Chapter 1: Introduction
5. The proinflammatory roles of HSP60s
HSP60 from several microorganisms such as bacteria, protozoa, fungi and helminthes have been shown to induce host immune response after infection with these organisms (Zugel and Kaufmann 1999). Antibodies specific to Mycobacterial tuberculosis HSP60 have been detected in patients with
tuberculosis and leprosy and also in mice (Young, O'Neill et al. 1988; Barrios, Tougne et al. 1994). In addition, the HSP60 of M. tuberculosis was demonstrated to activate secretion of pro-inflammation cytokines, IL1-β and TNF-α from human monocytes (Friedland, Shattock et al. 1993). HSP60 of Chlamydia pneumoniae have been demonstrated to cause acute pulmonary inflammation via TLR4 in mice and increase the level of IL-6 in the bronchoalveolar lavage fluid
(Bulut, Shimada et al. 2009). Bulut and colleagues have identified the Chlamydia trachomatis HSP60 could activate macrophages and endothelial cells
trough TLR4 to activate NF-κB and promote inflammation (Bulut, Faure et al.
2002). Similarly, HSP60 of Helicobacter pylori has been shown to induce series of pro-inflammation cytokine expression such as IL-1β, IL-8, and IL-6 through TLR2 and 4 from human monocytes and gastric epithelium cells (Gobert, Bambou et al. 2004; Lin, Ayada et al. 2005; Zhao, Yokota et al. 2007).
6. The antibody response to HSP60
Helicobacter pylori is associated with gastritis and peptic ulcer disease in
humans. H. pylori infection induces the host’s constitutional immune response against various antigens of this bacterium (Yunoki, Yokota et al. 2000). The detection of immunoglobulin G (IgG) antibodies to H. pylori is useful for the diagnosis of infection. Some investigators reported that the titers of these antibodies declined during therapy for H. pylori eradication (Veenendaal, Pena et al. 1991; Kosunen, Seppala et al. 1992; Perez-Perez, Brown et al. 1994; Wang, Chen et al. 1994; Cutler and Prasad 1996; Perez-Perez, Cutler et al. 1997).
Yunoki et al. measured the titers of IgG antibodies to the HSP60, urease, and whole-cell lysates of H. pylori in sera from patients with peptic ulcer during
antimicrobial treatment of H. pylori and then assessed its usefulness for the monitoring of eradication therapy. In recent years, it has also been accepted that host immune reactions play an important role in the pathogenesis of H. pylori infection (Ishii, Yokota et al. 2001). Although H. pylori is a noninvasive bacterium and is restricted to gastric epithelial cells, infection with H. pylori induces humoral and cellular immune responses in the gastric mucosa (Mattsson, Quiding-Jarbrink et al. 1998; Sommer, Faller et al. 1998). In the research of Ishii et al., rHpHSP60 was expressed, and the levels of antibodies to HSP60 in sera were measured in patients with gastric ulcer, duodenal ulcer, gastritis, and gastric MALT lymphoma to demonstrate the immunological role of HSP60 in H.
pylori-infected patients(Ishii, Yokota et al. 2001). Besides, to investigate
whether the antibodies against HpHSP60 involved in inflammatory reactions to further cause gastric disease is very important.
7. Fc receptor
Fc receptors are found on some cells of the immune system. These include phagocytes like macrophages and monocytes, granulocytes like neutrophils and eosinophils, and lymphocytes of the innate immune system or adaptive immune system (Sarfati, Fournier et al. 1992; Sulica, Chambers et al. 1995; Selvaraj,
Fifadara et al. 2004). They allow these cells to bind to antibodies that are attached to the surface of microbes or microbe infected cells, helping these cells to identify and eliminate microbial pathogens. The Fc receptors bind the antibodies at their Fc region, an interaction that activates the cell that possesses the Fc receptor (Raghavan and Bjorkman 1996). Activation of phagocytes is the most common function attributed to Fc receptors. Also Fcγ receptors (FcγR) trigger inflammatory reactions in response to immunoglobulin-opsonized pathogens and antigen-antibody complexes. Thus, in our study we will investigate whether the inflammatory reactions are associated with specific antibodies.
Chapter 2: Materials and Methods
Material
1. Reagent
The following reagents obtained were described as following: RPMI1640 and Dulbecco’s modified Eagle’s medium (DMEM) were from Invitrogen Inc.
(Gaithersburg, MD, USA). Fetal Bovine Serum (FBS) were from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Penicillin/ streptomycin/ amphotericin (PSA) were from Biological industries (Beithaemek, Israel). Kanoamycin and Tris were from MDBio Inc. (Rockville, MD, USA).
Isopropyl-beta-D-thiogalactopyranoside, NaCl, yeast extract, agar, Tis-HCl, Triton X-100, TEMED, imidazole, glycin, and 2-mercantoethanol were from Amresco Inc. (Solon, OH, USA). Sephadex G-25 Medium was from Amersham Bioscciences (Uppsala, Sweeden). Heparin , sodium dodecyl sulfate (SDS), 3,3´,5,5´-tetramethylbenzidine (TMB), dimethyl sulfoxide (DMSO), 3,3’-di-aminobenzidine (DAB) and ammonium persulfate (APS) were from Sigma-Aldrich (Steinheim, Germany). Goat anti-human IgG, IgA, IgM antibody was from Millipore Co. (Billerica, MA, USA). Goat anti-mouse IgG (H + L chain-specific)-HRP was from Jackson Immunoresearch Laboratories (West Grove, PA, USA). Nitrocellulose membrane (Hybond-ECL extra; Amersham,
Buckingham, UK). HAT and HT medium (Hybri-Max; Sigma Chemical Co., St.
Louis, MO).
2. Kit
Human IL-8, and TNF-α ELISA kit was obtained from R&D systems (Minneapolis, MN, USA). Coomasie PlusTM Protein Assay Reagent kit were from Pierce (Rockford, IL, USA).
3. Buffer
z 1╳ PBS
NaCl 8.18 g, KCl 0.2 g, Na2HPO4 1.41 g, KH2PO4 0.245 g in 1 L of DDW z LB solution
Tryptone 10 g, Yeast extract 5 g, NaCl 10 g in 1 L of DDW z LB agar
Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, Agar 15 g in 1 L of DDW z Alkaline lysis solution I
50 mM of Tris-HCl, 10 mM of EDTA, 100 µg/ml of RNase A in 100 ml of DDW
z Alkaline lysis solution II
0.02M of NaOH, 1% SDS in 100 ml of DDW z Alkaline lysis solution III
2.8 M of KOAc in 100 ml of DDW, pH = 5.1 z Buffer N2
KCl 34g, Tris-base 6.057g, 75 ml of Ethanol, 0.75 ml of TritoX100, in 500 ml of DDW, pH= 6.3
z Buffer N3
Tris-HCl 6.057 g, KCl 42.86 g, 79 ml of Ethanol in 500 ml of DDW, pH=
6.3
z Buffer N5
Tris-HCl 6.057 g, KCl 37.3 g, 79 ml of Ethanol in 500 ml of DDW, pH= 8.5 z Binding buffer for protein purification
20 mM of Na2HPO4, 0.5 M of NaCl, 40 mM of Imidazole in 500 ml of DDW, pH= 7.4
z Elution buffer for protein purification
20 mM of Na2HPO4, 0.5 M of NaCl, 500mM of Imidazole in 500 ml of DDW, pH= 7.4
z SDS-PAGE running buffer
Tris-HCl 15g, SDS 5g, Glycin 72g in 500 ml of DDW
z SDS-PAGE loading buffer
12mM Tris-HCl, pH 6.8, 0.4% SDS, 5% glycerol, 0.02% bromphenol blue z Transfer buffer
25mM Tris, 192mM glycine, 20% methanol, 0.0375% SDS (pH8.3)
4. Instrument
HisTrapTM HP column was from GE healthcare (Uppsala, Sweeden).
Sunrise remote control (TECAN). Fluorescence microscopy was from Olympus (Hicksville, NY, USA). FACScan flow cytometry (Becton Dickinson, Moutain View, CA). Semi-dry transfer cell obtained from Bio-Rad Laboratories (Hercules, CA, USA). ELISA plate (Nunc, Roskilde, Denmark).
5. Cell line
The human monocytic cell line (THP-1) and mouse myeloma cell line (F0) were obtained from the Bioresourece Collection and Research Center (Hsinchu, Taiwan).
6. Animal
Balb/c mice obtained from National Science Council (NSC) of Taiwan.
Rabbit obtained from Council of Agriculture, Executive Yuan, R.O.C.
7. Other
Escherichia coli (BL21 and DH5α) were obtained from Yeastern Biotech Co. H. pylori genome was from Department of Internal Medicine, College of Medicine, National Taiwan University.
Method
1. Cell culture condition
THP-1 were cultured in RPMI 1640 medium supplemented with 0.05 mM 2-mercantoethanol, 2g/L sodium bicarbonate, 50µg/ml of PSA, and 10%
heat-inactivated FBS. FO were cultured in DMEM medium supplemented with 1.5g/L sodium bicarbonate, 10% heat-inactivated fetal bovine serum (FBS), 50ug/ml of PSA. Hybridoma were cultured in medium DMEM supplemented with 1.5g/L sodium bicarbonate, 20% heat-inactivated FBS and 50µg/ml of PS.
2. Expression and purification of HpHSP60
Transformation of E. coli
Transformation was assayed by using competent cells of E. coli. DH5α was used for plasmid amplification, and BL21 was used for protein expression.
First, the competent cells were mixed gently with 1 ng DNA, and then incubated on ice for 30 min. After incubation, cells were heat shock at 42 ℃ for 90 seconds, and chilled on ice for 2 min. Next, place the competent cells to 250 µl LB and incubate at 37 ℃ with shaking 225 rpm for 1 hour. Cells were plated onto LB agar plate containing 30 µg/ml of kanamycin and incubate at 37 ℃ for
Midi plasmid DNA preparation
Pick up a single colony of transformed bacteria and transfer into 100 ml LB medium containing 30 µg/ml of kanamycin. Incubate the culture at 37 ℃ with vigorous shaking for 16 hours. The bacteria were recovered by centrifugation at 8000 rpm for 15 minutes. Remove the supernatant as dry as possible, and then resuspend the bacterial pellet with 8 ml of alkaline lysis solution I. Add 8 ml of Alkaline lysis solution II for lysis cells and mix the contents by inverting five times, then incubate for 3 min. Add 8 ml of ice-cold alkaline lysis solution III for neutralizing and gently invert the tube several times, then incubate on ice for 3 minutes. Centrifuge the bacterial lysates at 12000 rpm for 30 minutes. After washing NeucleoBond ion-exchange resin with 5 ml of buffer N2, the supernatant of bacteria lysates was added to the column;
following by 20 ml of buffer N3. The plasmid DNA was eluted by adding 5 ml of buffer N5 and then separating the eluted mixture into microfuge tubes. Next, add 700 µl of isopropanol to each tube for precipitating DNA and place the tube on ice for 10 minutes. Collect the precipitated DNA by centrifugation at 13000 rpm for 45 min and then remove the supernatant. The precipitated DNA was washed by adding 1 ml of 70 % ethanol, and then dissolved into DDW. Measure the absorbance at 260 and 280 nm and assay by using restriction enzyme
digestion for checking the DNA quantity and quality.
rHpHSP60 protein induction
E. coli (BL21) were transformed with 1ng of pET-HpHSP60 plasmid, and
then growth on LB plates containing 30 µg/ml of kanamycin at 37 ℃ for 16
hours. After incubation, pick up five colonies from the LB plates then inoculated into 100 ml of LB medium containing 30 mg/ml of kanamycin at 37 ℃ for 16 hours with vigorous shaking. Next, transfer the 100 ml of culture broth into 900 ml of LB medium containing 30 µg/ml of kanamycin and incubate at 37 ℃ with vigorous shaking until the value of OD600 reach the range from 0.6 to 0.8. Add 1.25 ml of IPTG (800mM) and continually incubate for 4 hours.
rHpHSP60 protein purification
After HpHSP60 induction, collect the bacterial by centrifugation at 8000 rpm for 20 min and then remove the supernatant. Resuspend the bacterial pellet with 30 ml of binding buffer, then disrupted by sonicate the whole cells mixture on ice for 15 min. After centrifugation, the supernatants were harvested. The rHpHSP60 were purified by HisTrapTMHP column according the manufacturer’s instructions. The purity of rHpHSP60 was examined by SDS-PAGE and confirmed with mass spectrometry analysis.
Gel Electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 1.5-mm-thick slab gel, using discontinuous system. The gel containing 10% (for non-reducing and reducing samples) polyacrylamide was used with a top stacking gel of 5% polyacrylamide. Approximately 10µg of the protein was loaded onto the gels and each tested sample was preheated at 100 ℃ for 10 min in a loading buffer without or with 140mM 2-mercaptoethanol. The samples were then run for about 1 hr at 100V and stained by Coomassie brilliant blue (Fig 1).
Western Blot Analysis
Following the SDS-PAGE, the gel was soaked briefly and instantly in the transfer buffer for 30 s. The gel was then immediately electrotransferred to a nitrocellulose membrane at 90 mA for 45 min in a semi-dry transfer cell. The membrane was immersed in 2% skim milk for 1 h with gentle shaking.
Following 3 x washes with PBS for 5 min, the membrane was then treated with polyclonal antibodies or mAb and developed with 3-3’-diaminobenzidine (3,3’,4,4’-tetra-amino-biphenyl) according to the method previously described (Yang and Mao 1999).
3. Detection of cytokines production in the THP-1 cells
THP-1 cells (5×105) were seeded in 24- well culture plates and incubated at 37℃, 5% CO2 atmosphere for 1hours. And 10µg HpHSP60 were treated into seeded cells for 24 hr. The supernatants with or without HpHSP60 stimulation were collected. TNF-α, IL-1β, IL-6, and IL-8 levels in the supernatants was measured by ELISA, according to the manufacturer’s specifications.
4. Measurement of patients’ sera antibody to HpHSP60
The serum samples were obtained from National Taiwan University Hospital. The serum samples derived from the patients which were diagnosis as Helicobacter pylori infection. According to the titer, samples were separated to 2
groups. And there were four symptoms including gastric cancer (HC), gastritis (HS), duodenal ulcer (HD) or peptic ulcer (HU). Serum antibodies to HpHSP60 were measured by enzyme-linked immunosorbent assay (ELISA). First, 96-well plates were coated with 100µL of HpHSP60 (1 mg/mL) and in phosphate-buffer saline overnight at 4 ℃ . After the wells were blocked with 300 µl of phosphate-buffer saline tween-20 containing 5% skim milk for 1 hour, plates were incubated with sera at a dilution of 1: 10,000 for anti-HpHSP60 antibody for 1 hour at room temperature and washed three times with phosphate-buffered
saline tween-20. Peroxidase-labeled goat anti-human IgG, IgA, IgM antibody was added (1:10,000), and the plate was incubated for 1 hour at room temperature. After plate was washed, each well was reacted with 3,3´,5,5´-tetramethylbenzidine solution for 20 minutes. After plate was reacted, the wells were added HCl to stop reaction. The optical density was measured at 450 nm on an ELISA plate reader.
5. Determination of TNF-α and IL-8 levels in sera condition by ELISA
THP-1 cells were seeded in 24- well culture plates with 0.2 ml of cell suspension (5×105/well) and incubated at 37℃, 5% CO2 atmosphere for 1hours.
And 5µg HpHSP60 were preincubated with sera (patients’ sera 1:250, anti-HpHsp60 mouse polyclonal antibodies 1:1000 and anti-HpHsp60 rabbit polyclonal antibodies 1:1000/200/100/50) in volume of 0.8ml for 30 min then treated into seeded cells for 24 hr. All sera were diluted then filtered by 0.22 nm filter. TNF-α, and IL-8 levels in the supernatants was measured by ELISA, according to the manufacturer’s specifications.
6. Production of monoclonal antibody
Animal care and use
The monoclonal antibody productions were utilized Balb/c mice with 5-7 weeks of age. The mice were fed in animal room from Chiao Tung University during the period of immunization. Feed and water were available daily. CO2. was used as a method of sacrificing and the other management was conducted according to guidelines established by NSC of Taiwan.
Preparation of mouse polyclonal antibodies against HpHSP60
Female Balb/c mice, aged 5-7 weeks, were used for immunization.
HpHSP60 in sterilized phosphate buffered saline (PBS), containing 0.12 M NaCl, 0.02 M phosphate, pH 7.4, was mixed and homogenized with an equal volume of complete Freund’s adjuvant by a three-way stopcock. Each mouse was initially given a total emulsion of 0.3 ml containing 100 µg of protein with 6 subcutaneous injections onto the back and an intraperitoneal injection. At day 7, an identical dose with incomplete adjuvant was given intraperitoneally followed by two intramuscular injections without adjuvant at day 14. Seven days following a final booster, blood was collected in 0.1% (wt/vol) EDTA and plasma was obtained. This plasma was used as a source for conventional polyclonal antibody against HpHSP60. Additional booster injections were given when necessary.
Preparation of rabbit polyclonal antibodies against HpHSP60
Female rabbit were used for immunization. HpHSP60 in sterilized phosphate buffered saline (PBS), containing 0.12 M NaCl, 0.02 M phosphate, pH 7.4, was mixed and homogenized with an equal volume of incomplete Freund’s adjuvant by a three-way stopcock. The rabbit was initially given a total emulsion of 2 ml containing 1 mg of protein with 6 subcutaneous injections onto the back. At day 30, an identical dose with incomplete adjuvant was given onto back followed by intramuscular injections at day 60. Then blood was collected in 0.1% (wt/vol) EDTA and plasma was obtained. This plasma was used as a source for conventional polyclonal antibody against HpHSP60. Additional booster injections were given when necessary. The titers of this antibody were over 1:20,000 as judged by an ELISA (described below).
Production of monoclonal antibody
After immunization, the titers of this antibody were over 1:6,400 as judged by an ELISA (described below). The spleen obtained was used for preparing hybridoma fusion. Monoclonal antibodies were produced according to the standard procedures (Mao, Rechtin et al. 1988; Mao, Rechtin et al. 1990). In brief, myeloma cell line (FO) was fused with spleen cells from immunized Balb/c mice at a ratio of 1:5. Fusion was carried out within 2 min at 37 ℃ using 1 ml of 50% (wt/vol) polyethylene glycol containing 10% (vol/vol) DMSO. Cell
mixture was then washed and resuspended in HAT medium containing approximately 1 x 105 FO cells per ml. The suspended cells were distributed as 100 µl per well in 96-well microtiter plates and incubated at 37℃ in a 5% CO2- incubator followed by an addition of 100 µl of fresh HAT medium after 7 days.
Subsequently, culture medium was assayed for the production of specific antibodies, between 14 and 21 days following the fusion, using a solid-phase ELISA described below. After primary screening, desired hybridomas were selected, expanded, and subcloned.
Enzyme linked immunosorbent assay
Initially, approximate 1 µg of HpHSP50 in 100 µl of PBS was coated on each well of an ELISA plate for screening hybridoma antibodies. Unbound proteins were washed with PBS 3 x and subsequently blocked by an addition of 300 µl of 2% (wt/vol) skim mlik for 1 hr. Following washes with PBS, 100 µl of hybridoma culture medium (2-3 weeks following the fusion) were added and incubated at room temperature for 60-90 min. Each well was washed 3 x with PBS containing 0.5% skim milk and 0.05% Tween-20. Bound antibodies were detected using a goat anti-mouse IgG (H + L chain-specific) conjugated with horseradish peroxidase (1:10,000) for 1 hr in PBS containing 0.5% skim milk and 0.05% Tween-20. Finally, each well was washed and developed with 0.04%
(wt/vol) 2,2-Azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) containing 0.01% (vol/vol) H2O2 in PBS.
Purification of HpHSP60 mAbs
Twice volume of 0.06 M sodium acetate buffer was added to hybridoma culture medium then added caprylic acid stirring at room temperature for 30 min.
Centrifuge at 4000 g for 30 min and retains the supernatant. By a 50% saturated ammonium, the precipitate was dissolved in 2 ml PBS followed by extensive dialyses against a 20 mM phosphate buffer at pH7.2. to remove the remaining ammonium sulfate.
7. Determination of TNF-α and IL-8 levels in mAbs condition by ELISA
THP-1 cells were seeded in 24- well culture plates with 0.9 ml of cell suspension (5×105/well) and incubated at 37℃, 5% CO2 atmosphere for 1hours.
And 5µg HpHSP60 were preincubated with mAb and non-related mAb (0.4, 2, 10, 50 µg) for 30 min in volume of 0.1 ml then treated into seeded cells for 24 hr.
On the other hand, the protocol of non-related mAb pretreating experiments, THP-1 cells were seeded in 24- well culture plates with 0.9 ml of cell suspension (5×105/well) contained with non-related mAb (50µg) and incubated at 37℃, 5% CO2 atmosphere for 1 h. And 5µg HpHSP60 were preincubated
with 5A8, 5A12 and 5B11 (10 µg) for 30 min in volume of 0.1 ml then added into seeded cells for 24 hr. TNF-α, and IL-8 levels in the supernatants was measured by ELISA, according to the manufacturer’s specifications.
Chapter 3: Results
1. The preparation of recombinant H. pylori HSP60
Several publishes have revealed that H. pylori HSP60 would stimulate IL-8 or IL-6 secretions in human monocytic cells or mouse macrophage respectively (Gobert, Bambou et al. 2004; Lin, Ayada et al. 2005). In this study, we cloned and expressed the recombinant HpHSP60 in E. coli expression system. The migration on SDS-PAGE revealed that the molecular weight of HpHsp60 is about 66 kDa (Fig. 1; left panel) and the rHpHSP60 could be recognized by mouse anti-HpHSP60 polyclonal antibodies. The results indicated that we successfully obtained rHpHSP60. Further, whether the rHpHSP60 proteins maintained their bioactivities to induce proinflammatory cytokines expressions on THP-1 cells as wild type of HpHSP60 was determined. The rHpHSP60 (10 µg/ml) were treated THP-1 cells for 24 h, and the supernatants were collected.
Pro-inflammatory cytokines, such as TNF-α, IL-1β, IL-6, and IL-8 in the supernatant were analyzed by ELISA. The productions of TNF-α, IL-1β, IL-6, and IL-8 in response to 10 µg/ml rHpHSP60 were significantly increased comparing to the samples without rHpHSP60 treatment. The mean value of IL-1β, IL-6, IL-8 or TNF-α in the culture medium was 101 ± 38, 277 ± 66, 16301 ± 1305 or 449 ± 153 pg/ml, respectively (Fig. 2A-2D). The results
indicated that the rHpHSP60 still had the capability to induce the releases of proinflammatory cytokines.
2. The patient sera with anti-HpHSP60 antibodies could enhance the expressions of TNF-α and IL-8.
The anti-HpHSP60 antibodies in sera of H. pylori-infected patients were measured by ELISA (Fig 3). According to the results, samples were divided into two groups: low titer group and high titer group (A: relative titer ratio < 2.2; B:
relative titer ratio > 2.2). The mean value ± SD of serum antibodies to H.
pylori-positive patients determined by an ELISA in group A (n = 8) and B (n = 8)
were 1.59 ± 0.36 and 2.82 ± 0.39.
To determine whether the anti-HpHSP60 antibodies in sera of H.
pylori-infected patients could lower the ability of HpHSP60 to stimulate
proinflammatory cytokine secretions, the patients’ sera from A and B groups were respectively incubated with rHpHSP60. The inductive amounts of TNF-α
proinflammatory cytokine secretions, the patients’ sera from A and B groups were respectively incubated with rHpHSP60. The inductive amounts of TNF-α