Chapter 2: Materials and Methods
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 or IL-8 were determined and the results showed no matter which sera all could increase the expressions of TNF-α (Fig. 4A; A group: 770.57 ± 147.30 and B group: 740.75 ± 105.75 pg/ml) or IL-8 (Fig. 4B; A group : 10943.35 ± 4935.40 and B group: 10280.29 ± 2148.42 pg/ml) than the samples without sera
treatments (TNF-α: 554.99 ± 35.33 and IL-8:4355.42 ± 331.12 pg/ml). There were no significant differences between low titer group (A group) and high titer group (B group). In addition, the patients’ sera alone without rHpHSP60 treatments could not result in any increases in the expressions of TNF-α or IL-8.
3. The effects of mouse and rabbit anti-HpHSP60 polyclonal antibodies on the abilities of HpHSP60 to induce expressions of TNF-α and IL-8.
To test the hypothesis that the anti-HpHSP60 polyclonal antibodies in H.
pylori-infected patients’ sera could enhance the abilities of HpHSP60 to
stimulate the expressions of TNF-α or IL-8 secretion, anti-HpHSP60 polyclonal antibodies derived from mice or rabbit immunized with rHpHSP60 were used to study the effects of polyclonal antibodies on the activities of HpHSP60 for inductions of proinflammatory cytokines. Combining with HpHSP60, all mouse anti-HpHSP60 sera significantly increased the expressions of TNF-α (1674.49 ± 188.12 pg/ml) or IL-8 (22402.75 ± 3341.75 pg/ml) comparing to the samples without sera treatment (TNF-α: 599.25 ± 19.93 and IL-8: 4587.82 ± 965.07 pg/ml) (Fig 5). While rHpHSP60 were treated with normal sera derived from naïve mice, TNF-α and IL-8 expressions were no significantly increase.
Similarly, the sera only without rHpHSP60 treatments could not cause any
significant TNF-α and IL-8 expressions.
All treatments with different doses of rabbit anti-HpHSP60 polyclonal antibodies facilitated rHpHSP60 to induce the expressions of TNF-α and IL-8, which were in accordance with the mouse sera (Fig. 6 A and B).
4. The effects of monoclonal antibodies on the abilities of HpHSP60 to induce expressions of TNF-α and IL-8.
So far, the previous results have revealed that sera against HpHSP60 no matter it were from patients, mice or rabbit can enhance the abilities of rHpHsp60 to stimulate TNF-α or IL-8 secretions. However, certain facts but not anti-HpHSP60 antibodies in serum may involve in or dominate this enhancive effect, which still could not be excluded. Thus, monoclonal antibodies against HpHSP60 were produced to verify the phenomenon. As shown in Table 1, from 390 hybridomas in a primary screening, 260 hybridomas could react with rHpHSP60 and 4 hybridomas were able to recognize the wild type HpHSP60.
Finally, 3 hybridomas were established and used in this study (designated as 5A8, 5A12 and 5B11) (Table 1).
Different dosages (0.4, 2, 10, 50 µg/ml) of three monoclonal antibodies were incubated with rHpHSP60 to treat THP-1 cells. All mAbs (5A8, 5A12 and
5B11) significantly increased the abilities of HpHSP60 to trigger the expressions of TNF-α comparing to the samples without mAb treatment (Fig 7A). In IL-8, almost doses of tree mAbs could significantly enhance the secretions, except of 0.4 µg/ml of 5A8, 50 µg/ml of 5A12 and 50 µg/ml of 5B11 (Fig 7B). In addition, these mAbs could not enhance the secretions of proinflmmatory cytokines by themselves.
The previous results indicated that the additional mAbs could cause the enhancive effect but the antigen-binding activities were essential for the enhancive effect or not still was unclear. Thus, the non-related Abs (anti-bovine haptoglobin) were prior interacted with rHpHSP60 and the results showed that TNF-α or IL-8 expressions were higher decreased as additions of non-related Abs were more (Fig. 8A and B). Further, whether the non-related mAb could affect the enhancive effect of specific anti-HpHSP60 mAbs on the cytokines expressions induced by HpHSP60 was examined. Figure 9A-C showed the abilities of 5A8, 5A12 and 5B11 to enhance expressions of TNF-α was significantly suppressed by non-related mAbs. Similarly, the non-related mAbs also decreased the specific anti-HpHSP60 mAbs’ enhancive effects on the IL-8 expressions (Fig. 10A-C).
Chapter 4: Discussion
In this study, we explored that the anti-HpHSP60 antibodies in patients’ sera could significantly enhance the ability of HpHSP60 to induce TNF-α or IL-8.
According the previous literatures, anti-HpHSP60 antibodies could be detected in H. pylori infected patients’ sera (Yunoki, Yokota et al. 2000; Ishii, Yokota et al.
2001; Tanaka, Kamada et al. 2009) and our results agreed well with these clinical examinations. Figure 3 pointed out that all H. pylori-infected patients had antibodies against HpHSP60 in their sera.
Furthermore, Dr. Perez-Perez et al. demonstrated that the titer of anti-HpHSP60 was correlated with the degree of gastric mucosal inflammation (Hussain, Shiratsuchi et al. 2000). As the knowledge from the literatures, extracellular HSPs are the most powerful ways of sending a ‘danger signal’ to the immune system in order to generate the responses that can help the organism manage an infection or disease and they are associating with the innate or adaptive immune systems activation (Ellis 1990; Young 1990). Furthermore, microbial HSP60s have been explored as an immunodominant antigen since it could induce powerful immune response after infection (Habich, Kempe et al.
2003). Bacterial HSPs such as HSP65 of Mycobacterial leprae have also been reported that they have the capability to induce releases of TNF-α, IL-6, and
IL-8 from human monocytic cells (Friedland, Shattock et al. 1993). Similarly, HpHSP60 has been reported to induce proinflammatory cytokines including IL-6, and IL-8 from human monocytic cells and/or gastric epithelium cells (Lin, Ayada et al. 2005; Zhao, Yokota et al. 2007), which is dominated by TLR-2 and TLR-4 pathways (Gobert, Bambou et al. 2004; Takenaka, Yokota et al. 2004).
Furthermore, some reports indicate that HSP60 is located on the surface of the bacteria (Yamaguchi, Osaki et al. 1996). These data indicate that hsp60 develops a tendency to be recognized by the host and may be closely related to H. pylori-induced inflammation. However, the role of anti-HpHSP60 antibodies in this inflammation is not clear. Ishii et al. have reported that the levels of IgG1 antibodies to HSP60 are elevated and it is closely associated with MALT lymphoma in H. pylori infected patients (Ishii, Yokota et al. 2001). Moreover, Hussain et al. also indicated the specific anti-PPD antibodies (IgG1) could up-regulate the activity of PPD to induce proinflammatory cytokines (Hussain, Shiratsuchi et al. 2000), which is similar as our finding on HpHSP60. In addition, different polyclonal anti-sera derived from different species confirm this effect again (Fig. 5 and 6), which indicated that the difference in species did not involved in the mechanism of enhancive effect on induction of proinflammatory cytokine release.
To sure the effect is because the anti-HpHSP60 antibodies but not the other factors in sera, we produced monoclonal antibodies against HpHSP60 to exclude the effects of non-specific anti-HpHSP60 antibodies or other factors in sera. Our data showed all purified mAbs (5A8, 5A12 and 5B11) could also cause higher levels of TNF-α or IL-8 expression in monocytes (Fig 7). Similar enhancive effect on pathogenesis by monoclonal antibodies is also explored for to H.
capsulatum HSP60 (Guimaraes, Frases et al. 2009). So far, our results indicated
the anti-HpHSP60 antibodies could result in this enhancive effect. Although these results revealed that different affinity of mAbs would cause different influences on cytokines expressions, seemingly the affinity is not the mechanism for the enhancive effect.
Our results indicated the specific anti-HpHSP60 antibodies could raise the capability of HpHSP60 to enhance the expressions of proinlammatory cytokines but the mechanisms were still unclear. Previous literatures have explored that HpHSP60 could induce expressions of proinflammatory cytokines through TLR-2 and TLR-4 pathways (Gobert, Bambou et al. 2004; Takenaka, Yokota et al. 2004). Although which factor(s) involved in the enhancive effects of specific antibodies did not completely identify, our results have indicated the non-related mAb could decrease the enhancive effect of specific mAbs (Fig 9 and 10).
Therefore, we proposed that Fc receptors may involve in the enhancive effects of specific antibodies. It has been reported that the Fc receptor can interact with the antibody’ Fc region (Raghavan and Bjorkman 1996) and the recent study has further found that Fcγ receptors can trigger inflammatory reactions in response to immunoglobulin-opsonized pathogens or antigen-antibody complexes (Liu, Masuda et al. 2005). Thus, prior combination of the anti-HpHSP60 antibody with HpHSP60 should posterior bind to Fc receptor, which could increase the probability of HpHSP60 to interact with TLR. Besides, the engagement of the Fc receptor could also trigger the signaling to contribute to the enhancive effect.
Finally, we indicated that the antibodies against HpHSP60 could enhance the ability of HpHSP60 to induce proinflammatory cytokines secretion in monocytes. In addition, we proposed that Fc receptor on monocyte involved in the enhancive effect of the anti-HpHSP60 antibodies. H. pylori infection causes the gastric diseases by induction of chronic inflammation. Although several virulent factors in this pathogenic microorganism have been identified, the pathogenic immunity induced by these factors are rare discussed. According our results, we proposed that patients’ the anti-HpHSP60 antibodies may bind to HpHSP60 to result in more serious inflammation and lead to more serious tissue damages in infectious sites.
Figures and Legends
Figure 1. SDS-PAGE and Western blot analyses of recombinant HpHSP60.
Left panel: Coomassie blue staining of rHpHSP60 run on a 10% SDS-PAGE under non-reducing and reducing conditions. Right panel: Western blot analysis of rHpHSP60 run on a 10% SDS-PAGE under non-reducing and reducing conditions. Lane M means the molecular marker and sample reacted without or with β-ME buffer was non-reducing conditions (lanes 1) and reducing conditions (lanes 2).
1 2
Figure 2. Production of proinflammatory cytokines in THP-1 cells. (A)
IL-1β, (B) IL-6, (C) IL-8 and (D) TNF-α secretion in THP-1 cells were measured in response to rHpHsp60. The THP-1 cells (5× 105) were treated and incubated with the rHpHsp60s (final concentration of 10 µg/ml) in 24-well plate for 24 h, and the cytokines in the medium were determined by ELISA. Results are representative three independent experiments. , ★ P＜0.001 compared to samples without rHpHsp60 treatment. (n = 3). Source from (Lin 2008)
(A) (B)
(C) (D)
Figure 3. The relative ratio of serum antibodies to HpHSP60 in H.
pylori-positive patients. The titer of anti-HpHSP60 antibodies from sera of H.
pylori-positive patients were analyzed using ELISA. And there are four
symptoms of samples including gastric cancer, gastritis, duodenal ulcer, and gastric ulcer. According to the titer, samples were divided into two groups including low titer group (A group, n = 8) and high titer group (B group, n = 8).
Results are shown as mean ± SD.
A B
(A.)
(B.)
-* **
HpHSP60 + + + Patients’ serum A B
-***
*
HpHSP60 + + Patients’ serum A B
+
Figure 4. The patients sera with anti-HpHSP60 antibodies could enhance the expressions of TNF-α and IL-8. The expressions of TNF-α (A.) and IL-8
(B.) were assessed by ELISA as described in material and method section. The patients’ sera of A and B groups were 1:250 diluted and incubated with HpHSP60 (5µg/ml) for 30 min then treated THP-1 cells (5×105 /well) for 24 hour. The horizontal lines of the data were expressed as the sample only with cells. Results are shown as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 as compare to samples without sera treatments.
(A.)
(B.)
HpHSP60 + + Polyclonal Abs - +
***
HpHSP60 + + Polyclonal Abs - +
***
Figure 5. The effect of mouse polyclonal antibodies on the abilities of HpHSP60 to induce expressions of TNF- α and IL-8. The expressions of
TNF-α (A.) and IL-8 (B.) were assessed by ELISA as described in material and method section. The mouse anti-HpHSP60 polyclonal antibodies were diluted (1:1000) and incubated with HpHSP60 (5µg/ml) for 30 min then treated THP-1 cells (5×105 /well) for 24 hour. The horizontal lines of the data were expressed as the sample only with cells. Results are shown as mean ± SD. ***p< 0.001 as compare to samples without sera treatments.
(A.)
Figure 6. The effect of rabbit polyclonal antibodies on the abilities of HpHSP60 to induce expressions of TNF- α and IL-8. The expressions of
Figure 6. The effect of rabbit polyclonal antibodies on the abilities of HpHSP60 to induce expressions of TNF- α and IL-8. The expressions of