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Toxoplasma gondii-derived heat shock protein 70 stimulates the maturation of human monocyte-derived dendritic cells
BBRC Biochemical and Biophysical Research Communications 322 (2004) 899–904 www.elsevier.com/locate/ybbrc
Toxoplasma gondii-derived heat shock protein 70 stimulates the maturation of human monocyte-derived dendritic cells Hyun Kyu Kanga,b, Ha-Young Leea,c, Youl-Nam Leea, Eun Jin Joc, Jung Im Kima,c, Fumie Aosaib, Akihiko Yanob, Jong-Young Kwaka,c, Yoe-Sik Baea,c,* a b
Medical Research Center for Cancer Molecular Therapy, Dong-A University, Busan 602-714, Republic of Korea Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan c Department of Biochemistry, College of Medicine, Dong-A University, Busan 602-714, Republic of Korea Received 29 July 2004
Abstract We investigated the role of Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) as a dendritic cell (DC) maturation-inducing molecule. TgHSP70 induced the maturation of human monocyte-derived dendritic cells as determined by increased levels of surface markers, namely, CD40, CD80, CD86, and HLA-DR. Moreover, TgHSP70 also reduced phagocytic activity and increased the allostimulatory capacity of DCs, suggesting the functional maturation of DCs by TgHSP70. Maturation of DCs by TgHSP70 also elicited a significant increase in IL-12 production in a polymyxin B-insensitive manner. TgHSP70 also stimulated extracellular signal-regulated kinase and p38 kinase pathways in DCs, and TgHSP70-induced IL-12 production was inhibited by SB203580 but not by PD98059, thus indicating the role of p38 kinase in the maturation of DCs by TgHSP70. This study demonstrates the role of TgHSP70 in the functional maturation of DCs and suggests TgHSP70 as a useful molecule for the development of a vaccine against T. gondii. 2004 Elsevier Inc. All rights reserved. Keywords: Toxoplasma gondii; HSP70; Dendritic cells; Maturation
Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) is known to play a role in the modulation of immune responses. TgHSP70 is a potent virulence factor in murine toxoplasma infection [1,2]. In terms of its mode of action, TgHSP70 has been suggested to inhibit the expression of nitric oxide synthase and to inhibit NF-jB activation [1]. Recently Mohamed et al. [3] demonstrated that DNA vaccination with TgHSP70 strongly enhanced the induction of protective immunity. Taken together, these results suggest a potential role of TgHSP70 as a modulator of adaptive immune response. However, the role of TgHSP70 in adaptive immune responses has not been elucidated.
*
Corresponding author. Fax: +82 51 241 6940. E-mail address: [email protected] (Y.-S. Bae).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.205
Dendritic cells (DCs) play a key role in the regulation of adaptive immune responses. DCs capture non-self antigen and present the processed antigen on the cellular surface with MHC complex, which is recognized by T cell receptor and results in T cell activation [4–6]. DCs are sparsely distributed throughout the body and are present in most tissues in an immature state, which shows a high capacity for antigen uptake and processing [5,6]. Once activated by inflammatory stimuli or infectious agents, immature DCs undergo maturation, a process that involves the acquisition of high levels of surface molecules including HLA-DR, CD40, CD83, and CD86 [7,8]. Mature DCs also acquire remarkable ability to produce a broad panel of cytokines, including IL-12 [4]. Various extracellular stimuli have been reported to stimulate the DC maturation. These include LPS and TNF-a [9,10].
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In this study, we aimed to investigate the effect of TgHSP70 on DC maturation. We examined the up-regulation of cell surface markers of DC and the functional activity of TgHSP70-stimulated DCs. DCs stimulated by TgHSP70 appeared morphologically and functionally to be mature DCs.
Liquid Scintillation Counter (TRI-CARB 2100 TR, Packard, Meriden, CT).
Results Morphological change of human monocyte-derived DCs by TgHSP70
Materials and methods Reagents and antibodies. RPMI 1640 was purchased from Invitrogen (Carlsbad, CA). Dialyzed fetal bovine serum and supplemented bovine calf serum were purchased from Hyclone Laboratory (Logan, UT). For FACS analysis, phycoerythrin (PE)-conjugated monoclonal antibodies against surface molecules (CD40, CD80, and CD86) and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against HLA-DR were obtained from Pharmingen/Becton–Dickinson (San Diego, CA). Fluorescein-conjugated dextran was purchased from Sigma (St. Louis, MO). LPS (from Escherichia coli strain 055:B5) was obtained from Sigma (St. Louis, MO). TgHSP70 was prepared as described before [11]. Generation of monocyte-derived DC. Peripheral blood was collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated by separation on a Histopaque-1077 gradient. After two washings with HanksÕ buffered saline solution (HBSS), without Ca2+ and Mg2+, PBMCs were suspended in RPMI medium containing 10% FBS and incubated for 60 min at 37 C to allow monocyte attachment to the culture dish. Attached monocytes were then collected as described previously [12]. Peripheral blood monocytes were differentiated to DCs by culturing the cells in a 6well plate in 2 ml of complete medium (RPMI 1640 supplemented with 10% FCS) supplemented with recombinant human GM-CSF (10 ng/ml; Pierce Endogen, Rockford, IL) and recombinant human IL-4 (10 ng/ml; Pierce Endogen, Rockford, IL). All cultures were incubated at 37 C in 5% humidified CO2. After 7 days of culture, DCs were stimulated with 100 ng/ml LPS or 1 lg/ml TgHSP70 for 48 h for maturation. FACS analysis. Cells were incubated with PE-labeled monoclonal antibodies against CD40, CD80, CD86, and FITC-labeled monoclonal antibody against HLA-DR for 30 min, washed with cold-PBS, and analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter, Miami, FL). Measurement of phagocytic activity. Phagocytosis was measured after 48 h treatments of vehicle, LPS, or TgHSP70. Cells were incubated in a 200 ll PBS containing 5% human serum with 1 mg/ ml fluorescein isothiocyanate (FITC)-dextran. After 30 min incubation at 37 C, cells were washed four times in ice-cold PBS and analyzed with a flow cytometer. For a control, cells from each culture condition were also maintained in the same solution for 30 min at 4 C. Cytokine assays. Human monocyte-derived dendritic cells (5 · 105 cells/ml) were treated with vehicle, LPS, or TgHSP70 in the presence or absence of PD98059 (50 lM), or SB203580 (20 lM) for 48 h. Culture supernatants were analyzed by ELISA for IL-12 (p70) according to the instructions from the manufacturer (BD Biosciences Pharmingen, San Diego, CA). Measurement of allostimulatory activity. T cells were purified from human PBMCs using nylon wool columns and were used as responders [13]. Human monocyte-derived dendritic cells, which were treated with vehicle, LPS, or TgHSP70 were used as stimulator cells. A total of 1 · 105 allogeneic T cells were added as responders to stimulator cells (1 · 104, 3 · 103, 1 · 103, or 3 · 102) in 96-well tissue culture plates containing 0.2 ml medium/well. Allostimulatory activity was measured for 3 days and the cells were pulsed with 0.5 lCi [3H]thymidine for the last 18 h. Radioactivity was counted using
To assess whether TgHSP70 cause morphological change in human immature DCs (iDCs), their morphology was observed after 48 h of TgHSP70 treatment. A morphological change of iDCs into mDCs was clearly observed in TgHSP70-treated cells (data not shown). These morphological changes in iDCs suggested that TgHSP70 induces iDC maturation. To further explore this possibility, we looked for phenotypic changes in TgHSP70-treated iDCs by using FACS and specific antibodies against HLA-DR molecules and co-stimulatory molecules (Figs. 1A and B). TgHSP70 treatment enhanced the expression of the HLA-DR molecules and CD80, CD86, and CD40, a response which could be compared with the phenotypic changes in iDCs treated with LPS (Fig. 1 and data not shown). Taken together, these data suggest that TgHSP70 promotes the maturation of iDCs. TgHSP70 reduced the phagocytic activity of DCs Immature DCs capture and process antigens as a consequence of their high endocytic activity, a feature that is lost during maturation [5,14]. Compared with LPS-treated cells, cells treated with TgHSP70 expressed higher levels of CD80 and CD86, which have been reported to be more highly expressed in mature DCs than in immature DCs [1]. This finding that the stimulation of iDCs with TgHSP70 induced higher expressions of CD80 and CD86 led us to investigate whether TgHSP70 stimulation elicits cellular maturation. When we stimulated iDCs with TgHSP70 the phagocytic activity of the cells significantly decreased, suggesting that the cells are mDCs (Fig. 2). This result indicates that the TgHSP70-mediated signaling pathway may be involved in DC maturation. TgHSP70 increases DC allostimulatory capacity DC function can be characterized in part by an ability to stimulate alloreactive T cells by MLR [1]. To determine whether DCs stimulated by TgHSP70 have allostimulatory activity, MLR was performed using TgHSP70-treated iDCs. As shown in Fig. 3, TgHSP70 has allostimulatory activity, which is comparable with that of LPS-treated iDCs at several responder: stimulator ratios. This result indicates that iDCs stimulated by TgHSP70 are functionally mature DCs. In view of the
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Fig. 2. TgHSP70 affects the phagocytic activity of human monocytederived dendritic cells. Human monocyte-derived dendritic cells were cultured for 48 h in the presence of vehicle (bold solid line), LPS (100 ng/ml) (dashed line), or TgHSP70 (1 lg/ml) (solid line) and their phagocytic activity was determined by flow cytometry analysis using FITC-conjugated dextran (A). The shaded area indicates unstained cells (A). Phagocytic activity was quantified by measuring mean fluorescence intensity (B). The results are representative of more than three independent experiments. Fig. 1. Human monocyte-derived dendritic cells treated with TgHSP70 show a mature dendritic cell phenotype. Human monocyte-derived dendritic cells were treated with vehicle alone (A) or treated with TgHSP70 (1 lg/ml) (B) for 48 h. Cell surface expressions of CD40, CD80, CD86, and HLA-DR were determined by flow cytometry analysis (A,B), and the surface expressions of these molecule are presented as mean fluorescence intensities (C). The results are representative of more than three independent experiments.
fact that mature DCs have reduced phagocytic activity as compared to immature DCs, this result correlates well with Fig. 2. TgHSP70-induced DC surface marker changes are polymyxin B-insensitive To assess whether TgHSP70 induce the maturation of DCs via LPS receptors, we investigated the effect of polymyxin B (a potent inhibitor of LPS). At first we found that polymyxin B alone did not affect CD86 expression in DCs (Fig. 4A). When polymyxin B was
Fig. 3. Allostimulatory capacity of human monocyte-derived dendritic cells treated with vehicle, LPS, or TgHSP70. Human monocyte-derived dendritic cells were cultured for 48 h in vehicle, LPS (100 ng/ml), or TgHSP70 (1 lg/ml). Cells were harvested and then used to stimulate 1 · 105 of allogeneic T cells purified from human PBMCs in a 96-well plate. Stimulation was done at several responder: stimulator ratios. Cells were pulsed with [3H]thymidine on day 3, and incorporation was measured after 18 h. Results represent means ± SE counts/min of triplicate results.
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Fig. 5. IL-12 production by TgHSP70-treated human monocytederived dendritic cells. Human monocyte-derived dendritic cells were stimulated with vehicle, LPS (100 ng/ml), or TgHSP70 (1 lg/ml) for 48 h in the absence or presence of polymyxin B (10 lg/ml), and supernatants were harvested from cultures and analyzed for the production of IL-12(p70) by ELISA. Data represent means ± SE of more than three experiments.
at 143.3 ± 1.46 pg/ml (Fig. 5). We also investigated the effect of polymyxin B on LPS- and on TgHSP70-induced IL-12 production. Preincubation of iDCs with 10 lg/ml of polymyxin B prior to LPS addition completely blocked IL-12 production by LPS (Fig. 5), but TgHSP70-induced IL-12 production was unaffected by polymyxin B (Fig. 5), indicating again that TgHSP70 stimulates IL-12 production via a different mechanism than that used by LPS. Fig. 4. Effect of polymyxin B on TgHSP70-induced dendritic cell maturation. Human monocyte-derived dendritic cells were treated with vehicle alone (A), LPS (100 ng/ml) (B), or TgHSP70 (1 lg/ml) (C) for 48 h in the absence or presence of polymyxin B (10 lg/ml). CD86 cell surface expressions were determined by flow cytometry analysis (A–C). Bold solid lines and thin solid lines represent polymyxin B-untreated and polymyxin B-treated cells, respectively. The dashed lines represent unstained control cells. The results are representative of more than three independent experiments.
added to culture, LPS-induced CD86 expression was almost completely inhibited (Fig. 4B), but TgHSP70-induced CD86 expression was unaffected (Fig. 4C). These results indicate that TgHSP70 induces DC maturation via a non-LPS mechanism. TgHSP70 induces the production of IL-12 in a polymyxin B-insensitive manner in DCs Since IL-12 is a key cytokine responsible for polarizing T cells toward the Th1 phenotype [15] and DC maturation, we assessed whether TgHSP70 alters cytokine production in iDCs. When iDCs were stimulated with TgHSP70, IL-12 secretion was significantly increased versus control cells (Fig. 5); the addition of 1 lg/ml TgHSP70 elicited 182.5 ± 5.60 pg/ml of IL-12 production (Fig. 5). As a positive control, we also examined the effect of LPS on IL-12 production in the cells. Stimulation of LPS in iDCs for 48 h caused IL-12 production
TgHSP70 stimulates mitogen-activated protein kinase activity in DCs Mitogen-activated protein kinase (MAPK) has been reported to mediate extracellular signals to the nucleus in various cell types [16]. In this study, we examined whether TgHSP70 stimulates MAPKs by Western blotting with anti-phospho-specific antibodies to each enzyme. When DCs were stimulated with TgHSP70 (at 1 lg/ml) for different times, the phosphorylation level of extracellular signal-regulated protein kinase (ERK) transiently increased, and showed maximal activity after 5–30 min of stimulation (Fig. 6A), and then returned to the baseline 60 min after stimulation (Fig. 6A). Another important MAPK, p38 kinase, was also transiently phosphorylated after TgHSP70 stimulation with kinetics resembling those of ERK phosphorylation (Fig. 6A). The stimulation of DCs with LPS (100 ng/ml) also caused ERK and p38 kinase phosphorylation in a manner similar to TgHSP70 (Fig. 6A). We also investigated the roles of the two MAPKs (ERK and p38 kinase) on TgHSP70-induced DC maturation using two different MAPK inhibitors. SB203580, a selective p38 kinase inhibitor, inhibited TgHSP70-induced IL-12 production by 53% (Fig. 6B) however, PD98059, a selective MEK inhibitor, did not affect TgHSP70-induced IL-12 production. These results indicate that the p38 kinasedependent signaling pathway, and not MEK-dependent
H.K. Kang et al. / Biochemical and Biophysical Research Communications 322 (2004) 899–904
Fig. 6. Role of MAPKs on TgHSP70-induced dendritic cell maturation. Human monocyte-derived dendritic cells were stimulated with LPS (100 ng/ml) or TgHSP70 (1 lg/ml) for different times. Thirty microgram aliquots of the cell lysates were subjected to SDS–PAGE. This was followed by immunoblot analysis with anti-phospho-ERK, -phosphop38 kinase antibodies (A). Western blot analysis was also performed with anti-ERK antibody (A) to confirm similar sample loadings. The data shown are representative of four independent experiments (A). Human monocyte-derived dendritic cells were pretreated with DMSO, 50 lM PD98059, or 20 lM SB203580 for 1 h and then treated with vehicle, LPS (100 ng/ml) or TgHSP70 (1 lg/ml) for 48 h (B). Supernatants were then collected, and IL-12 release was determined by ELISA. Data represent means ± SE of more than three experiments (B).
ERK pathway, is essentially involved in TgHSP70-induced DC maturation.
Discussion It has been demonstrated that T. gondii infection induces high levels of several cytokines, such as IL-12 and TNF-a. DCs are one of the major cells that produce IL-12 after pathogen-infection, and the injection of T. gondii antigen was also found to rapidly produce IL-12 in experimental animals [17]. In this study, we found that HSP70 derived from T. gondii stimulated IL-12 production in human DCs (Fig. 5). Our results suggest that TgHSP70 is a major DC stimulating antigen in T. gondii infection. HSP70 is a well-conserved protein in all organisms and is a major immunogen in infections caused by various pathogens. Mohamed et al. [3] showed that DNA vaccination with TgHSP70 induces protective immunity against T. gondii infection. Bearing in mind that DCs play a crucial role in immune responses against the vaccination of pathogen-derived
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antigens, our results on DCs maturation and IL-12 production by TgHSP70 suggest that HSP70 derived from T. gondii are critically involved in protective immunity against T. gondii by modulating DC activity. Since, LPS from E. coli was reported to induce the maturation of human monocyte-derived DCs [5], and TgHSP70 was prepared from E. coli, we tested for the possible contamination of LPS in TgHSP70. As shown in Fig. 4, DC maturation by TgHSP70 was not affected by polymyxin B. We also found that IL-12 production in TgHSP70-treated DCs was not inhibited by polymyxin B (Fig. 5). These results ruled out possible contamination by LPS in the TgHSP70 preparation from E. coli. Previous reports have demonstrated that Toll like receptors (TLR)-2 and TLR-4 are cellular receptors for HSP70 [18,19]. Moreover, TLR-4 was reported to be an essential molecule for TgHSP70-induced B cell activation in an experimental mice model [11]. Recently Mun et al. [20] also demonstrated that TLR-2 is an essential molecule for protective immunity against T. gondii infection in an experimental mouse model. These findings provide clues as to the identity of the molecule involved in TgHSP70-induced human monocyte-derived DC maturation. But, the roles of TLR-2 and TLR-4 on TgHSP70-induced DC maturation require clarification. Some reports have suggested the involvement of several other molecules, such as CD14, CD91, and CD40, on HSP70-mediated cellular response [21–23]. Moreover, investigations of the putative roles of CD14, CD91, and CD40 on HSP70-induced DC maturation are required. It is well known that DC maturation can be regulated by MAPKs [24,25]. To investigate the intracellular signaling involved in TgHSP70-induced DC maturation, we checked the effect of TgHSP70 on the phosphorylation of MAPKs. As shown in Fig. 6, the stimulation of DCs with TgHSP70 induced the phosphorylation of two MAPKs (ERK and p38 kinase) in DCs. To elucidate the signaling pathway leading to robust IL-12 production in TgHSP70-treated DCs, we used MAPK inhibitors to assess the amounts of IL-12 produced. Unlike the results obtained using PD98059, IL-12 production in the presence of SB203580 was significantly reduced in TgHSP7-treated DCs (Fig. 6B). These results are in agreement with previous reports of the positive effects of the p38 kinase signaling pathway on IL-12 production, and its influence on DC maturation [24]. However, PD98059 did not affect TgHSP70-induced IL-12 production in DCs (Fig. 6B). These results suggest that p38 kinase signaling but not ERK signaling has a positive effect on TgHSP70-induced IL-12 production in DCs. In summary, this study demonstrates that mature DCs are generated from human monocyte-derived DCs by TgHSP70. Since TgHSP70 has been reported to be a major virulent factor of T. gondii and to be essential for the induction of protective immunity against the
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pathogen, our results on the role of TgHSP70 as a DC maturation-inducing molecule provide clues about the action mechanism of TgHSP70 in immune response. Further studies are needed to elucidate the precise cellular receptor for TgHSP70 and the molecular details of its action mechanism.
[12]
[13]
Acknowledgment This work was supported by the Korea Science and Engineering Foundation through the Medical Science and Engineering Research Center for Cancer Molecular Therapy at Dong-A University.
[14]
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Toxoplasma gondii-derived heat shock protein 70 stimulates the maturation of human monocyte-derived dendritic cells Hyun Kyu Kanga,b, Ha-Young Leea,c, Youl-Nam Leea, Eun Jin Joc, Jung Im Kima,c, Fumie Aosaib, Akihiko Yanob, Jong-Young Kwaka,c, Yoe-Sik Baea,c,* a b
Medical Research Center for Cancer Molecular Therapy, Dong-A University, Busan 602-714, Republic of Korea Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan c Department of Biochemistry, College of Medicine, Dong-A University, Busan 602-714, Republic of Korea Received 29 July 2004
Abstract We investigated the role of Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) as a dendritic cell (DC) maturation-inducing molecule. TgHSP70 induced the maturation of human monocyte-derived dendritic cells as determined by increased levels of surface markers, namely, CD40, CD80, CD86, and HLA-DR. Moreover, TgHSP70 also reduced phagocytic activity and increased the allostimulatory capacity of DCs, suggesting the functional maturation of DCs by TgHSP70. Maturation of DCs by TgHSP70 also elicited a significant increase in IL-12 production in a polymyxin B-insensitive manner. TgHSP70 also stimulated extracellular signal-regulated kinase and p38 kinase pathways in DCs, and TgHSP70-induced IL-12 production was inhibited by SB203580 but not by PD98059, thus indicating the role of p38 kinase in the maturation of DCs by TgHSP70. This study demonstrates the role of TgHSP70 in the functional maturation of DCs and suggests TgHSP70 as a useful molecule for the development of a vaccine against T. gondii. 2004 Elsevier Inc. All rights reserved. Keywords: Toxoplasma gondii; HSP70; Dendritic cells; Maturation
Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) is known to play a role in the modulation of immune responses. TgHSP70 is a potent virulence factor in murine toxoplasma infection [1,2]. In terms of its mode of action, TgHSP70 has been suggested to inhibit the expression of nitric oxide synthase and to inhibit NF-jB activation [1]. Recently Mohamed et al. [3] demonstrated that DNA vaccination with TgHSP70 strongly enhanced the induction of protective immunity. Taken together, these results suggest a potential role of TgHSP70 as a modulator of adaptive immune response. However, the role of TgHSP70 in adaptive immune responses has not been elucidated.
*
Corresponding author. Fax: +82 51 241 6940. E-mail address: [email protected] (Y.-S. Bae).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.205
Dendritic cells (DCs) play a key role in the regulation of adaptive immune responses. DCs capture non-self antigen and present the processed antigen on the cellular surface with MHC complex, which is recognized by T cell receptor and results in T cell activation [4–6]. DCs are sparsely distributed throughout the body and are present in most tissues in an immature state, which shows a high capacity for antigen uptake and processing [5,6]. Once activated by inflammatory stimuli or infectious agents, immature DCs undergo maturation, a process that involves the acquisition of high levels of surface molecules including HLA-DR, CD40, CD83, and CD86 [7,8]. Mature DCs also acquire remarkable ability to produce a broad panel of cytokines, including IL-12 [4]. Various extracellular stimuli have been reported to stimulate the DC maturation. These include LPS and TNF-a [9,10].
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H.K. Kang et al. / Biochemical and Biophysical Research Communications 322 (2004) 899–904
In this study, we aimed to investigate the effect of TgHSP70 on DC maturation. We examined the up-regulation of cell surface markers of DC and the functional activity of TgHSP70-stimulated DCs. DCs stimulated by TgHSP70 appeared morphologically and functionally to be mature DCs.
Liquid Scintillation Counter (TRI-CARB 2100 TR, Packard, Meriden, CT).
Results Morphological change of human monocyte-derived DCs by TgHSP70
Materials and methods Reagents and antibodies. RPMI 1640 was purchased from Invitrogen (Carlsbad, CA). Dialyzed fetal bovine serum and supplemented bovine calf serum were purchased from Hyclone Laboratory (Logan, UT). For FACS analysis, phycoerythrin (PE)-conjugated monoclonal antibodies against surface molecules (CD40, CD80, and CD86) and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody against HLA-DR were obtained from Pharmingen/Becton–Dickinson (San Diego, CA). Fluorescein-conjugated dextran was purchased from Sigma (St. Louis, MO). LPS (from Escherichia coli strain 055:B5) was obtained from Sigma (St. Louis, MO). TgHSP70 was prepared as described before [11]. Generation of monocyte-derived DC. Peripheral blood was collected from healthy donors, and peripheral blood mononuclear cells (PBMCs) were isolated by separation on a Histopaque-1077 gradient. After two washings with HanksÕ buffered saline solution (HBSS), without Ca2+ and Mg2+, PBMCs were suspended in RPMI medium containing 10% FBS and incubated for 60 min at 37 C to allow monocyte attachment to the culture dish. Attached monocytes were then collected as described previously [12]. Peripheral blood monocytes were differentiated to DCs by culturing the cells in a 6well plate in 2 ml of complete medium (RPMI 1640 supplemented with 10% FCS) supplemented with recombinant human GM-CSF (10 ng/ml; Pierce Endogen, Rockford, IL) and recombinant human IL-4 (10 ng/ml; Pierce Endogen, Rockford, IL). All cultures were incubated at 37 C in 5% humidified CO2. After 7 days of culture, DCs were stimulated with 100 ng/ml LPS or 1 lg/ml TgHSP70 for 48 h for maturation. FACS analysis. Cells were incubated with PE-labeled monoclonal antibodies against CD40, CD80, CD86, and FITC-labeled monoclonal antibody against HLA-DR for 30 min, washed with cold-PBS, and analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter, Miami, FL). Measurement of phagocytic activity. Phagocytosis was measured after 48 h treatments of vehicle, LPS, or TgHSP70. Cells were incubated in a 200 ll PBS containing 5% human serum with 1 mg/ ml fluorescein isothiocyanate (FITC)-dextran. After 30 min incubation at 37 C, cells were washed four times in ice-cold PBS and analyzed with a flow cytometer. For a control, cells from each culture condition were also maintained in the same solution for 30 min at 4 C. Cytokine assays. Human monocyte-derived dendritic cells (5 · 105 cells/ml) were treated with vehicle, LPS, or TgHSP70 in the presence or absence of PD98059 (50 lM), or SB203580 (20 lM) for 48 h. Culture supernatants were analyzed by ELISA for IL-12 (p70) according to the instructions from the manufacturer (BD Biosciences Pharmingen, San Diego, CA). Measurement of allostimulatory activity. T cells were purified from human PBMCs using nylon wool columns and were used as responders [13]. Human monocyte-derived dendritic cells, which were treated with vehicle, LPS, or TgHSP70 were used as stimulator cells. A total of 1 · 105 allogeneic T cells were added as responders to stimulator cells (1 · 104, 3 · 103, 1 · 103, or 3 · 102) in 96-well tissue culture plates containing 0.2 ml medium/well. Allostimulatory activity was measured for 3 days and the cells were pulsed with 0.5 lCi [3H]thymidine for the last 18 h. Radioactivity was counted using
To assess whether TgHSP70 cause morphological change in human immature DCs (iDCs), their morphology was observed after 48 h of TgHSP70 treatment. A morphological change of iDCs into mDCs was clearly observed in TgHSP70-treated cells (data not shown). These morphological changes in iDCs suggested that TgHSP70 induces iDC maturation. To further explore this possibility, we looked for phenotypic changes in TgHSP70-treated iDCs by using FACS and specific antibodies against HLA-DR molecules and co-stimulatory molecules (Figs. 1A and B). TgHSP70 treatment enhanced the expression of the HLA-DR molecules and CD80, CD86, and CD40, a response which could be compared with the phenotypic changes in iDCs treated with LPS (Fig. 1 and data not shown). Taken together, these data suggest that TgHSP70 promotes the maturation of iDCs. TgHSP70 reduced the phagocytic activity of DCs Immature DCs capture and process antigens as a consequence of their high endocytic activity, a feature that is lost during maturation [5,14]. Compared with LPS-treated cells, cells treated with TgHSP70 expressed higher levels of CD80 and CD86, which have been reported to be more highly expressed in mature DCs than in immature DCs [1]. This finding that the stimulation of iDCs with TgHSP70 induced higher expressions of CD80 and CD86 led us to investigate whether TgHSP70 stimulation elicits cellular maturation. When we stimulated iDCs with TgHSP70 the phagocytic activity of the cells significantly decreased, suggesting that the cells are mDCs (Fig. 2). This result indicates that the TgHSP70-mediated signaling pathway may be involved in DC maturation. TgHSP70 increases DC allostimulatory capacity DC function can be characterized in part by an ability to stimulate alloreactive T cells by MLR [1]. To determine whether DCs stimulated by TgHSP70 have allostimulatory activity, MLR was performed using TgHSP70-treated iDCs. As shown in Fig. 3, TgHSP70 has allostimulatory activity, which is comparable with that of LPS-treated iDCs at several responder: stimulator ratios. This result indicates that iDCs stimulated by TgHSP70 are functionally mature DCs. In view of the
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Fig. 2. TgHSP70 affects the phagocytic activity of human monocytederived dendritic cells. Human monocyte-derived dendritic cells were cultured for 48 h in the presence of vehicle (bold solid line), LPS (100 ng/ml) (dashed line), or TgHSP70 (1 lg/ml) (solid line) and their phagocytic activity was determined by flow cytometry analysis using FITC-conjugated dextran (A). The shaded area indicates unstained cells (A). Phagocytic activity was quantified by measuring mean fluorescence intensity (B). The results are representative of more than three independent experiments. Fig. 1. Human monocyte-derived dendritic cells treated with TgHSP70 show a mature dendritic cell phenotype. Human monocyte-derived dendritic cells were treated with vehicle alone (A) or treated with TgHSP70 (1 lg/ml) (B) for 48 h. Cell surface expressions of CD40, CD80, CD86, and HLA-DR were determined by flow cytometry analysis (A,B), and the surface expressions of these molecule are presented as mean fluorescence intensities (C). The results are representative of more than three independent experiments.
fact that mature DCs have reduced phagocytic activity as compared to immature DCs, this result correlates well with Fig. 2. TgHSP70-induced DC surface marker changes are polymyxin B-insensitive To assess whether TgHSP70 induce the maturation of DCs via LPS receptors, we investigated the effect of polymyxin B (a potent inhibitor of LPS). At first we found that polymyxin B alone did not affect CD86 expression in DCs (Fig. 4A). When polymyxin B was
Fig. 3. Allostimulatory capacity of human monocyte-derived dendritic cells treated with vehicle, LPS, or TgHSP70. Human monocyte-derived dendritic cells were cultured for 48 h in vehicle, LPS (100 ng/ml), or TgHSP70 (1 lg/ml). Cells were harvested and then used to stimulate 1 · 105 of allogeneic T cells purified from human PBMCs in a 96-well plate. Stimulation was done at several responder: stimulator ratios. Cells were pulsed with [3H]thymidine on day 3, and incorporation was measured after 18 h. Results represent means ± SE counts/min of triplicate results.
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Fig. 5. IL-12 production by TgHSP70-treated human monocytederived dendritic cells. Human monocyte-derived dendritic cells were stimulated with vehicle, LPS (100 ng/ml), or TgHSP70 (1 lg/ml) for 48 h in the absence or presence of polymyxin B (10 lg/ml), and supernatants were harvested from cultures and analyzed for the production of IL-12(p70) by ELISA. Data represent means ± SE of more than three experiments.
at 143.3 ± 1.46 pg/ml (Fig. 5). We also investigated the effect of polymyxin B on LPS- and on TgHSP70-induced IL-12 production. Preincubation of iDCs with 10 lg/ml of polymyxin B prior to LPS addition completely blocked IL-12 production by LPS (Fig. 5), but TgHSP70-induced IL-12 production was unaffected by polymyxin B (Fig. 5), indicating again that TgHSP70 stimulates IL-12 production via a different mechanism than that used by LPS. Fig. 4. Effect of polymyxin B on TgHSP70-induced dendritic cell maturation. Human monocyte-derived dendritic cells were treated with vehicle alone (A), LPS (100 ng/ml) (B), or TgHSP70 (1 lg/ml) (C) for 48 h in the absence or presence of polymyxin B (10 lg/ml). CD86 cell surface expressions were determined by flow cytometry analysis (A–C). Bold solid lines and thin solid lines represent polymyxin B-untreated and polymyxin B-treated cells, respectively. The dashed lines represent unstained control cells. The results are representative of more than three independent experiments.
added to culture, LPS-induced CD86 expression was almost completely inhibited (Fig. 4B), but TgHSP70-induced CD86 expression was unaffected (Fig. 4C). These results indicate that TgHSP70 induces DC maturation via a non-LPS mechanism. TgHSP70 induces the production of IL-12 in a polymyxin B-insensitive manner in DCs Since IL-12 is a key cytokine responsible for polarizing T cells toward the Th1 phenotype [15] and DC maturation, we assessed whether TgHSP70 alters cytokine production in iDCs. When iDCs were stimulated with TgHSP70, IL-12 secretion was significantly increased versus control cells (Fig. 5); the addition of 1 lg/ml TgHSP70 elicited 182.5 ± 5.60 pg/ml of IL-12 production (Fig. 5). As a positive control, we also examined the effect of LPS on IL-12 production in the cells. Stimulation of LPS in iDCs for 48 h caused IL-12 production
TgHSP70 stimulates mitogen-activated protein kinase activity in DCs Mitogen-activated protein kinase (MAPK) has been reported to mediate extracellular signals to the nucleus in various cell types [16]. In this study, we examined whether TgHSP70 stimulates MAPKs by Western blotting with anti-phospho-specific antibodies to each enzyme. When DCs were stimulated with TgHSP70 (at 1 lg/ml) for different times, the phosphorylation level of extracellular signal-regulated protein kinase (ERK) transiently increased, and showed maximal activity after 5–30 min of stimulation (Fig. 6A), and then returned to the baseline 60 min after stimulation (Fig. 6A). Another important MAPK, p38 kinase, was also transiently phosphorylated after TgHSP70 stimulation with kinetics resembling those of ERK phosphorylation (Fig. 6A). The stimulation of DCs with LPS (100 ng/ml) also caused ERK and p38 kinase phosphorylation in a manner similar to TgHSP70 (Fig. 6A). We also investigated the roles of the two MAPKs (ERK and p38 kinase) on TgHSP70-induced DC maturation using two different MAPK inhibitors. SB203580, a selective p38 kinase inhibitor, inhibited TgHSP70-induced IL-12 production by 53% (Fig. 6B) however, PD98059, a selective MEK inhibitor, did not affect TgHSP70-induced IL-12 production. These results indicate that the p38 kinasedependent signaling pathway, and not MEK-dependent
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Fig. 6. Role of MAPKs on TgHSP70-induced dendritic cell maturation. Human monocyte-derived dendritic cells were stimulated with LPS (100 ng/ml) or TgHSP70 (1 lg/ml) for different times. Thirty microgram aliquots of the cell lysates were subjected to SDS–PAGE. This was followed by immunoblot analysis with anti-phospho-ERK, -phosphop38 kinase antibodies (A). Western blot analysis was also performed with anti-ERK antibody (A) to confirm similar sample loadings. The data shown are representative of four independent experiments (A). Human monocyte-derived dendritic cells were pretreated with DMSO, 50 lM PD98059, or 20 lM SB203580 for 1 h and then treated with vehicle, LPS (100 ng/ml) or TgHSP70 (1 lg/ml) for 48 h (B). Supernatants were then collected, and IL-12 release was determined by ELISA. Data represent means ± SE of more than three experiments (B).
ERK pathway, is essentially involved in TgHSP70-induced DC maturation.
Discussion It has been demonstrated that T. gondii infection induces high levels of several cytokines, such as IL-12 and TNF-a. DCs are one of the major cells that produce IL-12 after pathogen-infection, and the injection of T. gondii antigen was also found to rapidly produce IL-12 in experimental animals [17]. In this study, we found that HSP70 derived from T. gondii stimulated IL-12 production in human DCs (Fig. 5). Our results suggest that TgHSP70 is a major DC stimulating antigen in T. gondii infection. HSP70 is a well-conserved protein in all organisms and is a major immunogen in infections caused by various pathogens. Mohamed et al. [3] showed that DNA vaccination with TgHSP70 induces protective immunity against T. gondii infection. Bearing in mind that DCs play a crucial role in immune responses against the vaccination of pathogen-derived
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antigens, our results on DCs maturation and IL-12 production by TgHSP70 suggest that HSP70 derived from T. gondii are critically involved in protective immunity against T. gondii by modulating DC activity. Since, LPS from E. coli was reported to induce the maturation of human monocyte-derived DCs [5], and TgHSP70 was prepared from E. coli, we tested for the possible contamination of LPS in TgHSP70. As shown in Fig. 4, DC maturation by TgHSP70 was not affected by polymyxin B. We also found that IL-12 production in TgHSP70-treated DCs was not inhibited by polymyxin B (Fig. 5). These results ruled out possible contamination by LPS in the TgHSP70 preparation from E. coli. Previous reports have demonstrated that Toll like receptors (TLR)-2 and TLR-4 are cellular receptors for HSP70 [18,19]. Moreover, TLR-4 was reported to be an essential molecule for TgHSP70-induced B cell activation in an experimental mice model [11]. Recently Mun et al. [20] also demonstrated that TLR-2 is an essential molecule for protective immunity against T. gondii infection in an experimental mouse model. These findings provide clues as to the identity of the molecule involved in TgHSP70-induced human monocyte-derived DC maturation. But, the roles of TLR-2 and TLR-4 on TgHSP70-induced DC maturation require clarification. Some reports have suggested the involvement of several other molecules, such as CD14, CD91, and CD40, on HSP70-mediated cellular response [21–23]. Moreover, investigations of the putative roles of CD14, CD91, and CD40 on HSP70-induced DC maturation are required. It is well known that DC maturation can be regulated by MAPKs [24,25]. To investigate the intracellular signaling involved in TgHSP70-induced DC maturation, we checked the effect of TgHSP70 on the phosphorylation of MAPKs. As shown in Fig. 6, the stimulation of DCs with TgHSP70 induced the phosphorylation of two MAPKs (ERK and p38 kinase) in DCs. To elucidate the signaling pathway leading to robust IL-12 production in TgHSP70-treated DCs, we used MAPK inhibitors to assess the amounts of IL-12 produced. Unlike the results obtained using PD98059, IL-12 production in the presence of SB203580 was significantly reduced in TgHSP7-treated DCs (Fig. 6B). These results are in agreement with previous reports of the positive effects of the p38 kinase signaling pathway on IL-12 production, and its influence on DC maturation [24]. However, PD98059 did not affect TgHSP70-induced IL-12 production in DCs (Fig. 6B). These results suggest that p38 kinase signaling but not ERK signaling has a positive effect on TgHSP70-induced IL-12 production in DCs. In summary, this study demonstrates that mature DCs are generated from human monocyte-derived DCs by TgHSP70. Since TgHSP70 has been reported to be a major virulent factor of T. gondii and to be essential for the induction of protective immunity against the
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pathogen, our results on the role of TgHSP70 as a DC maturation-inducing molecule provide clues about the action mechanism of TgHSP70 in immune response. Further studies are needed to elucidate the precise cellular receptor for TgHSP70 and the molecular details of its action mechanism.
[12]
[13]
Acknowledgment This work was supported by the Korea Science and Engineering Foundation through the Medical Science and Engineering Research Center for Cancer Molecular Therapy at Dong-A University.
[14]
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