3-1 Pro-inflammatory cytokines production
Several publishes revealed that H. pylori HSP60 would stimulate IL-8 or IL-6 secretion in human monocytic cells and mouse macrophage respectively (29, 30). The level of IL-1β and tumor necrosis factor-α will increase in the infected gastric tissues (135).
To examine whether the cytokines could be induced in THP-1 cells with rHpHSP60 treatment, THP-1 cells co-incubated with rHpHSP60 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 production of TNF-α, IL-1β, IL-6, and IL-8 in response to 10 μg/ml rHpHSP60 is significantly increased compared to the control. The concentration of IL-1β, IL-6, IL-8, and TNF-α in the culture medium was 101 ± 38 pg/ml, 277 ± 66 pg/ml, 16301 ± 1305 pg/ml, and 449 ± 153 pg/ml, respectively (Fig. 3a-3d). Among these cytokines, IL-8 which is a neutrophil attraction factor was greatly present in the culture medium. However, it has poorly effect on monocytes activation. According to the previous report (80), we know that the level of TNF-α induced by THP-1 cells in this system was able
to induce monocytes activation. IL-6 is involved in activation of adaptive immunity dominantly. IL-1β, a product of phagocytic cells, has been reported that 20 ng/ml of IL-1 could cause maximal upregulation of monocyte phagocytosis (136). However, a little amount
of IL-1β was detected in the supernatant.
3-2 Release of TNF-α from THP-1 cells incubated with rHpHSP60 or heated rHpHSP60
We know that LPS is a general factor to stimulate TNF-α secretion from immune cells.
Commercial recombinant protein from E. coli system would always contaminate LPS. In order to exclude the effect of rHpHSP60 contaminating with LPS to result in TNF-α secretion, we heated the protein at 95 ℃ for 1 h to cause proteolysis. As shown in Fig. 4, proteolysis induced by heating to 95 ℃ abolished TNF-α secretion. Thus, this evidence indicated that the observed effect of rHpHSP60 on TNF-α expression was not due to heat-stable LPS (Fig.
4).
3-3 Kinetic of cytokine protein expression
Friedland et al. showed that mycobacterial 65-kD heat shock protein induced a maximum expression of TNF-α at 4 h, IL-6 was obviously present at 8 h, and a great amount
of IL-8 was detected at 24 h in THP-1 cells (136). However, GroEL, the 60 kDa HSP of Escherichia coli, time-dependent induced TNF-α, IL-6, IL-1α, and ICAM-1 expression (137).
Chlamydial HSP 60 induces TNF-α production by mouse macrophages and it was secreted at a maximum at 6 h, and TNF-α expression kept in a stable level until 72 h (126).
In order to further confirm the cytokine expression kinetic, we treated THP-1 cells with
10 μg/ml rHpHSP60 and time-course collected the supernatants. The supernatants were collected at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. The amounts of cytokines were then analyzed by ELISA. IL-1β was slightly present at 2 h and kept low level until 24 h, whereas the amount of IL-6 and IL-8 were more than IL-1β in 24 h. The level of IL-8 was
obviously appeared at 2 h. All of these three cytokines were time-dependent increase (Fig.
5a-5c). However, a dissimilar trend of TNF-α expression was observed. A little amount of TNF-α was measured at 1 h, and immediately increased at 2 h, then at 4 h reached a peak (Fig.
5d). This result showed that TNF-α was earliest secreted by THP-1 cells, and afterward other cytokines just appeared. According to this phenomenon, we speculated that TNF-α was the first cytokine THP-1 cells contacted after rHpHSP60 stimulation. Therefore, TNF-α might have a greater effect on THP-1 cells than other cytokines.
3-4 Kinetic of TNF-α mRNA expression
In the present results, we can see that TNF-α was earliest secreted. It was measured at 1h,
and immediately reached maximal amounts between 2 and 4 h. In following experiment, we further investigated that the gene expression kinetic of TNF-α in THP-1 cells with rHpHSP60 treatment. As shown in Fig. 6, the level of TNF-α mRNA expression was slightly increased at 30 min, and reached a peak during 1 and 2 h after rHpHSP60 treatment, as compared to the control. This result revealed that there was a rapid, transient TNF-α mRNA accumulation
between 1 and 2 h after THP-1 cells by rHpHSP60 stimulation.
3-5 Detection of monocytic activation
Since THP-1 cells preferentially secreted TNF-α which was quite associated with monocytes activation. In the following experiments, we further examined THP-1 cells by detecting its endocytosis activity and mature surface marker expression after rHpHSP60 stimulation.
3-5.1 Endocytosis ability of THP-1 cells by rHpHSP60 treatment
THP-1 cells were stimulated with rHpHSP60 for 16 h and then incubated with FITC–dextran for 2 h in order to assess whether the engulf ability of monocytes influenced by rHpHSP60. As shown in Fig. 7, the fluorescence intensity was obviously decrease post-treatment rHpHSP60. The capacity of THP-1 cells to ingest the particles was significantly suppressed by rHpHSP60. The mean fluorescence intensity (MFI) value decreased from 47 ± 10 (untreated cells) to 30 ± 6 (rHpHSP60 treated cells), 43% inhibition was observed.
3-5.2 Surface marker expression on THP-1 cells
THP-1 cells were treated for 16 h with 10 μg/ml rHpHSP60, and the surface marker expression of CD40, CD80, CD86, MHC I and MHC II, being the monocytic activation markers, were evaluated by flow cytometric analysis. As shown in Fig. 8a, a considerable
level of CD40 was up-regulated in cells with rHpHSP60 treatment, and the mean fluorescence intensity (MFI) of treated cells was increased three folds than untreated cells. The expression of costimulatory molecules, such as CD80, CD86 was obviously increased by rHpHSP60 stimulation. The expression of MHC I seemed not influenced by rHpHSP60. Interestingly, the MHC II expression showed a significant down-regulation with the rHpHSP60 treatment.
MHC II present antigens to CD4+ T-helper cells and then control differentiation of B cells in antibody producing B-cell blasts (138). Histograms showed that a clear up-regulation was observed for CD40 and CD80 molecules whereas MHC II expression was obviously down regulated (Figure. 8b)
3-6 Effect of recombinant human TNF-α on CD40 expression and endocytotic activity of THP-1 cells
To examine if TNF-α can trigger THP-1 cells activation, we studied the effects of recombinant human TNF-α (rhTNF-α) on CD40 expression and endocytotic activity of THP-1 cells. Cells were respectively treated with 0.1, 0.5, and 1 ng/ml rhTNF-α for 24 h. As shown in Fig. 9, rhTNF-α significantly up-regulated CD40 expression under a dose of 0.1 ng/ml. The dose-dependent up-regulation of CD40 expression by rhTNF-α was observed. To observe the effect of rhTNF-α on endocytotic activity, we treated THP-1 cells with seven concentrations which were 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 2 ng/ml respectively for 16 h. The
endocytosis level was expressed by relative median fluorescence intensity (RMF). To calculate RMF, we defined the endocytotic ability of cells without rhTNF-α treatment as 100
%. The RMF of cells treated with 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 2 ng/ml rhTNF-α were 99.8 ±
6 %, 94.2 ± 5 %, 90.8 ± 2 %, 86.9 ± 4 %, 86.6 ± 9 %, 87.9 ± 7 %, and 79.4 ± 6 %, respectively. The data showed that rhTNF-α can dose-dependent decrease the endocytotic activity of THP-1cells (Fig. 10).
3-7 Effect of treatment with TGF-β1 and TNF-α on endocytotic activity and CD40 expression of THP-1 cells
TGF-β1 is a multifunctional cytokine that plays a central role in the pathogenesis of
several chronic infectious diseases. A range of macrophage deactivating properties for TGF -β1 has been described previously (139). The TGF-β1-specific staining immunohistochemistry of patients infected with H. pylori from National Taiwan University Hospitalalso showed a high level of TGF-β1 expression at the infection area (Fig. 11). We further studied the effect of synergy of TGF-β1 and TNF-α on endocytotic activity and CD40 expression of THP-1 cells. First, to test the effect of TGF-β1 combined with TNF-α on endocytotic activity, cells incubated with 1ng/ml rhTNF-α alone, 1ng/ml rhTNF-α + 0.5 ng/ml TGF-β1, 1ng/ml rhTNF-α + 1 ng/ml TGF-β1, 1ng/ml rhTNF-α + 10 ng/ml TGF-β1, and 1 ng/ml TGF-β1 for 16 h. FITC-dextran assay was used to test its engulf ability and the
median fluorescence intensity was then analyzed by flow cytomestry. The data was expressed as relative median fluorescence intensity (RFI), and the cells without TNF-α and TGF-β1 identified as 100 %. The RFI of cells treated with 1ng/ml rhTNF-α alone was 88.2 ± 7 %. The RFI of cells incubated with 1ng/ml rhTNF-α and 0.5, 1, and 10 ng/ml TGF-β1 were 80.8 ± 8
%, 76.7 ± 15 %, and 69.6 ± 10 %, respectively. This result clearly showed that TGF-β1 dose-dependent synergized with TNF-α to suppress the endocytotic activity of THP-1 cells (Fig. 12).
Under the same condition, we also tested the CD40 expression on THP-1 cells. As shown in Fig. 13, the data showed as the relative mean fluorescence intensity, and cells without TNF-α and TGF-β1 identified as 100 %. The value of cells with 1ng/ml rhTNF-α treatment was 118.6 ± 15 %, and TGF-β1 can dose-dependent inhibit the TNF-α mediated CD40 expression (1ng/ml rhTNF-α + 0.5 ng/ml TGF-β1: 102 ± 26 %, 1ng/ml rhTNF-α + 1 ng/ml TGF-β1: 90.6 ± 24 %, 1ng/ml rhTNF-α + 10 ng/ml TGF-β1: 87.7 ± 28 %, and 1 ng/ml TGF-β1: 82.5 ± 15 %). This result revealed that TGF-β1 can influence TNF-α-mediated THP-1 cells maturation.