Chapter 7 Introduction (2)
7.2 TGF-β
7.2.8. b Regulation monocytes/macrophage functions by TGF-β1
Monocytes/macrophages play a central role in many cellular reactions, like
immune activation and modulation of inflammatory responses and wound repair process.
Macrophages could phagocytose microbes and produce inflammatory mediators upon microbial challenge. TGF-β1 could inhibit phagocytosis of IgG-coated particle in
human THP-1 cells by downregulation of expression of two of the IgG receptors, FcγRI
and FcγRIII, and of the common γ-subunit [150]. Reducing endocytosis activity of
monocytes-macrophages would have negative influences on antigen uptake, and the cytotoxic activity of macrophage. TGF-β can also suppress the antigen presentation of
macrophages by reducing the expression of MHC class II, the costimulatory molecule
CD40 and the inflammatory cytokine IL-12p40 [151, 152]. Furthermore, TGF-β1
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suppress IFN-γ-induced NO production by multiple mechanisms. TGF-β1 inhibits of
iNOS mRNA transcription and enhancement of iNOS protein degradation through suppressing the IFN-γ-induced signal transducer and activator of transcription 1 (STAT1)
phosphorylation and activation [153]. It was also shown that TGF-β1 inhibits
LPS-induced septic shock in the mouse. TGF- β1 arrested LPS-induced hypotension
and decreased mortality. Blockade of TGF-β1 signaling pathway resulted in
overreaction to endotoxin stimulation [154, 155]. TGF-β could inhibit LPS-stimulated
mRNA and protein levels of iNOS, TNF-α and IL-1β in vitro and in vivo [156].
Toll-like receptors (TLRs) are types of receptors involved in the recognition of
microbes. LPS is recognized by TLR4 with the assistance of CD14. TGF-β inhibits
activation protein1 (AP-1)-mediated CD14 expression in LPS-stimulated macrophage.
MyD88 is a key adapter molecule downstream of TLRs. TGF-β would promote MyD88
degradation in LPS-stimulated macrophage. Therefore, TGF-β attenuate TLR4
signaling and suppress LPS induced activation of macrophages [154, 157] . 7.2.8.c Regulation of dentritic cells by TGF-β1
Immature dentritic cells (DCs) located in peripheral nonlymphoid organs to filter
the foreign antigens and pathogens. These immature DCs are highly specialized in
antigen uptake and processing in nonlymphoid tissues. DCs from these nonlymphoid
peripheral organs are capable of migrating to T-cell rich areas of secondary lymphoid
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organs such as draining lymph nodes or spleen. These migrated DCs up-regulate major
histocompatibility complex (MHC) class I and II molecules as well as T-cell
costimulatory molecules such as CD80 and CD86. Thus, they lose potent antigen uptake
and processing capacity, and in turn acquire potent T-cell stimulatory capacity. Several studies have shown that TGF-β1 inhibits in vitro activation and maturation of DCs.
TGF- β1 inhibits upregulation of critical T-cell costimulatory molecules on the surface
of DCs and reduces the antigen-presenting capacity of DCs. Thus, in addition to direct inhibitory effects of TGF-β1 on effector T lymphocytes, inhibitory effects on APCs like
DC and macrophage may critically contribute to immunosuppressive effects of TGF- β1
[158]. In contrast to these negative regulatory effects of TGF- β1 on function and
maturation of DCs, TGF-β1 positive stimulate development and maturation of certain
subpopulations of immature DCs. Recent studies established that TGF- β1stimulation is
absolutely required for the development of epithelial Langerhans cells (LCs) from
hematopoietic progenitor cells in vitro and in vivo. As mentioned above, these results suggest TGF-β1 has stage-specific effects on DCS.
7.2.8.d Regulation of CD4+ and CD8+ T lymphocytes cell functions by TGF-β1
TGF-β1 is an important immunomodulatory cytokine that regulates the
differentiation, proliferation, and apoptosis of CD4+ T cells. In the present study, TGF-β1 knockout mice exhibit a striking expansion of CD4+ T cells in the liver by 11
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days of age, accompanied by CD4+ T cell-dependent necroinflammatory liver disease.
Further results indicate that CD4+ T cells in TGF-β1-/- mice are activated by and respond to self-Ags present in the periphery, and define a key role for TGF-β1 in the
peripheral regulation of Ag-specific CD4+ T cell responses [159].
CD8+ T cells often differentiate into highly cytotoxic cells. A cytotoxic T cell (also
known as TC, CTL or killer T cell) are capable of inducing the death of infected somatic
or tumor cells; they kill cells that are infected with viruses (or other pathogens), or are otherwise damaged or dysfunctional. TGF-β1 potently suppressed CTL differentiation
of human cord blood naïve CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. TGF-β1–null mice
showed severe autoimmune inflammation in most organs, characteristics involved that
infiltrations of large amount of inflammatory cells, elevated levels of nuclear
autoantibodies and activated peripheral lymphocytes in tissue lesions. Inactivation of TGF-β type II receptor (TβRII) in CD4+ and CD8+ T cells in vivo leads to autoimmune
inflammatory disease and spontaneous T-cell activation, showed the importance of TGF-β for these cells to control autoimmunity and T-cell homeostasis [160]. Another
aspect also reflects that TGF-β1 associated with CTL activity closely. TGF- β1 on CTL
cytotoxic activity is consistent with enhanced tumor eradication in mice with T cells lacking TGF- β signaling. Tumors secrete large amounts of TGF-β1, which limits
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tumor-infiltrating CD8+ T cells to fail to differentiate into effector or effector memory
cells, leading to premature cell death.
7.2.8.e Regulation of regulatory T lymphocytes functions by TGF-β1
There are lots of study suggesting that TGF-β1 closely associated with the
maturation and bio-functions of regulatory T lymphocytes. It is widely accepted that
CD4+CD25+ regulatory T lymphocytes are critical to maintain immune homeostasis and
immunological tolerance. Naturally occurring CD4+CD25+ regulatory T cells (Treg cells)
mediates immune suppression to limit immunopathogenesis associated with chronic inflammation, persistent infection and autoimmune disease. TGF-β1 not only mediate
cell-contact dependent suppression between regulatory T cells and CD4+CD25- or CD8+
Tcells, but also involved in conversion of CD4+CD25- T cells to CD4+CD25+ Treg
cells [161]. In the present study, CD4+CD25+ Treg cells suppress the CD4+CD25- T cells proliferation and B cell immunoglobulin production but anti-TGF-β would abolish the
cell-contact suppression. Further studies suggest that high level of active form of membrane–bound TGF-βl expressed on CD4+CD25+ Treg cells would responsible to the
suppression activity [162]. In addition to direct suppressive effect on CD4+ cells, Treg
cells also modulate the immune response through APC. Either soluble or surface bound TGF-β expression by Treg cells could deliver a suppressive signal to DCs, and these
DCs would become tolerogenic with phenotypical and functional alterations and may
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not prime the naïve T cells. In addition to suppression effect of Treg cells, TGF-β also
involved in conversion Treg cells. This effect not only occurs in suppression of
autoimmune disease but also occur in tumor evasion of the immune system.
Tumor-derived TGF-β are responsible for converting CD4+CD25- into CD4+CD25+ Treg
cells and suppress the immune response in the tumor microenvironments. These conversions would facilitate tumor progression. Neutalization of TGF-β abrogated this
conversion both in vitro and in vivo and reduces the tumor burden in mice [163].
7.2.8.f Regulation of B lymphocytes functions by TGF-β1
B lymphocytes play a large role in the humoral immune response. The antigen
receptor on B lymphocytes is a cell-associated immunoglobulin. On activation by
antigen, B cells differentiate into cells producing antibody of the same antigen specificity as this receptor. Exogenous TGF-β inhibits cytokine-induced proliferations
and stimulates apoptosis of human B cells. TGF-β may function as an autocrine growth
inhibitor that limits B lymphocyte proliferation [164, 165]. In addition, TGF-β is also an important regulator of B cell activation and differentiation. TGF-β stimulates activated
murine and human B lymphocytes to isotype switch to IgA. Mice with a blockade of TGF-β signaling in B cells are almost devoid of serum IgA [166]. In addition to
enhance IgA production, in vivo studies reveal a general inhibitory function for TGF-β
on antibody production with the exception of IgA [167, 168]. Secretory IgA is
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important for the prevention of microbial infection in mucosa. The discovery that TGF-β stimulates isotype switching to IgA suggests that alterations in TGF-β
expression could play a role in the pathogenesis of conditions associated with
alterations in IgA.
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7.3 Strategy & Specific Aims
First, we investigate the effects of propofol on mouse cells. From the study of mouse cells, we speculated that propofol has potential to induced TGF-β1. No studies
have examined the correlations between propofol and TGF-β1 expression. Since
propofol was one of most used anesthetic agents in hospital, we focus our study about
the effects of propofol on human beings within clinical dosages.
To investigate the effects of propofol in clinical concentrations on the immunosuppression by determining the potent immunosuppressive TGF-β1 expression
levels in patient sera, the consequential increase of TGF-β1 in sera induced lead the
study to seek which responsive cell expresses TGF-β1 after propofol treatment. Human
peripheral blood mononuclear cells (PBMC), vein endothelial cells (HUVECs),
lymphocytes (Jurkat) and monocytes (THP-1) were candidates and tested in the study.
In addition, whether the propofol-induced TGF-β1 is effective to decrease the activity of
monocytes is also verified.
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Chapter 8 Material and Methods (2)
8.1 Materials 8.1.1 Reagents:
The propofol emulsion was purchased from Fresenius Kabi, Germany whose trade
name was Fresofol (the license number of health department, Taiwan: 022868) and the
content include 1% propofol, soyben oil, purified egg phosphatide, glycerol, oleic acid,
sodium hydroxide and water for injections. LPS, Dextran-fluorescein isothiocyanate and
SB431542 was purchase from Sigma, Louis, MO.
8.1.2 Animal:
Six-eight week female BALB-C mice were purchased from the National
Laboratory Animal Center, Taipei, Taiwan, R.O.C.
8.1.3 Plasmid:
pNF-kB/hrGFP and pAP-1/hrGFP plasmids containing the NF-κB and AP-1 transcription binding site, respectively, followed by a hrGFP reporter gene, were
purchased from Stratagene, Garden Grove, CA.
8.1.4 Bacterial strains
Escherichia coli DH5 strain were purchased from Invitrogen, Carlsbad, CA and used for plamids amplications.
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8.1.5 Patient serum and human white blood cells concentrate acquirement:
The study was approved by the Human Research Ethics Committee of the Tzu Chi
Medicine Center Hospital, Hualien, Taiwan. Sera from 24 trauma patients and
heparinized white blood cells concentrates were taken from 3 healthy volunteers after
receiving institutional approval and written, informed consent from the participants. All
medical procedures followed the standard guidelines. The sera of patients from different
ages, sex, department, diagnosis and operations (Table 3) were collected within 48 days
after surgical operation and divided into two groups.
Propofol-received group: which received a daily intravenous injection of propofol
for a minimal of 2 days (n=14).
No-propofol-received group: which did not receive any intravenous injection of
propofol (n=10).
8.1.6 Cell lines:
z P338/D1 (Mouse macrophage-like cell line; ATCC number: CCL-46.)
z EL-4 (Mouse T lymphocyte cell line, ATCC number: TIB-39)
z HEK293 (Human embryonic kidney cells; ATCC number: CRL-1573)
z THP-1 (Huamn monocytic cells; ATCC number: TIB-202.)
z JURKAT (Human T lymphocyte; ATCC number: TIB-152)
z HUVEC: Primary cultures of human umbilical vein endothelial cells;
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All cell lines except were obtained from BCRC (Food Industry and Development
Institute, Hsinchu, Taiwan). HEVEC were kindly provided by Dr. Ko-Jiunn Liu (Nation
Health Research Institute, Taipei, Taiwan). P338/D1 and JURKAT cultured with
RPMI1640 supplemented with 10% fetal bovine serum (Biological industries, Beit
Haemek, Israel) and 1% penicillin-streptomycin amphotericin B (Biological industries);
HEK293 cultured with Dulbecco's Modified Eagle's Medium (Gibco, Invitrogen Co.,
Carlsbad, CA) supplemented with 10% FBS and 1% antibiotics PSA THP-1 was
cultured with RPMI1640 with 10mM HEPES, Sodium pyruvate and 2-Mecaptomethnol,
10% FBS and 1% PSA. HUVECs were grown in Medium 199 (Gibco, Invitrogen Co.)
supplemented with 20% fetal bovine serum, 25 units/ml heparin (Sigma,) and 30 μg/ml
endothelial cell growth supplements (ECGS; Sigma), according to the instructions of
the American Type Culture Collection (Manassas, VA). Passages between 3 and 7 were
used for the subsequent experiments.
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8.2 Methods
8.2.1 Spleen preparation and culture
BALB-C mice were sacrificed by CO2 asphyxiation. The spleen was taken, minced
with DMEM, and filtered with a mesh. Soup was centrifuged at 1,200 rpm at 12℃ for 5
min. The supernatant was removed and 5 mL ACK buffer (150 mM NH4Cl, 10mM
KHCO3, 0.1 mM Na2-EDTA) was added. After 5 min incubation, the mixture was
centrifuged (at 1200rpm at 12 for 5 min) and washed twice with PBS to remove ACK ℃
buffer. 5 mL of RPMI 1640 was added to resuspend the cells. 2 x 106 cells were then
incubated with treatment of LPS and/or propofol. The supernatant of each group was
collected after 48 hr and stored at -80 . ℃
8.2.2 Midi-preparation of plasmid DNA
One ml of pre-cultured E. coli transformed with specific plasmids was added into
100 ml LB (1% tryptone, 0.5% yeast extract, 1% NaCl in sterile ddH2O) broth with
antibiotics and incubated at 37℃ with shaking (~225 rpm) for 12-16 hr. The broth was
centrifuged at 8,000 rpm at 4℃ for 15 min. After discarding supernatant, 4 ml Buffer S1
(50 mM Tris-HCl, 10 mM EDTA, 100 μg/ml RNase A, pH8.0) was added and the
solution was vortexed to dispense the pellet. Then 4 ml of Buffer S2 (200 mM NaOH,
1% SDS) was added. The lysate was mixed gently by inverting the tube 6~8 times and
incubated at room temperature for 2~3 min. The solution was mixed with the 4 ml
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pre-cooled Buffer S3 (100 mM Tris, 15% ethanol, 900 mM KCl, 0.15% Triton X100,
adjusted to pH 6.3) and inverted gently 6~8 times until a homogeneous suspension
containing an off-white flocculate was formed. The suspension was incubated on ice for
5 min. A NucleoBond AX 100 column (MACHEREY-NAGEL, Düren, Germany) was
equilibrated with 2.5 ml Buffer N2 (100 mM Tris, 15% ethanol, 900 mM KCl, 0.15%
Triton X100, adjusted to pH 6.3 with H3PO4). The solution of bacterial lysate was
centrifuged at 12,000 rpm at 4℃. The lysate was removed and the supernatant was
loaded into the NuceloBond column. After the fluid flowing away by gravity flow, ten
ml of Buffer N3 (100 mM Tris, 15% ethanol, 1M KCl, adjusted to pH6.3 with H3PO4)
was added to wash the column and repeat this step once again. Plasmid DNA was eluted
with 5 ml of Buffer N5 (100 mM Tris, 15% ethanol, 1M KCl, adjusted to pH 8.5 with
H3PO4). Then 3.5 ml isopropanol was added to precipitate the eluted plasmid DNA. The
mixture was incubated on ice for 10 min and centrifuged at 13,000 rpm for 30 min at
4℃. The supernatant was discarded. One ml 70% ethanol was added and stored at -20℃
or the solution was centrifuged at 13,000 rpm for 10 min, discarded ethanol carefully and the pellet was dissolved in 20 μl DWW for further application.
8.2.3 Transcription factor activity assay
According to the manufacturer’s instruction, pNF-κB/hrGFP and pAP-1/hrGFP
were transfected by Lipofectamine 2000 (Invitrogen) into Balb/3T3 cells seeded in the
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6-well plate, respectively. Twenty-four hrs later, cells were passaged by versene (0.2g
EDTA-4Na/L in PBS; PBS: 137 mM NaCl, 10 mM Na2HPO4, 2.7 mM KCl, 1.8 mM
KH2PO4, Ph 7.4) and re-seeded into a 24-well plate. The transfectants were treated with LPS (14 μg/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. In
each plate, control plasmid pCMV/hrGFP was transfected into the target cells to
measure the transfection efficiency, which was about 60 %.
8.2.4 Mouse cytokine measurement
100 μl of capture antibody (0.8 μg/mL) was added into each well of an ELISA
plate (Costar, Cambridge, MA) and the plate was incubated overnight. Wash buffer
(0.05% Tween 20 in PBS, pH7.2~7.4) was applied three times. 300 μl of Block buffer
was added and the plate was incubated for 1 hr at room temperature. Wash buffer was
applied three times. 100 μl of samples were added into each well and incubated at room
temperature for 2 hr. The plate was then washed with wash buffer for three times. 100 μl
of detection antibody (150 ng/mL) was added into each well. Samples were incubated at
room temperature for 2 hr, and then washed three times with wash buffer. 100 μl of
Tetramethylbenzidine substrate (CLINICAL, Mansfield, MA) was added into each well
and the plate was incubated at room temperature for 20 min. To stop the reaction, 50 μl
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of stop solution (1N HCl) was added and the quantification was determined by the
ELISA reader at the absorbance wavelength of 450 nm.
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
patients and ten no-propofol-received patients; each sample was measured in two