℃. The supernatant was removed and
the cells were washed again with PBS. Finally, the cells were suspended in RPMI1640
(with 10 % FBS and 1 % PSA.) for the study.
8.2.6 Human TGF-beta measurement
Human peripheral blood mononuclear cells, Jurkat and THP-1 were seeded with
the condition that 2x106 cells in 1 ml growth medium respectively. Each culture
co-incubated with the propofol for 24 h, yielding a final concentration of 0.45, 2 and 6.5
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μg/ml, which was about the maximum therapeutic dosages of propofol for postoperative
nausea and vomiting [112], for sedation [111], and for anesthesia [109, 110]. HUVECs were seeded in a gelatin-coated plate, conditioned with 600μl, 3x105 cells, and
co-incubated with 6.5 μg/ml propofol. After 24 h incubation, the supernatants were
collected as day1 group or the same amount of propofol as stated previously was added
again and the supernatants were collected after incubating for another 24 h, categorized as the day2 group. The human TGF-β1 existed in the cell culture supernatant and human
sera were measured by TGF-β1 Emax ImmunoAssay System (Promega, Madison, WI)
and operated as indicated by the manufacturer. The treated process of samples is stated
briefly below. The human sera were diluted in DPBS and sample buffer. The cell culture
supernatants were not diluted. The samples were acidified to approximately pH 2.6 and then to approximately pH 7.6 to measured the total amount TGF-β1. The amounts of
active TGF-β1 were measured without acidification.
8.2.7 Endocytosis activity measurement:
THP-1 cells (3x105, 300μl) were seeded in RPMI1640 without FBS, and 300 μl
condition medium of HEVEC from the day2 group was added for 16 h. Co-culturing the
condition medium of HUVEC without propofol pretreated served as the control group and co-culturing the condition medium of HUVEC with propofol (6.5μg/ml) pretreated
served as the propofol group. The cells were harvested and centrifuged (1500 rpm,4℃,
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5 min). The supernatant was discarded and the 180 μl coolly fresh mediums were added
to suspend the pellets. The cells were incubated at 0 ℃ for 5 min and 20 μl stock
dextran-fluorescein isothiocyanate solution (dextran-FITC, 10mg/ml) was added and
incubated at 0 or 37℃ for 2 h. The cells were washed three times by PBS and analyzed
with flow cytometry (BD Biosciences, San Diego, CA). The FL-1 total fluorescent
intensities at 0 ℃ represented the nonspecific dextran-FITC uptake of THP-1 cells.
Thus, specific dextran-FITC uptake was represented as FL-1 total fluorescent intensities
at 37 ℃ discount FL-1 total fluorescent intensities at 0 ℃. The relative endocytosis
activity × 100% = specific dextran-FITC uptake of sample / specific dextran-FITC
uptake of control.
The patient sera of each group (propofol and no-propofol received) were mixed
respectively. 3 x 105 THP-1 cells were seeded and cultured in different conditions for 16 h stated below: (a) Control group: THP-1 cells were cultured in 600 μl serum-free
RPMI 1640. (b) SB431542 group: THP-1 cells were cultured in 600 μl serum-free
RPMI 1640 with 15 μM SB431542. (c) Propofol group: THP-1 cells were cultured in
300 μl serum-free RPMI 1640 mixed with 300 μl human sera from the
propofol-received patients. (d) Propofol with SB431542 group: THP-1 cells were cultured in 300 μl serum-free RPMI 1640 mixed with 300 μl human sera from the
propofol-received patients and 15 μM SB431542. (e) No-propofol group: THP-1 cells
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were cultured in 300 μl serum-free RPMI 1640 mixed with 300 μl human sera from the
no-propofol-received patients. (f) No-propofol with SB431542 group: THP-1 cells were cultured in 300 μl serum-free RPMI 1640 mixed with 300 μl human sera from the
no-propofol-received patients and 15 μM SB431542. Then, the dextran-FITC uptake
activities were assayed as described above.
8.2.8 Statistical analysis
All in figures are expressed as mean ± SE. To measure the human TGF-β1
expression in patient sera, the sample size consisted of fourteen propofol-received
patients and ten no-propofol-received patients; each sample was measured in two
independent ELISA and duplicated. Kruskal Wallis test were used to compare the
difference between groups. All other data were compared by student-test. The data of TGF-β1 expression in the condition medium human peripheral blood mononuclear cells
were obtained from 2 independent experiments and duplicated in each group (n=4). The data of TGF-β1 expression in the condition medium Jurkat, THP-1 and HUVEC were
obtained from 3 independent experiments and duplicated in each group (n=6). The data
of the endocytosis activity assay of THP-1 co-cultured with condition medium of
HUVECs were obtained from 2 independent experiments and duplicated in each group
(n=4). The data of the endocytosis activity assay of THP-1 co-cultured with patient sera
were obtained from 3 independent experiments and duplicated in each group (n=6).
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Other results were performed at least 2 independent experiments. All statistical
significant was set at p<0.05.
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Chapter 9 Results (2)
9.1 The study of propofol action on mouse cells
9.1.1 Propofol reduced TNF-α and IL-1β release in mouse splenocyes at the presence of LPS.
As mentioned above, propofol was believed to have immuno-modulation effect.
Here we used mouse-derived splenocytes to evaluate the effect of propofol on mouse
immune cells. Six-week BALB-C mice were sacrificed by CO2 asphyxiation and
harvested the spleen cells with germfree manipulation as stated in methods. 2 x 106 cells
in 1ml were seeding with LPS and/or propofol and incubated 48hrs. The supernatants of
each group were collected and the cytokine expressions were measured by ELISA. The mouse TNF-α and IL-1β expression were measured because TNF-α and IL-1β were
thought as dominant proinflammatory cytokine during early stage of LPS-induced immune response. The results showed that LPS could strongly stimulate IL-1β (Fig.4)
and TNF-α (Fig.5) expression; however, 30μg/mL propofol suppressed the expression
of IL-1β and TNF-α significantly within the same concentration of LPS. At the same
experiment condition, 14μg/mL and 30μg/mL propofol alone would inhibit the
IL-1β (Fig.4) expression of mouse splenocyes compared to control group. Another
interesting thing we observed was that propofol itself would stimulate TNF-α expression (Fig.5) compared to control group at both 14μg/mL and 30μg/mL
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group.
9.1.2 Propofol reduced IL-10 release in mouse-derived splenocyes at the presence of LPS.
From the present study and our results, propofol would decrease the
proinflammatory cytokine secretions of splenocyes when co-cultured with LPS. Thus,
the sequential issue we concerned was that propofol down-regulate the proinflammatory
cytokine by direct or indirect way. The direct way could be performed that propofol
affect the intracellular physiology of the immune cells, like suppress the transcription
factor activities associated to proinflammatory cytokine expression. The indirect way could be performed by inhibitory cytokine like IL-10 or TGF-β which induced by
propofol. To investigate the mechanism of propofol action on immune-modulation, the IL-10 and TGF-β expression in supernatant of the mouse-derived splenocytes were
measured. The cultured condition was the same as mentioned above (results 9.1.1). The
results showed that LPS also induced IL-10 expression at this cultured condition (2 x
106 cell in 1ml medium, 37℃, 48 hrs), but propofol decreased IL-10 expression in
mouse splenocytes at the presence of LPS. In addition, propofol alone induced IL-10 expression at the concentration of 30μg/ml (Fig.6). The result implicated that the
anti-inflammatory effect of propofol on mouse-derived splenocytes may not act through
the effect of IL-10.
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9.1.3 However, propofol enhanced TNF-α release in mice macrophage cell line P338/D1 at the presence of LPS.
In this study, we also investigated the effect of propofol on mouse macrophage
P338/D1. Although some articles showed that propofol would suppress the
inflammatory effect on LPS-activated mouse macrophage RAW 264.7 cells [122], our data showed that propofol would amplify the TNF-α expression induced by LPS in
mouse macrophage P338/D1 (Fig.8). Furthermore, propofol itself induced TNF-α
expression of P338/D1 compared to control group. It seemed that propofol would induce the TNF-α secretion and augment LPS-induced TNF-α secretion in P338/D1.
9.1.4 Propofol induced TGF-β release in mouse-derived splenocytes at the presence of LPS.
TGF-β is another well-known inhibitory cytokine responsible to immune
suppression, thus we also investigated the expression level of TGF-β of mouse
splenocytes under the effects of propofol. The cultured condition was the same as mentioned above, and total amounts of TGF-β were measured after acid treatment
followed the manufacturer indicated. The results showed that LPS alone could not induce TGF-β compared to control group, however, propofol could increase total
amounts of TGF-β whether at the presence of LPS or not (Fig.7). The result implied that
TGF-β might be responsible for the anti-inflammatory effect of propofol in mouse
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splenocytes.
9.1.5 Propofol did not affect the TGF-β expression level in mouse macrophage cell P338/D1 and T lymphocyte cell EL-4.
Due to mouse splenocytes were mixture of many type of immune cells, identifying which kinds of cells responded to propofol to secreted TGF-β was the aim which we did
our effort to. Mouse cell P338/D1 and EL-4 were used to investigate the effect of
propofol on the TGF-β expressions in mouse macrophages and T lymphocytes. 2x106
cells were seeding in 1ml growth medium with or without propofol and incubated 48hrs.
The results of ELISA showed the total amount of TGF-β did not statistically
significantly affected by propofol significantly both in P338/D1 (Fig.9A) and EL-4 (Fig.9B) at 14 or 30 μg/ml. We did not exclude the possibility that higher concentrations
of propofol would affect the TGF-β expression in P338/D1 and EL-4, but the effects of
extremely high dose of propofol would not be discuss in this study.
9.1.6 Propofol suppressed the activities of NF-κB and AP-1 at the presence of LPS.
In addition to the cytokine secretions affected by propofol, another part we
concerned was that transcription level of immune cells affected by propofol. To
investigate the intracellular transcription factor regulation by propofol at the presence of
LPS, what we needed to do was chosen some representative transcription factor during LPS-induced response. The present study has shown that transcription factor NF-κB and
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AP-1 were activated at the presence of LPS and the activities of NF-κB and AP-1 were
highly related to the LPS-induced inflammatory response like TNF-α production and
endocytosis activity of macrophages. Here we examine the propofol effects on NF-κB
and AP-1 at the presence of LPS by transcription factor activity assay. The reporter gene
was transfected into Balb-3T3. We chose Balb-3T3 to perform this study because
Balb-3T3 has toll-like receptor 4 expressed on its surface and has good transfection efficiency with Lipofectamine 2000. In addition, transcription factor NF-κB and AP-1
are critical intracellular regulators of macrophages function in the innate immunity.
According to the manufacturer’s instruction, pNF-kB/hrGFP and pAP-1/hrGFP were
transfected into Balb/3T3 cells seeded in the 6-well plate, respectively. Twenty-four hrs
later, cells were passaged by versene (0.2g EDTA-4Na/L in PBS) and re-seeded into a
24-well plate. The aim of this operation is prevention the wrong results or large
deviations due to difference of transfection efficiency between each wells. The transfectants were treated with LPS (14μg/ml) and co-incubated without or with
propofol for 16 hr, respectively. The transfectants were harvested and analyzed by flow
cytometer. Specific FL-1 fluorescent intensities, representing the activities of the
transcriptional factors, were calculated and compared to each control group. The control
group was transfected respectively with pNF-kB/hrGFP or pAP-1/hrGFP without other
treatments except growth medium In our experiments, LPS increased the activities of
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NF-κB in cells (Fig.10A) and slightly enhanced the activities of AP1 (Fig.10B).
However, 30μg/ml propofol reduced the activities of NF-κB and AP-1 at the presence
of LPS (Fig.11). Previous literatures have reported that LPS activates NF-κB and AP1
pathways to enhance the TNF-α and IL-1β expression and release. Thus our result
would illustrate that the intracellular regulation occurred when propofol reduce inflammatory response like TNF-αand IL-1β expression at the stimulation of LPS.
9.1.7 Propofol promotes the activities of NF-κB and AP-1 without LPS stimulation.
We also examine the propofol’s effects on NF-κB and AP-1 activities without LPS
stimulation. According to the inhibitory effect of propofol on macrophages, NF-κB and
AP-1 activities would be downregulated with reasonable speculation. However, distinctive results showed that propofol would promote NF-κB activity at the
concentration of 30μg/ml (Fig. 10A) and promoted AP-1 activity at the concentration of
14μg/ml (Fig.10B). The reasonable explanation of this result could be due to the
complicated effect of propofol. The complicated effect of propofol may result from
different concentration, timing, or cell types of action. Compare the results of 3.1.4 and
3.1.7 (Fig.8 and Fig.11), it seemed propofol could reduce the inflammatory response
induced by LPS in mouse cells; however propofol itself might activated inflammatory response, especially in macrophage-like cells by activation of NF-κB and AP-1.
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9.2 The study of propofol action on human TGF-β secretion and immune modulation
9.2.1 Intravenous injection of propofol during medical procedure increased both total amount and active form of TGF-β in patient serum.
In order to study the effect of propofol used in hospitals, we were in corporation
with Tzu Chi Medicine Center Hospital, Hualien, Taiwan to obtain the sample. All
medical procedures were followed the standard operations. The sera of patients form
different diseases and medical processes are collected within 2 days after surgery and
divides into with propofol group which did receive intravenous injection of propofol
(n=14) and without propofol group which did not receive intravenous injection of
propofol (n=10). The sera were diluted and assayed by ELISA according to the
manufacturer’s instruction. The results of ELISA were retreat to the origin
concentrations of each sample. The results show that successive two days propofol injections significantly increased the total amounts of TGF-β in patient sera in propofol
group compared to without propofol group (Fig.12A). Interestingly, the active form TGF-β1 also significantly increased in propofol group for two days propofol injections
(Fig.12B). According to these results, propofol used in hospitals could increase both total and active TGF-β1 in patients and implied that there were some matters needing
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attention.
9.2.2 Clinical dosages of propofol have no significant effect on total amounts of TGF-β1 but slightly raised the amount of active TGF-β1 in the condition mediums of human peripheral blood mononuclear cells.
The next issue we interested was what type of cells responded to intravenous injections of propofol to increased production of TGF-β1. As mentioned above, almost
all types of human cells could secrete TGF-β1, and our first candidate was human
peripheral blood mononuclear cells (PBMCs) due to directly contact to propofol. After
separations of human peripheral blood mononuclear cells from white blood cells
concentrate of healthy donors, 2x106 cells were cocultured with propofol and incubated
at 37℃. In order to mimic the situation of propofol used in hospitals, we collected the
supernatants 24 hrs after treatments (Day 1) or added propofol again and collected
supernatants another 24 hrs after treatments (Day 2). The cells cultured without
propofol were considered as control group. The results showed that propofol did not affect the total amount of TGF-β1 at all (Fig. 13A); whereas a fine change was observed
(Fig. 13B) in which 6.5 μg/ml propofol treatment could slightly increased the active
TGF-β1 expression when compared to control.
9.2.3 Clinical dosages of propofol slightly reduced total amounts of TGF-β1 but raised the amount of active TGF-β1 in the condition mediums of human Jurkat cells.
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Because human PBMCs were mixtures of many kinds of immune cells, the next
step we wanted study what types of immune cells responded to propofol to increase the active TGF-β1 production, as showed in human PBMC. Because T lymphocytes were
the largest populations in human PBMCs, we chose the human T lymphocytic cell line
JURKAT for study. The same cultured conditions were taken, 2x106 JURKAT cells were
co-cultured with propofol and incubated at 37℃ for 24hrs. The results showed the total
amounts of TGF-beta were slightly reduced only when treated with 6.5μg/ml, the
maximum concentrations used in clinical (Fig 14A). We also found that active TGF-β1
in the condition medium increased in a very slight manner when treated with 6.5μg/ml
(20.85±3.24 pg/ml compared to 13.13±4.2 pg/ml of control group) (Fig 14B).
9.2.4 Clinical dosages of propofol reduced total amounts of TGF-β1 but raised the amount of active TGF-β1 in THP-1 cells in a dose dependent manner.
Since there were not significantly differences of TGF-β1 secretions between Jurkat
cells with or without propofol, our next target was monocytic cells. 2x106 human
monocytic cells THP-1 cells were co-cultured with propofol and incubated at 37℃ for
24hrs. Interestingly, the results showed that total TGF-β in the THP-1 condition medium
decreased by propofol in a dose dependent manner Fig15A). The active form of TGF-β1
decreased propofol groups (Fig15B). These implied that propofol did not affect the TGF-β1 secretions but affect the activation of TGF-β in the cultured mediums of human
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monocytic cells THP-1.
9.2.5 Propofol inhibited the endocytosis activities of THP-1.
Since active form TGF-β increased propofol co-cultured mediums in THP-1, we
supposed it may have suppressive effects on THP-1. Here we used dextran-FITC uptake
to measure the endocytosis activity of THP-1. In order to control the THP-1 endocytosis
activity with normal growth conditions, we took a preliminary experiment to speculate
the correlation between concentrations of dextran-FITC and uptake of dextran-FITC in
THP-1. 5x105 THP-1cells were cultured in growth mediums for 24 hrs and harvested
the cells by centrifugation. Remove the supernatant and add different amounts of cooled
fresh medium to suspend the cells and 10mg/ml dextran-FITC (adjust the final volume equal to 200 μl). Incubate at 37℃ for 2 hrs and assay FL-1 fluorescent intensities by
flow cytometer. The data showed that FL-1 fluorescent intensity of gated THP-1 cells
increased in a dose-dependent manner with dextran-FITC concentrations (Fig.16.) Because it showed plateau at 15, 20 and 30 μl dextran-FITC, we chose the 1mg/ml as
our experiment conditions following (20μl of 10 mg/ml dextran-FITC solution added
into 180 μl cell suspensions).
With the same conditions, 5x105 THP-1 cells were co-cultured with different
dosages of propofol for 48 hrs (retreat in 24hrs.) The cells were harvested and incubated
with 1mg/ml dextran-FITC at 37 ℃ or 0 ℃for two hours uptake. The results showed
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that the relative endocytosis activities of THP-1 decreased in a dose-dependent manner
by propofol (Fig.17). The results that propofol could act on THP-1 and inhibited its
activities were fitted with the present studies that propofol could directly inhibit
monocytes / macrophages activities.
9.2.6 Clinical dosages of propofol raised total amounts of TGF-β1 in HUVECs.
In addition to human peripheral blood mononuclear cells, we also considered whether blood vessel endothelial cells would be induced to secrete TGF-β1 by propofol.
5 x 105 HUVECs were seeding and treated with 6.5 μg/ml propofol. The supernatant was collected after 24 hrs and 48 hrs as stated above. The results total TGF-β1 increased
day 2 propofol compared to control groups. However, the levels of active TGF-β1 did
not vary both in day 1 and day 2 propofol groups (Fig18). Thus, the above results
not vary both in day 1 and day 2 propofol groups (Fig18). Thus, the above results