Heat-shock proteins (HSPs) also called stress proteins, are a group of proteins present in both prokaryotic and eukaryotic cells. They are induced when a cell undergoes distinc types of environmental stress. Extracellular HSPs are the most powerful ways of sending a ‘danger signal’ to the immune system in order to generate a response that can help the organism manage an infection or disease and also been reported that are closely associated with the innate or adaptive immune systems activation (140, 141). Bacterial HSPs have also been reported to have the capability to activate human monocytes and macrophages. The 60 kDa heat-shock protein (HSP60), an immunepotent antigen of H. pylori, induces IL-8 secretion from human gastric epithelial cells and monocytic cells (28, 29). Gobert et al. also showed that H. pylori HSP60 can induce IL-6 secretion from macrophages via Toll-like receptors (30).
H. pylori infection leads to gastric inflammation, and the gastric mucosal levels of the
pro-inflammatory cytokines IL-1β, IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) have been reported to be increased in H. pylori-infected subjects (142, 143). Recruitment and activation of immune cells in the underlying mucosa involves chemokines such as IL-8 and pro-inflammatory cytokines secreted by mononuclear phagocytes (TNF-α, IL-1 and IL-6) as part of non-specific immunity (135).
In this study, we detected that the expression of pro-inflammatory cytokines, including
IL-1β, IL-6, IL-8, and TNF-α in THP-1 cells with H. pylori HSP60 stimulation (Fig. 3). All
of these cytokines were significantly increased compared to the cell without rHpHSP60 treatment. IL-1β is a soluble factor secreted by stimulated monocytes (Mo) was measured as 101 ± 38 pg/ml, however, Simms et al. revealed that 20 ng/ml of IL-1 can just cause maximal up-regulation of monocyte phagocytosisv (136). The production of IL-6 in the gastric mucosa is consistently induced by H. Pylori infection and correlate with the development of chronic gastritis (144, 145). The quantities of IL-6 released into culture medium were considerable less than that observed following treatment murine macrophages with rHpHSP60 (30). This phenomenon may be caused by different cell types or the level of LPS involved in the recombinant HpHSP60. A great amount of IL-8 was measured in the culture medium. IL-8 is a pro-inflammatory cytokine, the actions of which are reported to include neutrophil and T-lymphocyte chemotaxis, neutrophil activation and enhanced expression of neutrophil adhesion molecules (146), while IL-8 is certainly not involved in THP-1 cells activation (80).
In contrast, Miyazawa et al. showed that TNF-α can directly activate THP-1 cells to up-regulate the expression of co-stimulatory molecules at concentrations ranging from 39.1 pg/ml to 2500 pg/ml (80). According to this result, we can speculate the concentration of TNF-α measured in the culture medium was able to induce THP-1 cells activation. To exclude LPS contamination from the observed effect, we heated the protein at 95 ℃ for 1 h and TNF-α secretion was abolished. The observed effect of rHpHSP60 on TNF-α expression
was not due to heat-stable LPS (Fig. 4).
Interestingly, the cytokine expression kinetic showed that IL-1β, IL-6, and IL-8 were time-dependent increase whereas TNF-α was secreted to a maximum at 4 h, and it was earlier
present than other cytokines. When THP-1 cells stimulated with mycobacterial 65-kD heat shock protein, the secretion of TNF-α also reached a maximum at 4 h, but IL-6 was obviously present at 8 h, and the expression of IL-8 seems not time-dependent increase (123).
Keratinocytes stimulated with GroEL, the 60 kDa HSP (HSP 60) of E. coli, would time-dependent secret TNF-α, IL-1α, IL-6, and ICAM-1 (137). These reports revealed that the HSP60 of various species even can induce the same cytokines production while the kinetic of cytokine expression was not exactly similar. In the result of kinetic of cytokine expression showed that TNF-α was the earliest secreted cytokine, it may have a great effect on THP-1 cells. TNF-α which is a pro-inflammatory cytokine associates with monocytes activation (Fig.
5). HSPs from pathogens might induce a localized accumulation of self HSP; in turn, would provide a stimulus for autoreactive T-cell proliferation, thereby triggering a cycle of events which may contribute to the pathological damage associated with autoimmune diseases (147, 148). Wei Chen et al. showed that human hsp60 is a danger signal to the innate immune system (115). Human hsp60 induces TNF-α secretion in monocyte-derived macrophage (149), whereas another study suggested that rhHSP60 does not induce TNF-α release from macrophages (150). We found that both rhHSP60 and rHpHSP60 have ability to induce
THP-1 cells to secret TNF-α (Fig. 1 and Fig 14). But the cell counts was not the same between these two experiments. Cells were used to detect human HSP60-induced TNF-α se cretion just at the concentration of 105/ml so that the level of TNF-α secreted from THP-1 cells was low (Fig. 14). Highly homologous between prokaryotic and eukaryotic cells hsp60 are strongly immunogenic and immune responses to microbial hsp60 are speculated to initiate chronic inflammatory diseases in which autoimmune responses to human hsp60 may be central to pathogenesis (151). In this regard, the immune system could be triggered by bacterial antigen, GroEL for example (bacterial hsp60), which share a high degree of homology with self hsp60 proteins, resulting in an aberrant immune response and chronicity of inflammation. Tabata et al. showed that affinity-serum purified antibodies to human hsp60 and P. gingivalis GroEL from selected patients reacted with P. gingivalis GroEL and human hsp60, respectively. They suggested that molecular mimicry between GroEL of the periodontopathic bacterium P. gingivalis and autologous human hsp60 may play some role in immune mechanisms in periodontitis (152). It has been revealed that Chlamydial HSP 60 frequently colocalizes with human HSP 60 in plaque macrophages in human atherosclerotic lesions. Chlamydial and human HSP 60 induce TNF-α and MMP production by macrophages.
Chlamydial HSP 60 might mediate the induction of these effects by C pneumoniae. Induction of such macrophage functions provides potential mechanisms by which chlamydial infections may promote atherogenesis and precipitate acute ischemic events (126). Our colleagues also
detected whether the cross-reaction would occur when THP-1 cells stimulated by rHpHSP60.
We further detected whether human heat shock protein 60 would be secreted from THP-1 cells after rHpHSP60 stimulation. The result showed that human hsp60 was not significantly induced from THP-1 cells after rHpHSP60 treatment (Fig. 15). The OD value of human hsp60 analyzing of 10 μg /ml rHpHSP60 + THP-1 cells and PBS + THP-1 cells was 0.091 ± 0.012 and 0.095 ± 0.005 respectively. In this sandwich ELISA system, the OD value of 1 μg recombinant human hsp60 was 0.574 ± 0.048 as the positive control. According to this result, we suggest that rHpHSP60 can not induce human HSP60 secretion from THP-1 cells. Next, we examined the limitation of human heat shock protein 60 could be detected by the sandwich ELISA system (Fig 16). The data showed that the OD value of 0, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, and 1 μg was 0.087 ± 0.003, 0.087 ± 0.021, 0.086 ± 0.001, 0.09 ± 0.019, 0.083 ±
0.002, 0.013 ± 0.021, 0.195 ± 0.03, and 1.237 ± 0.006 respectively. The concentration of human hsp60 has to reach 1 μg/ml or cannot be detected by the sandwich ELISA system. We concluded that 10 μg rHpHSP60 induced lower than 1 μg human hsp60 or not induction of
human hsp60 expression. Thus, we can completely exclude rHpHSP60 cross reacting with human hsp60 to induce TNF-α secretion. The effect we observed was resulting from rHpHSP60.
Monocytes express a variety of cell surface proteins that are thought to play an important role in antigen presentation and the cell contact-dependent interaction of monocytes with
other leukocytes. In this study, the expression of co-stimulatory molecules, such as CD40, CD80, and CD86, are increased of rHpHSP60 stimulation (Fig. 8). HLA-ABC (MHC I) was not significantly affected by rHpHSP60. Interestingly, the expression of HLA-DR (MHC II) was down-regulated by rHpHSP60 treatment (Fig. 8), as previously observed with heat-killed
Mycobacterium bovis and Escherichia coli (153). CD40 binds to the CD40 ligand (CD40L)
on T cells. In addition, CD40-CD40L interactions may also play an important role in the cell contact-dependent interaction that occurs between activated helper T cells and monocytes/macrophages during antigen presentation (154). In vitro, ligation of the monocyte CD40 surface protein with the T cell-derived CD40L increases the production of cytokines such as IL-1, TNF, and IL-6 through an ERK1/2-dependent pathway (154, 155). Nevertheless, the CD40-CD40L interactions would result in the up-regulation of matrix metalloproteinases which both facilitate the movement of the cells out of the vasculature into surrounding stroma and to sites of inflammation as well as accelerate the breakdown of ECM during chronic inflammatory diseases (156). The co-stimulatory ligands CD80 and CD86 play a crucial role in the initiation and maintenance of an immune response. The interaction betweenCD28 expressed on T cells and its counter-receptors CD80 (B7-1)and CD86 (B7-2) expressed on specialized APC provides the mostimportant co-stimulatory signal (157, 158). In our results, the up-regulation of CD40, CD80, and CD86 was observed in THP-1 cells stimulated by rHpHSP60. The expression of MHC I seemed not influenced by rHpHSP60. However, the
down-regulation of HLA-DR (MHC II) was showed after rHpHSP60 treatment. Cytosolic proteins like viral proteins, or bacterial proteins with access to the host’s cytosol, are degraded by the proteasome and bind to MHC class I molecules in the endoplasmic reticulum, leading to cytolytic CD8+ T cell responses. Antigens sequestered from the cytosol in endocytic or phagocytic compartments encounter their partners through fusion with endocytic vesicles containing MHC class II molecules. MHC class II is believed to play a central role in initiating the immune response by presenting the foreign antigen to T helper cells. MHC II present antigens to CD4+ T-helper cells and then control differentiation of B cells in antibody producing B-cell. Patients or mice failing to produce proper MHC II–peptide complexes will not produce efficient antibody responses to infection (138). According to these reports, the antigen presenting capacity of THP-1 cells seems diminished by rHpHSP60, and the adaptive immune response could not be exactly activated consequently.
Monocytes play a pivotal role as professional phagocytic other than antigen-presenting cells in the innate immunity. In our study, the engulf ability of THP-1 cells was manifestly inhibited after stimulation THP-1 cells with rHpHSP60 for 16 h (Fig. 7). The accurately mechanism was still unknown. But we can try to explain why the expression of MHC class II was down-regulated. Antigen presentation process that involves the uptake of complex forms of antigens. Then, antigens sequestered from the cytosol in endocytic or phagocytic compartments encounter their partners through fusion with endocytic vesicles containing
MHC class II molecules, leading to CD4+ T cell responses (159, 160).
The findings above show that H. pylori HSP60 can stimulate THP-1 cells to produce pro-inflammatory cytokines, including IL-1β, IL-6, IL-8, and TNF-α; hence it was quite correlated with the occurrence of H. pylori-associated gastric inflammation. However, the activity of THP-1 cells seems be weakened by H. pylori HSP60. Although the expression of co-stimulatory molecules (CD40, CD80, and CD86) was up-regulated, the MHC class II was contrarily down-regulated. Presumably the adaptive immune response would be influenced consequently. Furthermore, the engulf capacity of THP-1 cells was obviously abated by H.
pylori HSP60. We tried to figure out whether these phenomenons were associated with TNF-α,
the rhTNF-α was used in the following experiments. Treatment with rhTNF-α can augment
CD40 expression in a dose-dependent manner, but down-regulated the endocytotic activity of THP-1 cells (Fig 9 and Fig 10). It seems that TNF-α can promote THP-1 cells maturation but suppress it’s engulf ability under our systems. There were few studies directly using rhTNF-α to stimulate monocytes and examine the effect of rhTNF-α on endocytotic activity. According to previous reports, many publishes revealed that TNF-α was able to augment the monocytes antibacterial activity in vitro experiments with human monocytes (161, 162). Zerlauth et al.
showed that TNF-α plays a crucial role in the activation of monocytes for growth restriction of intracellular microbes, including Mycobacterium avium intracellulare and L.
monocytogenes (161). However, the studies which prove the TNF-α can enhance endocytotic
activity of THP-1 cells are really deficient. Roilides et al. revealed that incubation of human monocytes with TNF-α at 0.001 to 10 ng/ml for 2 days had no effect on the percent phagocytosis of conidia (163), however, Kathleen et al. showed that the effect of TNF-α on
mature macrophages from mouse or human was to reduce the clearance of apoptotic cells rather than to enhance the uptake and the conclusion of the study was TNF-α stimulation of mature macrophages induces oxidant production through cPLA2 activation and arachidonic acid release leading to increased active Rho and decreased efferocytic function (164). On the other hand, TNF-mediated skewing of monocyte differentiation toward DCs could be observed at a concentration of 1 ng/ml TNF and reached a peak at 10 ng/ml, however, the phagocytosis ability of various phagocytic cells was monocyte-derived macrophage ＞ monocytes ＞ immature dendritic cells ＞ polymorphonuclear neutrophilic leukocytes ＞ matute dendritic cells (165). We tried to explain the effect of TNF-α on THP-1 cells we observed was that if TNF-α earlier contact to THP-1 cells, it may skew cells to differentiate to dendritic cells phenotype and then cells turn to a professional antigen presenting cells and phagocytosis ability was weakened.
TGF-β1 is a multifunctional cytokine that plays a central role in the pathogenesis of several chronic infectious diseases. The ability of TGF-β1 to interfere with the activation and oxidative mechanisms of macrophages (139) has been implicated in promoting persistent infection by providing a survival advantage to pathogens. In this regard, the ability of
pathogens to elicit TGF-β1 expression has been hypothesized as a potential virulence trait (107, 166). The TGF-β1-specific staining of immunohistochemistry showed that gastric epithelium of people long-term infected with H. pylori expressed a high level of TGF-β1 (Fig.
11). As for the pro-inflammatory cytokines, the frequencies of TGF-β1-specific cells were also higher in the H. pylori-infected subjects than in the uninfected subjects (45). In this study, we further investigated the immune cells at the late stage of inflammation region where filled with pro-inflammatory cytokines and regulatory cytokines, such as TNF-α and TGF-β1 respectively. Monocytes require 1 to 10 ng/ml of TGF-β to inhibit peroxide secretion and microbial killing. TGF-β can impair human monocyte functions on release of H2O2 and 02-, adherence and phagocytosis. Moreover, increased levels of TGF-β have been reported in the
monocytes and granulomatous lesions of tuberculosis patients (167). Several pathogens have been found that they can utilize TGF-β1 to escape from host immune attack, such as
Trypanosoma cruzi (168), Mycobacterium avium (99), and the Mycobacterium tuberculosis
strain (100). Our results showed that TNF-α alone (the concentration between 0.4 ng ~ 2 ng) could suppress endocytotic activity of THP-1 cells, and when synergized with TGF-β1 in a
dose-dependent mannar, the inhibition effect was more obviously (Fig. 12). It seems like TNF-α synergized with TGF-β1 to diminish the endocytotic activity of THP-1 cells.
Treatment of both HL60 and U937 cells with TNF-α induced a dose-dependent increase in expression of TGF-β receptors, suggesting that the synergy between TNF-α and TGF-β1 may
result from upregulation of TGF-β1 receptor expression by TNF-α (169). Thus, we reasonably supposed that TNF-α may favorably augment TGF-β1-mediated suppression effect on endocytosis ability of THP-1 cells. On the other hand, we also found that TGF-β1 would suppress TNF-α-mediated CD40 expression on THP-1 cells in a dose-dependent manner (Fig. 13). In terms of statistics, the value of this data was not significant between each condition but it can still give us a hint among this result.
In conclusion, H. pylori HSP60 can induce THP-1 cells to produce pro-inflammatory cytokines; including IL-1β, IL-6, IL-8 and TNF-α which are quite associated with gastric inflammation occurrence. Among these cytokines, TNF-α was earliest secreted by THP-1
cells and it may have a greater effect on cells than other cytokines. We can completely exclude from contaminating with LPS or cross-reacting with human hsp60 causing TNF-α expression. Although pro-inflammatory cytokines, especially TNF-α have been reported to activate immune activity, however, an inhibition effect of endocytosis activity was observed in our result. The costimulatory molecules (CD40, CD80, CD86) was up-regulated on THP-1 cells surface, whereas the expression of MHC class II was decrease. This effect would influence the CD4+ T cell responses. Treatment THP-1 cells with recombinant human TNF-α surprisingly found that endocytosis activity of THP-1 cells was decrease in a dose dependent manner, whereas the CD40 expression was up-regulation on cell surface as our expected. This study suggested that rHpHSP60 induces proinflammatory cytokines secretion
but diminishes monocytes activation.
Table 1
Table 1. Oligonucleotides for quantitative real-time PCR. F and R indicate forward and reverse primers, respectively; numbers indicate the sequence position.
Figure 1. Identification of recombinant H. pylori hsp60 plasmid by restriction enzyme digestion. Lane 1: Recombinant plasmid without restriction enzyme, Lane 2: Recombinant plasmid digested with EcoR I and Xho I. Lane 3: Nucleotide marker.
Insert:
Hphsp60
(a)
(b)
Figure 2. 1 SDS-PAGE and western blot analysis of the recombinant protein expressed in BL21. (a) SDS-PAGE analysis of the recombinant H. pylori protein expression. Lane 1:
molecular weight marker (20,30,43,67, and 94 kD),Lane 2: Control strain BL21 (pET) before induction, Lane3: Control strain BL21(pET) after 4 h induction with IPTG, Lane 4:
Purified recombinant protein Hsp60 by HisTrapTM HP column, Lane 5: Purified recombinant protein Hsp60 passing through Sephadex G-25 gel filtration column. (b) Western bolt analysis of the recombinant H. pylori protein expression. Lane 1: Control strain BL21(pET) before induction,Lane 2: Control strain BL21(pET) after 4 h induction with IPTG, Lane3: Purified recombinant protein Hsp60 by HisTrapTM HP column,Lane 4: Purified recombinant protein Hsp60 passing through Sephadex G-25 gel filtration column.
(a) (b)
(c) (d)
Figure 3. Production of pro-inflammatory cytokines in THP-1 cells. (a) IL-1β, (b) IL-6, (c) IL-8 and (d) TNF-α secretion in THP-1 cells in response to rHpHSP60. Recombinant heat shock protein 60 was added to medium containing 5×105 cells to a final concentration of 10 μg/ml incubated in 24-well plate for 24 h. Cytokines were measured by ELISA in 24 h culture medium. Results are representative at least three independent experiment. ★, P < 0.001 compared to without rHpHSP60 treatment. (n=3)
Figure 4. Release of TNF-α from THP-1 cells incubated with rHpHSP60 or heated rHpHSP60.
THP-1 cells stimulated with rHpHSP60 or rHpHSP60 heating to 95 ℃ for 1 h. After 16 h, the production of TNF-α analyzed by ELISA. TNF-α expression due to rHpHSP60 but not to LPS is sensitive to heating 95 ℃ for 1 h, further indicating that the observed effect of rHpHSP60 is not a consequence of contamination by LPS. ★, P < 0.01
★
(a) (b)
(c) (d)
Figure 5. Kinetics of pro-inflammatory cytokine secretion in THP-1 cells. (a) IL-1β, (b) IL-6, (c) IL-8, and (d) TNF-α secretion by THP-1 cells stimulated with 10 μg/ml rHpHSP60.
Cytokines were measured in culture supernatants at eight different time-points. Results are representative at least three independent experiment. ★, P <0.01 compared to without HpHSP60 treatment. (n=3)
Figure 6. Kinetic of TNF-α mRNA gene expression in THP-1cells. THP-1 cells stimulated by 10 μg/ml rHpHSP60 and the cells were collected at 5 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. TNF-α mRNA expression in THP-1 cells was analyzed by quantitative real-time PCR.
Maximal TNF-α mRNA significantly expression between 1 and 2 h post-stimulation.★, P
< 0.01 compared to without rHpHSP60 treatment. (n= 4)
(a)
(b)
Figure 7. Endocytotic ability of THP-1 cells treated with rHpHSP60. THP-1 cells were cultured with or without rHpHSP60 for 16 h. and then incubated with FITC- dextran for 2 h at 37 ℃ or at 0 ℃ as a control for endocytosis. Cells were analyzed by flow cytometry. (a) The solid histogram indicated cells without rHpHSP60 treatment and the thick-lined histogram indicated cells with rHpHSP60 treatment. (b) The mean fluorescence intensity of cells is reported. Data are expressed as means ± standard deveations from three experiments. ★,P<
0.05 compared to untreated cells. (n=3)
★
Untreated rHpHSP60 rHpHSP60 treated
(a)
CD40 CD80
CD86 MHC I
MHC II
(b)
NC CD40
CD80 CD86
MHC I MHC II
Figure 8. Surface marker expression on THP-1 cells treated with rHpHSP60. Flow cytometric
Figure 8. Surface marker expression on THP-1 cells treated with rHpHSP60. Flow cytometric