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Cell Growth & Differentiation Vol. 13, 27-38, January 2002
© 2002 American Association for Cancer Research

Expression of Toll-like Receptors 2 and 4 and CD14 during Differentiation of HL-60 Cells Induced by Phorbol 12-Myristate 13-Acetate and 1{alpha}, 25-Dihydroxy-Vitamin D31

Changlin Li2, Yibing Wang2, Li Gao, Jingsong Zhang, Jie Shao, Shengnian Wang, Weiguo Feng, Xingyu Wang, Minglie Li and Zongliang Chang3

Laboratory of Immune Cells Signaling, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences [C. L., Y. W., J. Z., J. S., S. W., W. F., X. W., Z. C.], and Shanghai Cancer Institute [L. G., M. L.] Shanghai 200031, China


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Macrophages form a crucial bridge between the innate and adaptive immune response. One of their most important functions is to recognize infectious microorganisms. Toll-like receptors (TLRs) are key elements in pathogen recognition, and among them, TLR2 and TLR4 are most discussed. However, expression patterns of TLRs during myeloid cell differentiation to macrophage are unknown. In this study, we examined differentiation in the model human myeloid cell line, HL-60, treated with phorbol 12-myristate 13-acetate (PMA) or VitD3. Expression of TLR2, TLR4, and CD14 were measured by reverse transcription-PCR, RNase protection assay, and fluorescence-activated cell sorter assays. After treatment by PMA (1, 10, and 100 nM) for 12, 24, and 48 h, expression of TLR2 and CD14 mRNA was increased in a time- and dose-dependent manner. However, VitD3 only induced expression of CD14 but not TLR2 in HL-60 cells. TLR4 was expressed constitutively before differentiation and increased slightly after that. Thus, PMA-mediated differentiation of HL-60 cells to macrophages is associated largely with TLR2 expression and, to a much lesser extent, with TLR4. Furthermore, up-regulation of TLR2 and CD14 mRNA expression by PMA was abrogated by a protein kinase C inhibitor, Calphostine C, suggesting the up-regulation of TLR2 and CD14 mRNA is dependent on the activation of protein kinase C. Coexpression of CD14/TLR2 and/or CD14/TLR4 may be essential but not sufficient for the production of tumor necrosis factor-{alpha} in response to lipopolysaccharide in our system.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Innate and adaptive immunity contribute to host responses to meet the pathogen challenge. Macrophages are effector cells of the innate immune system and play a pivotal role in initiating and maintaining the immune response. Macrophages produce numerous biologically active molecules, including cytokines such as TNF-{alpha},4 radicals such as superoxide anions (O2-) or nitric oxide (NO), and eicosanoids such as prostaglandin E2 (1) . These biological activities, however, occur only after cell maturation and activation. Thus, transition of immature macrophages into a mature stage is essential for the initiation of above responses to pathogens.

The permanent promyelocytic leukemia cell line, HL-60, has been used as a model for study of granulocyte/monocyte/macrophage differentiation (2) . This cell line can be induced to differentiate into monocytic cells by incubation with VitD3 (3) or into macrophage-like cells by induction with phorbol esters, such as PMA (4 , 5) , or into granulocytic cells by incubation with RA (6) .

Recognition of pathogens by macrophages enabled the presence of certain conserved structural features of the microbes called pathogen-associated molecular patterns, such as glycolipid of the outer membrane of Gram-negative bacteria, LPS (7) . The interaction of LPS with macrophages is known to occur via CD14 and is strongly enhanced by LPS-binding protein (8) . CD14, however, is a glycosyl-phosphatidylinsitol-archored membrane protein that is unable to transduce an LPS signal attributable to a lack of the cytoplasmic domain.

In 1997, the first human homologue of Drosophila Toll was cloned (9) and has recently been designated TLR4. After that, six other TLR proteins were identified (10, 11, 12) , and now, at least 10 members of this family can be found in the GenBank (13) . The discovery of TLRs introduced the possibility that TLR proteins may serve as transducers in LPS signaling. As a kind of the plasma mammalian receptor, TLRs are now considered as gifts left by evolution to recognize pathogen-associated molecular patterns (7) . However, there is some controversy regarding the relative roles of TLR2 and TLR4 in the cellular response to various pathogens, especially LPS (14, 15, 16, 17, 18) .

Although the expression of TLR2 and TLR4 induced by LPS is reported in several articles (18, 19, 20, 21, 22, 23, 24) , there is still little known about their expression patterns during the course of macrophage differentiation. In the present study, we evaluated and compared the expression patterns of TLR2, TLR4, and CD14 in PMA- and VitD3-induced HL-60 cells. Additionally, the functions of differentiated HL-60 cells in phagocytosis and cytokine release were examined. In addition, an inhibitor of PKC was used to determine whether the expression of these proteins is associated with PKC activity and discussed the possible signal transduction pathway.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
PMA and VitD3 Induced the Monocytic/Macrophage Differentiation of HL-60 Cells.
VitD3- and PMA-induced differentiation of HL-60 cells into monocytic cells and macrophage-like cells have been already reported (3, 4, 5) . We first examined and confirmed the effect of PMA and VitD3 on cell adhesion and spreading, which are hallmarks of macrophage differentiation (5) . HL-60 cells (>90%) exhibited cell adhesion and spreading after a 24-h PMA treatment. VitD3- induced cells were not accompanied by the loss of proliferative capacity and shape change as compared with the untreated HL-60 cells (data not shown). These morphological characteristics are consistent with previous reports (3, 4, 5) . We also examined whether the differentiation competence of HL-60 cells in our case gained the capacity of phagocytosis and induced CD14 expression. As shown in Fig. 1Citation , differentiation with PMA markedly enhanced phagocytic activity as shown by ingestion of fluorescent beads. An increase in phagocytic activity was observed as early as 24 h and gradually increased at 48 and 72 h. However, VitD3-induced differentiated cells showed little increase of phagocytosis even after 72-h incubation with beads.



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Fig. 1. Effect of PMA- and VitD3-induced differentiation on phagocytosis. Cells were cultured in the presence of PMA (10 nM) or VitD3 (100 nM) as described in "Materials and Methods." After the indicated time, cells were washed and incubated in fresh medium supplemented with 10% FCS containing fluorescent beads. The histogram shows the ratio of Geometric Means of the tested samples to negative controls without added beads. Values show means ± SD of three independent experiments.

 
As shown in Fig. 2Citation , there was little expression of CD14 mRNA in undifferentiated HL-60 cells. In the presence of 1, 10, and 100 nM PMA or 1, 10, 100, and 200 nM VitD3 for 24 h, the expression of CD14 mRNA was increased in a dose-dependent manner (Fig. 2A)Citation . In addition, treatment of HL-60 cells with PMA (10 nM) or VitD3 (100 nM) for 12, 24, and 48 h, respectively, resulted in the mRNA increase of CD14 with a time-dependent manner (Fig. 2B)Citation . Compared with CD14 mRNA, CD14 protein was clearly evident as early as 12 h after induction with PMA and 24 h after induction with VitD3 (Fig. 3C)Citation . On the basis of these results, we concluded that the model of differentiation to monocyte/macrophage was well established.



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Fig. 2. Expression of TLR2, TLR4, and CD14 genes induced by PMA or VitD3 in HL-60 cells: dose (A) and time (B) response. In A, HL-60 cells were induced by PMA or VitD3 at different doses as indicated. After 24-h stimulation, total cellular RNA was purified, and RT-PCR was performed as described in "Materials and Methods." The PCR products were separated by electrophoresis on 7% PAGE and visualized by sliver staining. In B, HL-60 cells were induced by PMA or VitD3 at different times as indicated. Expression of TLR2, TLR4, and CD14 mRNA was analyzed by PCR after reverse transcription of total RNA. Results are representative of three different experiments.

 




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Fig. 3. Cell surface expression of TLR2 (A), TLR4 (B), or CD14 (C) in differentiated HL-60 cells by flow cytometry. The HL-60 cells were induced by PMA (10 nM, left column) or VitD3 (100 nM, right column) for indicated time before collection. Cells were stained with goat antihuman TLR2 antibodies and then FITC-conjugated mouse antigoat antibodies (A) or rabbit antihuman TLR4 antibodies and then FITC-conjugated goat antirabbit antibodies (B) or FITC-conjugated mouse antibodies against CD14 (C). The vertical axis represents cell events. Histograms from one representative experiment out of five are shown.

 
Expression Pattern of TLR2 and TLR4 Induced by PMA and VitD3.
Because the above data established that PMA or VitD3 mediated monocyte/macrophage differentiation of HL-60 cells, we examined whether expression patterns of TLR2 and TLR4 mRNAs and proteins were differently regulated during differentiation. Interestingly, it was PMA and not VitD3 that was able to induce the TLR2 gene expression in a time- and dose-dependent manner (Fig. 3ACitation and Fig. 4Citation ). The significant induction of TLR2 in PMA-induced HL-60 was detected at 12 h (~2-fold; Fig. 4ACitation , Lane 3) at the mRNA level and 24 h on the cell surface (Fig. 3A)Citation . However, there was little change of TLR2 expression in VitD3-induced HL-60 cells (Fig. 3ACitation and Fig. 4Citation ). In fact, down-regulation could be seen at mRNA level (Fig. 4)Citation . The expression of mRNA and protein of TLR4 appears to be constitutive and slightly increased in response to PMA (Fig. 3BCitation and Fig. 4Citation ). These results showed that different expression patterns of TLR2 and TLR4 were exhibited by PMA- or VitD3-induced HL-60 cells.



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Fig. 4. Different expression patterns of TLR2 and TLR4 mRNA in PMA-induced and VitD3-induced HL-60 cells. In A, HL-60 cells were cultured with medium alone as control (Ctl, Lane 1), with various doses of PMA (1, 10, 50, and 100 nM) for 24 h (Lanes 2–5) or with various doses of VitD3 (1, 10, 100, and 200 nM) for 24 h (Lanes 6–9). Total RNA was prepared, and then RPA was performed as described in "Materials and Methods." One representative result of three experiments is shown. In B, HL-60 cells were cultured with medium alone (Ctl, Lane 1) as control, with 10 nM PMA for 6, 12, 24, and 48 h (Lanes 2–5), or with 10 nM VitD3 for 6, 12, 24, and 48 h (Lanes 6–9). Total RNA was prepared, and then RPA was performed as described in "Materials and Methods." Finally, the gel was dried, exposed, and quantitated in GIS gel image system. Densitometric analyses were presented as the relative ratio of TLR2:L32 mRNA or TLR4:L32 mRNA. The relative ratio of Ctl was arbitrarily selected as 1. A representative result of three experiments is shown. The probe set was included as a degradation control.

 
The Influence of PKC Inhibitor on TLR2 and TLR4 Gene Expression.
The experiments above provided evidence that the expression pattern of TLR2 and TLR4 was influenced differently by PMA in differentiated HL-60 cells. PMA is a well-known activator for PKC, mimicking the physiological role of diacylaglycerol (25 , 26) . To determine whether PKC was involved in the expression of TLR2 and TLR4, a PKC inhibitor, CC, was used for our investigation. When HL-60 cells were incubated in the presence of CC (50, 500, and 1000 nM), expression of TLR2 and TLR4 was inhibited in a dose-dependent manner (Fig. 5)Citation . The inhibition of TLR2 and TLR4 was already seen at a concentration of 500 nM, and their expression was totally blocked at a higher CC concentration (1000 nM). It is also reported that prolonged exposure to PMA is not necessary to induce macrophage-like differentiation, and significant macrophage-like differentiation occurs in HL-60 cells after PMA exposure as short as 20 min (6) . To evaluate whether the order for the addition of activator and inhibitor might influence the generation of TLR2 and TLR4, experiments for the addition of CC (500 nM) 30 min earlier or later than that of PMA (10 nM) or VitD3 (100 nM) were done. Analysis of the mRNA demonstrated that pretreatment of CC for 30 min blocked the TLR2 expression (Fig. 5B)Citation . The level of protein was also influenced (Fig. 5A1)Citation . However, the addition of CC after PMA treatment for 30 min had little effect on PMA-induced TLR2 expression (Fig. 5A2)Citation . It could be postulated that the initial challenge with PMA determines the expression of TLR2, and it indicates that there may be a positive feedback loop independent of long-time PMA exposure. It is interesting to note that the expression of TLR4 was influenced by the presence of CC regardless of the time of addition of CC (Fig. 5, A1 and A2)Citation , suggesting that the constitutive expression of TLR4 mRNA requires PKC activity.




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Fig. 5. Effect of CC on expression of TLR2 and TLR4 molecules. In A1, HL-60 cells were pretreated with CC (500 nM) for 30 min and induced by PMA (10 nM) for 48 h. In A2, CC was added 30 min later, then PMA and cells were induced for 48 h. Surface expression of TLR2 (left column) or TLR4 (right column) was detected by antibody reaction and flow cytometry as described in "Materials and Methods." The vertical axis represents cell events. Histograms from one representative experiment out of five are shown. In B, Lane 1 represents untreated cells. HL-60 cells were induced with 10 nM PMA or 100 nM VitD3 (Lanes 2 and 3) for 48 h. HL-60 cells treated with 500 nM CC alone for 48 h were showed in Lane 4. Besides, PMA-induced or VitD3-induced HL-60 cells were incubated with three different doses of CC (50, 500, and 1000 nM) for 48 h (Lanes 5–10). Total RNA was then extracted with TRIzol reagent, and RPA was performed as described. One of three independent experiments with similar results is shown.

 
TNF-{alpha} Release.
One feature of monocytes/macrophages is TNF-{alpha} production in response to stimulation with LPS through TLRs. To study the response of differentiated HL-60 cells to LPS, we compared the level of TNF-{alpha} release in normal HL-60 and differentiated HL-60 cells. The supernatant fluids of the cells were collected, and the TNF-{alpha} release was assayed by ELISA. As shown in Fig. 6Citation , undifferentiated HL-60 cells alone ("Ctl") did not release significant amounts of TNF-{alpha}. Stimulation of undifferentiated HL-60 cells with LPS or PMA alone (24, 48, or 72 h) still did not induce the release of TNF-{alpha}. However, the level of TNF-{alpha} was greatly enhanced in PMA-induced cells: it was ~5 ng/2.5 x 105 cells/ml (~100-fold higher than in undifferentiated HL-60 cells) at 24 h after stimulation with LPS (100 ng/ml). Secreted TNF-{alpha} reached the maximum at 48 h and remained high for >=72 h.



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Fig. 6. TNF-{alpha} production of PMA- or VitD3- induced HL-60 cells in response to LPS. HL-60 cells were pretreated with PMA (10 nM) or VitD3 (100 nM) for the indicated times. Then, LPS (100 ng/ml) was or was not added, and cells were incubated for another 24 h. Values are means ±SD of three independent experiments.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
During the last 3 years, there have been significant advances in our understanding of the biology of TLRs. Although TLR2 and TLR4 have generated considerable interest because of their ability to respond to conserved pathogen components, such as LPS (14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24) , there was very little information available regarding the expression pattern of these two proteins in HL60 cells differentiating on treatment with of PMA and VitD3. Here we characterized for the first time TLR2, TLR4, and CD14 expression pattern by using a PMA- and VitD3-mediated differentiation model of human myeloid cell line, HL-60 cells.

As it is known that LPS can trigger TNF-{alpha} release in human monocytes (1) , we hypothesized that TLR2 or TLR4 or both would be expressed during PMA- and VitD3-induced differentiation. However, we were surprised to find that PMA induced the expression of both genes, though at a different level, whereas VitD3 had no effect on their up-regulation. The mechanism responsible for this difference is unclear at present. It would be interesting to clarify the signaling mechanisms involved in the differential effect of VitD3 or PMA. Cell surface expression data paralleled mRNA expression with a little time lag, indicating a process of protein translation was rapidly followed by protein transportation. It is interesting that the expression of TLR4 was constitutive and increased slightly in response to PMA when compared with TLR2 in HL-60 cells differentiated by PMA, demonstrating that the expression event of TLR2 appears at a later stage than that of TLR4 during differentiation of myeloid cells to macrophages. These distinct effects may represent selective biochemical effects superimposed on the differentiation program induced in HL-60 cells.

During the preparation of this manuscript, Liu et al. (19) reported that TLR2 mRNA expression was enhanced in HL-60 cells treated with VitD3 (50 nM) during a 4-day culture. Similar up-regulation by RA of TLR4 expression was reported by Mita et al. (27) . Their results showed that TLR4 expression was slightly induced in RA-treated HL-60 cells (HL-60- derived granulocytic cells) but was strongly induced in IFN-{gamma}-treated HL-60 cells (HL-60-derived monocytic cells) after 3 days in culture (27) . The reason for the apparent discrepancy between our and these reported data could be the use of different inducers, as well as the length of treatment leading to different signaling pathways in regulation of TLR2 and TLR4.

Currently, there is some evidence suggesting that the levels of the expression of TLR4 and TLR2 are differentially regulated in monocytes (23 , 28 , 29) , human endothelial cells (21) , mouse macrophages (22) , and murine or rat liver and hepatocytes (30 , 31) . Furthermore, TLR4 can up-regulate TLR2 expression (32) . These results seem to be generally consistent with our results.

A specific inhibitor, CC, was used to investigate a possible relationship between PKC’s activity and the expression of TLR2 and TLR4. Interestingly, the order in which these reagents were added was important for PMA-induced TLR2 expression. The surface expression of TLR2 was partially brought back to the normal level by the pretreatment with CC, whereas adding the inhibitor 30 min later had little effect on TLR2 surface expression. This suggests that the first short-time challenge, which was sufficient to induce HL-60 differentiation as reported before (6) , might have also been sufficient to induce TLR2 expression in our case. It further suggests that PKC signaling pathways, after first short-time challenge by PMA, may play an important role in the commitment of HL-60 cells toward macrophage-like cell differentiation.

NF-{kappa}B is a critical and pleiotropic transcriptional factor in control of gene expression (33) and differentiation (34) of immune cells. Very recently, two reports indicated that up-regulation of TLR2 gene expression was associated with NF-{kappa}B activation (19 , 35) . This observation suggests that NF-{kappa}B might play an essential role in TLR2 gene expression during cell differentiation.

It is clear from our data that TNF-{alpha} release was strongly enhanced in PMA-differentiated cells but not in VitD3- induced HL-60 monocytic cells in response to LPS, although the latter bears CD14 and TLR4 molecules as pattern recognition receptors for LPS (17 , 36) . The reason for failure of VitD3-induced HL-60 cells to produce TNF-{alpha} is unknown. Additional research in this area is likely to provide us new information.

Taken together, this is the first report that TLR2 and TLR4 expression was up-regulated during the process of PMA-induced HL-60 differentiation. We demonstrated that TLR2, TLR4, and CD14 expression patterns were different in PMA- or VitD3-induced HL-60 differentiation. Our data suggest that different levels of TLR2 and TLR4 expression in myeloid cell differentiation may contribute to monocyte maturation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture and Differentiation.
Human HL-60 promyelocytic leukemia cells (American Type Culture Collection, Manassas, VA) were cultured in RPMI 1640 with 2.05 mM L-Glutamine (HyClone) containing 10% FCS (Life Technologies, Inc.). Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. Differentiation was achieved by resuspending the cells at 5 x 105 cells/ml in culture medium with the addition of PMA or VitD3 (both from Sigma Chemical Co.) at the indicated concentration. In some cases, cells were pretreated with CC (Sigma Chemical Co.) to inhibit PKC activity.

Phagocytosis.
5 x 105 cells/ml HL-60 cells were seeded into dishes with fluorescent microspheres (Duke Scientific, Palo Alto, CA), 150 beads/cell, after treatment with or without PMA (10 nM) or VitD3 (100 nM) for a desired time. Cells were recollected after 40 min and centrifuged at 250 x g for 5 min at 4°C. The supernatant containing the free beads was removed. Then, cells were washed with ice-cold PBS five times and resuspended in 300 µl of PBS. The data from flow cytometry were analyzed with WinMDI 2.8 software.

RT-PCR Analysis.
The procedure followed Qi et al. (37) with little modification. Total RNA was isolated from HL-60 cells using TRIzol reagent (Life Technologies, Inc.). Reverse transcription reaction was performed with RNA PCR Kit (Takara Shuzo Co., Ltd.) at 42°C for 1 h, 99°C for 5 min, and 4°C for 5 min. PCR amplification was performed with Taq polymerase (Promega) for 28 cycles at 95°C for 40 s, 54°C for 40 s, and 72°C for 1 min. PCR primers for TLR2, TLR4, CD14, and glyceraldehyde-3-phosphate dehydrogenase were from Zhang et al. (38) , Murray et al. (39) , and Tokunaga et al. (40) , respectively. The sequences of these primers are listed in Table 1Citation .


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Table 1 Oligonucleotide primers used for gene expression analysis by RT-PCR and RPA

 
Plasmid Construction.
Subclones of TLR2, TLR4, and ribosomal protein L32 (RPL32) were generated by RT-PCR with oligonucleotide primers flanked 5' with HindIII (sense primer) and EcoRI (antisense primer) sites (Table 1)Citation . The primers were designed to generate fragments of a length that could be conveniently separated on a standard polyacrylamide sequencing gel. After PCR, the amplified fragments were digested with HindIII/EcoRI and then ligated into pSP72 (Promega) and sequenced. The orientation of the fragment allows antisense RNA synthesis from flanking T7 RNA polymerase promoter.

RPA.
The RPA was performed using a previously described method (41) . For the synthesis of a radiolabeled antisense RNA probe set for the TLR2, TLR4, and RPL32, in vitro transcription kit (PharMingen) was used to transcribe an equimolar pool of HindIII linearized templates (80-ng total). Probe was dissolved (6 x 105 cpm/µl) in hybridization buffer (PharMingen), and 2 µl of this were added to tubes containing 16 µg of target RNA dissolved in 8 µl of hybridization buffer. All the following procedures were performed as described before (41) .

Protein Expression Analysis.
TLR4 and TLR2: HL-60 cells were resuspended at 1 x 106 cells/ml in PBS and blocked with goat antihuman IgG for 15 min at 4°C (1.8 µg/100 µl; Sino-American Biotechnology, Co.) when analysis of TLR2 expression was carried out. The cells were washed one time with PBS and incubated with goat antihuman TLR2 antibody (dilution 1:30; Santa Cruz Biotechnology, Inc.) or rabbit antihuman TLR4 antibody (dilution 1:200; Santa Cruz Biotechnology, Inc.) in PBS. For preparing negative control, cells were reacted only with goat antihuman IgG when analysis of TLR2 was carried out and PBS when analysis of TLR4 was carried out, respectively. Samples were incubated at 4°C for 1 h. After washing three times with PBS, mouse antigoat FITC-conjugated IgG (dilution 1:120; Santa Cruz Biotechnology, Inc.) or goat antirabbit FITC-conjugated IgG (dilution 1:100; Sino-American Biotechnology, Co.) were added as the second antibody, incubated at 4°C for 30 min in a dark chamber, and rewashed as above. The samples were resuspended in 400 µl of PBS and analyzed by flow cytometer. CD14: HL-60 cells were resuspended at 1 x 106 cells/ml in PBS and stained with mouse monoclonal antihuman CD14 antigen FITC-conjugated antibody (DIACLONE Co.) for 60 min at 4°C. Samples were prepared according to the manufacturer’s instruction. The data from flow cytometry were analyzed by WinMDI 2.8 software.

TNF-{alpha} Release.
HL-60 cells were incubated with PMA or VitD3 for the indicated times (24, 48, or 72 h) in 24-well culture plates (Nunclon, Roskilde, Denmark) at a density of 25 x 104 cells/ml. After that, LPS (100 ng/ml, 055:B5; Sigma Chemical Co.) was added for another 24 h, and the supernatants were recollected for measuring the amount of released TNF-{alpha} by a commercial ELISA kit (Human TNF-{alpha} Set; PharMingen).


    Acknowledgments
 
We thank the staff of the Flow Cytometry Facility of the Institute of Biochemistry and Cell Biology for assistance. We also thank Profs. Yeh Ming for her helpful discussion, T. L. Whiteside of the Department of Pathology, University of Pittsburgh, Pittsburgh, PA, and T. R. Kozel of the Department of Microbiology, University of Nevada, Reno, NV, for expert advice in the preparation and revision of this manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the grant from the Ministry of Science and Technology, China (G1999054201), and the National Natural Science Foundation of China (39870717). Back

2 C. L. and Y. W. contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at the Laboratory of Immune Cells Signaling, Room 1104, Cell Biology Building, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, P. R. China. Phone: 86-21-64315030, extension 2061; Fax: 86-21-64331090; E-mail: immusig{at}sunm.shcnc.ac.cn Back

4 The abbreviations used are: TNF-{alpha}, tumor necrosis factor-{alpha}; VitD3, 1{alpha}, 25-dihydroxyvitamin D3; TLR, Toll-like receptor; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; LPS, lipopolysaccharide; CC, calphostin C; RPA, RNase protection assay; RA, retinoic acid; NF-{kappa}B, nuclear factor {kappa}B; RT-PCR, reverse transcription-PCR. Back

Received for publication 7/23/01. Revision received 11/ 1/01. Accepted for publication 11/ 5/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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